Clustering	NN	O	O
of	NN	O	O
missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
gene	NN	O	O
in	NN	O	O
a	NN	O	O
sporadic	NN	O	B-Disease
T	NN	O	I-Disease
-	NN	O	I-Disease
cell	NN	O	I-Disease
leukaemia	NN	O	I-Disease
.	NN	O	O

Ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
is	NN	O	O
a	NN	O	O
recessive	NN	O	B-Disease
multi	NN	O	I-Disease
-	NN	O	I-Disease
system	NN	O	I-Disease
disorder	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
at	NN	O	O
11q22	NN	O	O
-	NN	O	O
q23	NN	O	O
(	NN	O	O
ref	NN	O	O
.	NN	O	O
3	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
risk	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
,	NN	O	O
especially	NN	O	O
lymphoid	NN	O	B-Disease
neoplasias	NN	O	I-Disease
,	NN	O	O
is	NN	O	O
substantially	NN	O	O
elevated	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
and	NN	O	O
has	NN	O	O
long	NN	O	O
been	NN	O	O
associated	NN	O	O
with	NN	O	O
chromosomal	NN	O	O
instability	NN	O	O
.	NN	O	O

By	NN	O	O
analysing	NN	O	O
tumour	NN	O	B-Disease
DNA	NN	O	O
from	NN	O	O
patients	NN	O	O
with	NN	O	O
sporadic	NN	O	B-Disease
T	NN	O	I-Disease
-	NN	O	I-Disease
cell	NN	O	I-Disease
prolymphocytic	NN	O	I-Disease
leukaemia	NN	O	I-Disease
(	NN	O	O
T	NN	O	B-Disease
-	NN	O	I-Disease
PLL	NN	O	I-Disease
)	NN	O	O
,	NN	O	O
a	NN	O	O
rare	NN	O	O
clonal	NN	O	B-Disease
malignancy	NN	O	I-Disease
with	NN	O	O
similarities	NN	O	O
to	NN	O	O
a	NN	O	O
mature	NN	O	B-Disease
T	NN	O	I-Disease
-	NN	O	I-Disease
cell	NN	O	I-Disease
leukaemia	NN	O	I-Disease
seen	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
demonstrate	NN	O	O
a	NN	O	O
high	NN	O	O
frequency	NN	O	O
of	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
in	NN	O	O
T	NN	O	B-Disease
-	NN	O	I-Disease
PLL	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
marked	NN	O	O
contrast	NN	O	O
to	NN	O	O
the	NN	O	O
ATM	NN	O	O
mutation	NN	O	O
pattern	NN	O	O
in	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
,	NN	O	O
the	NN	O	O
most	NN	O	O
frequent	NN	O	O
nucleotide	NN	O	O
changes	NN	O	O
in	NN	O	O
this	NN	O	O
leukaemia	NN	O	B-Disease
were	NN	O	O
missense	NN	O	O
mutations	NN	O	O
.	NN	O	O

These	NN	O	O
clustered	NN	O	O
in	NN	O	O
the	NN	O	O
region	NN	O	O
corresponding	NN	O	O
to	NN	O	O
the	NN	O	O
kinase	NN	O	O
domain	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
highly	NN	O	O
conserved	NN	O	O
in	NN	O	O
ATM	NN	O	O
-	NN	O	O
related	NN	O	O
proteins	NN	O	O
in	NN	O	O
mouse	NN	O	O
,	NN	O	O
yeast	NN	O	O
and	NN	O	O
Drosophila	NN	O	O
.	NN	O	O

The	NN	O	O
resulting	NN	O	O
amino	NN	O	O
-	NN	O	O
acid	NN	O	O
substitutions	NN	O	O
are	NN	O	O
predicted	NN	O	O
to	NN	O	O
interfere	NN	O	O
with	NN	O	O
ATP	NN	O	O
binding	NN	O	O
or	NN	O	O
substrate	NN	O	O
recognition	NN	O	O
.	NN	O	O

Two	NN	O	O
of	NN	O	O
seventeen	NN	O	O
mutated	NN	O	O
T	NN	O	B-Disease
-	NN	O	I-Disease
PLL	NN	O	I-Disease
samples	NN	O	O
had	NN	O	O
a	NN	O	O
previously	NN	O	O
reported	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
allele	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
no	NN	O	O
mutations	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
p53	NN	O	O
gene	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
this	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
is	NN	O	O
not	NN	O	O
frequently	NN	O	O
altered	NN	O	O
in	NN	O	O
this	NN	O	O
leukaemia	NN	O	B-Disease
.	NN	O	O

Occasional	NN	O	O
missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
ATM	NN	O	O
were	NN	O	O
also	NN	O	O
found	NN	O	O
in	NN	O	O
tumour	NN	O	B-Disease
DNA	NN	O	O
from	NN	O	O
patients	NN	O	O
with	NN	O	O
B	NN	O	B-Disease
-	NN	O	I-Disease
cell	NN	O	I-Disease
non	NN	O	I-Disease
-	NN	O	I-Disease
Hodgkins	NN	O	I-Disease
lymphomas	NN	O	I-Disease
(	NN	O	O
B	NN	O	B-Disease
-	NN	O	I-Disease
NHL	NN	O	I-Disease
)	NN	O	O
and	NN	O	O
a	NN	O	O
B	NN	O	B-Disease
-	NN	O	I-Disease
NHL	NN	O	I-Disease
cell	NN	O	O
line	NN	O	O
.	NN	O	O

The	NN	O	O
evidence	NN	O	O
of	NN	O	O
a	NN	O	O
significant	NN	O	O
proportion	NN	O	O
of	NN	O	O
loss	NN	O	O
-	NN	O	O
of	NN	O	O
-	NN	O	O
function	NN	O	O
mutations	NN	O	O
and	NN	O	O
a	NN	O	O
complete	NN	O	O
absence	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
copy	NN	O	O
of	NN	O	O
ATM	NN	O	O
in	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
mutated	NN	O	O
tumours	NN	O	B-Disease
establishes	NN	O	O
somatic	NN	O	O
inactivation	NN	O	O
of	NN	O	O
this	NN	O	O
gene	NN	O	O
in	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
sporadic	NN	O	B-Disease
T	NN	O	I-Disease
-	NN	O	I-Disease
PLL	NN	O	I-Disease
and	NN	O	O
suggests	NN	O	O
that	NN	O	O
ATM	NN	O	O
acts	NN	O	O
as	NN	O	O
a	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
.	NN	O	O

As	NN	O	O
constitutional	NN	O	O
DNA	NN	O	O
was	NN	O	O
not	NN	O	O
available	NN	O	O
,	NN	O	O
a	NN	O	O
putative	NN	O	O
hereditary	NN	O	O
predisposition	NN	O	O
to	NN	O	O
T	NN	O	B-Disease
-	NN	O	I-Disease
PLL	NN	O	I-Disease
will	NN	O	O
require	NN	O	O
further	NN	O	O
investigation	NN	O	O
.	NN	O	O
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
protein	NN	O	O
kinase	NN	O	O
is	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
modulation	NN	O	O
of	NN	O	O
the	NN	O	O
Ca2	NN	O	O
+	NN	O	O
homeostasis	NN	O	O
in	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
cells	NN	O	O
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
the	NN	O	O
most	NN	O	O
prevalent	NN	O	O
muscular	NN	O	B-Disease
disorder	NN	O	I-Disease
in	NN	O	O
adults	NN	O	O
,	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
-	NN	O	O
repeat	NN	O	O
expansion	NN	O	O
in	NN	O	O
a	NN	O	O
gene	NN	O	O
encoding	NN	O	O
a	NN	O	O
protein	NN	O	O
kinase	NN	O	O
(	NN	O	O
DM	NN	O	B-Disease
protein	NN	O	O
kinase	NN	O	O
;	NN	O	O
DMPK	NN	O	O
)	NN	O	O
and	NN	O	O
involves	NN	O	O
changes	NN	O	O
in	NN	O	O
cytoarchitecture	NN	O	O
and	NN	O	O
ion	NN	O	O
homeostasis	NN	O	O
.	NN	O	O

To	NN	O	O
obtain	NN	O	O
clues	NN	O	O
to	NN	O	O
the	NN	O	O
normal	NN	O	O
biological	NN	O	O
role	NN	O	O
of	NN	O	O
DMPK	NN	O	O
in	NN	O	O
cellular	NN	O	O
ion	NN	O	O
homeostasis	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
compared	NN	O	O
the	NN	O	O
resting	NN	O	O
[	NN	O	O
Ca2	NN	O	O
+	NN	O	O
]	NN	O	O
i	NN	O	O
,	NN	O	O
the	NN	O	O
amplitude	NN	O	O
and	NN	O	O
shape	NN	O	O
of	NN	O	O
depolarization	NN	O	O
-	NN	O	O
induced	NN	O	O
Ca2	NN	O	O
+	NN	O	O
transients	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
content	NN	O	O
of	NN	O	O
ATP	NN	O	O
-	NN	O	O
driven	NN	O	O
ion	NN	O	O
pumps	NN	O	O
in	NN	O	O
cultured	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
cells	NN	O	O
of	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
and	NN	O	O
DMPK	NN	O	O
[	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
]	NN	O	O
knockout	NN	O	O
mice	NN	O	O
.	NN	O	O

In	NN	O	O
vitro	NN	O	O
-	NN	O	O
differentiated	NN	O	O
DMPK	NN	O	O
[	NN	O	O
-	NN	O	O
/	NN	O	O
-	NN	O	O
]	NN	O	O
myotubes	NN	O	O
exhibit	NN	O	O
a	NN	O	O
higher	NN	O	O
resting	NN	O	O
[	NN	O	O
Ca2	NN	O	O
+	NN	O	O
]	NN	O	O
i	NN	O	O
than	NN	O	O
do	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
myotubes	NN	O	O
because	NN	O	O
of	NN	O	O
an	NN	O	O
altered	NN	O	O
open	NN	O	O
probability	NN	O	O
of	NN	O	O
voltage	NN	O	O
-	NN	O	O
dependent	NN	O	O
l	NN	O	O
-	NN	O	O
type	NN	O	O
Ca2	NN	O	O
+	NN	O	O
and	NN	O	O
Na	NN	O	O
+	NN	O	O
channels	NN	O	O
.	NN	O	O

The	NN	O	O
mutant	NN	O	O
myotubes	NN	O	O
exhibit	NN	O	O
smaller	NN	O	O
and	NN	O	O
slower	NN	O	O
Ca2	NN	O	O
+	NN	O	O
responses	NN	O	O
upon	NN	O	O
triggering	NN	O	O
by	NN	O	O
acetylcholine	NN	O	O
or	NN	O	O
high	NN	O	O
external	NN	O	O
K	NN	O	O
+	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
we	NN	O	O
observed	NN	O	O
that	NN	O	O
these	NN	O	O
Ca2	NN	O	O
+	NN	O	O
transients	NN	O	O
partially	NN	O	O
result	NN	O	O
from	NN	O	O
an	NN	O	O
influx	NN	O	O
of	NN	O	O
extracellular	NN	O	O
Ca2	NN	O	O
+	NN	O	O
through	NN	O	O
the	NN	O	O
l	NN	O	O
-	NN	O	O
type	NN	O	O
Ca2	NN	O	O
+	NN	O	O
channel	NN	O	O
.	NN	O	O

Neither	NN	O	O
the	NN	O	O
content	NN	O	O
nor	NN	O	O
the	NN	O	O
activity	NN	O	O
of	NN	O	O
Na	NN	O	O
+	NN	O	O
/	NN	O	O
K	NN	O	O
+	NN	O	O
ATPase	NN	O	O
and	NN	O	O
sarcoplasmic	NN	O	O
reticulum	NN	O	O
Ca2	NN	O	O
+	NN	O	O
-	NN	O	O
ATPase	NN	O	O
are	NN	O	O
affected	NN	O	O
by	NN	O	O
DMPK	NN	O	O
absence	NN	O	O
.	NN	O	O

In	NN	O	O
conclusion	NN	O	O
,	NN	O	O
our	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
DMPK	NN	O	O
is	NN	O	O
involved	NN	O	O
in	NN	O	O
modulating	NN	O	O
the	NN	O	O
initial	NN	O	O
events	NN	O	O
of	NN	O	O
excitation	NN	O	O
-	NN	O	O
contraction	NN	O	O
coupling	NN	O	O
in	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
.	NN	O	O
.	NN	O	O

Constitutional	NN	O	O
RB1	NN	O	O
-	NN	O	O
gene	NN	O	O
mutations	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
isolated	NN	O	O
unilateral	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
most	NN	O	O
patients	NN	O	O
with	NN	O	O
isolated	NN	O	O
unilateral	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
,	NN	O	O
tumor	NN	O	B-Disease
development	NN	O	O
is	NN	O	O
initiated	NN	O	O
by	NN	O	O
somatic	NN	O	O
inactivation	NN	O	O
of	NN	O	O
both	NN	O	O
alleles	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
some	NN	O	O
of	NN	O	O
these	NN	O	O
patients	NN	O	O
can	NN	O	O
transmit	NN	O	O
retinoblastoma	NN	O	B-Disease
predisposition	NN	O	O
to	NN	O	O
their	NN	O	O
offspring	NN	O	O
.	NN	O	O

To	NN	O	O
determine	NN	O	O
the	NN	O	O
frequency	NN	O	O
and	NN	O	O
nature	NN	O	O
of	NN	O	O
constitutional	NN	O	O
RB1	NN	O	O
-	NN	O	O
gene	NN	O	O
mutations	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
isolated	NN	O	O
unilateral	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
analyzed	NN	O	O
DNA	NN	O	O
from	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
and	NN	O	O
from	NN	O	O
tumor	NN	O	B-Disease
tissue	NN	O	O
.	NN	O	O

The	NN	O	O
analysis	NN	O	O
of	NN	O	O
tumors	NN	O	B-Disease
from	NN	O	O
54	NN	O	O
(	NN	O	O
71	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
76	NN	O	O
informative	NN	O	O
patients	NN	O	O
showed	NN	O	O
loss	NN	O	O
of	NN	O	O
constitutional	NN	O	O
heterozygosity	NN	O	O
(	NN	O	O
LOH	NN	O	O
)	NN	O	O
at	NN	O	O
intragenic	NN	O	O
loci	NN	O	O
.	NN	O	O

Three	NN	O	O
of	NN	O	O
13	NN	O	O
uninformative	NN	O	O
patients	NN	O	O
had	NN	O	O
constitutional	NN	O	O
deletions	NN	O	O
.	NN	O	O

For	NN	O	O
39	NN	O	O
randomly	NN	O	O
selected	NN	O	O
tumors	NN	O	B-Disease
,	NN	O	O
SSCP	NN	O	O
,	NN	O	O
hetero	NN	O	O
-	NN	O	O
duplex	NN	O	O
analysis	NN	O	O
,	NN	O	O
sequencing	NN	O	O
,	NN	O	O
and	NN	O	O
Southern	NN	O	O
blot	NN	O	O
analysis	NN	O	O
were	NN	O	O
used	NN	O	O
to	NN	O	O
identify	NN	O	O
mutations	NN	O	O
.	NN	O	O

Mutations	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
21	NN	O	O
(	NN	O	O
91	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
23	NN	O	O
tumors	NN	O	B-Disease
with	NN	O	O
LOH	NN	O	O
.	NN	O	O

In	NN	O	O
6	NN	O	O
(	NN	O	O
38	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
16	NN	O	O
tumors	NN	O	B-Disease
without	NN	O	O
LOH	NN	O	O
,	NN	O	O
one	NN	O	O
mutation	NN	O	O
was	NN	O	O
detected	NN	O	O
,	NN	O	O
and	NN	O	O
in	NN	O	O
9	NN	O	O
(	NN	O	O
56	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
tumors	NN	O	B-Disease
without	NN	O	O
LOH	NN	O	O
,	NN	O	O
both	NN	O	O
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
a	NN	O	O
total	NN	O	O
of	NN	O	O
45	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
tumors	NN	O	B-Disease
of	NN	O	O
36	NN	O	O
patients	NN	O	O
.	NN	O	O

Thirty	NN	O	O
-	NN	O	O
nine	NN	O	O
of	NN	O	O
the	NN	O	O
mutations	NN	O	O
-	NN	O	O
including	NN	O	O
34	NN	O	O
small	NN	O	O
mutations	NN	O	O
,	NN	O	O
2	NN	O	O
large	NN	O	O
structural	NN	O	O
alterations	NN	O	O
,	NN	O	O
and	NN	O	O
hypermethylation	NN	O	O
in	NN	O	O
3	NN	O	O
tumors	NN	O	O
-	NN	O	O
were	NN	O	O
not	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
corresponding	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
DNA	NN	O	O
.	NN	O	O

In	NN	O	O
6	NN	O	O
(	NN	O	O
17	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
36	NN	O	O
patients	NN	O	O
,	NN	O	O
a	NN	O	O
mutation	NN	O	O
was	NN	O	O
detected	NN	O	O
in	NN	O	O
constitutional	NN	O	O
DNA	NN	O	O
,	NN	O	O
and	NN	O	O
1	NN	O	O
of	NN	O	O
these	NN	O	O
mutations	NN	O	O
is	NN	O	O
known	NN	O	O
to	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
reduced	NN	O	O
expressivity	NN	O	O
.	NN	O	O

The	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
constitutional	NN	O	O
mutation	NN	O	O
was	NN	O	O
not	NN	O	O
associated	NN	O	O
with	NN	O	O
an	NN	O	O
early	NN	O	O
age	NN	O	O
at	NN	O	O
treatment	NN	O	O
.	NN	O	O

In	NN	O	O
1	NN	O	O
patient	NN	O	O
,	NN	O	O
somatic	NN	O	O
mosaicism	NN	O	O
was	NN	O	O
demonstrated	NN	O	O
by	NN	O	O
molecular	NN	O	O
analysis	NN	O	O
of	NN	O	O
DNA	NN	O	O
and	NN	O	O
RNA	NN	O	O
from	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
.	NN	O	O

In	NN	O	O
2	NN	O	O
patients	NN	O	O
without	NN	O	O
a	NN	O	O
detectable	NN	O	O
mutation	NN	O	O
in	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
,	NN	O	O
mosaicism	NN	O	O
was	NN	O	O
suggested	NN	O	O
because	NN	O	O
1	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
showed	NN	O	O
multifocal	NN	O	O
tumors	NN	O	B-Disease
and	NN	O	O
the	NN	O	O
other	NN	O	O
later	NN	O	O
developed	NN	O	O
bilateral	NN	O	B-Disease
retinoblastoma	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
conclusion	NN	O	O
,	NN	O	O
our	NN	O	O
results	NN	O	O
emphasize	NN	O	O
that	NN	O	O
the	NN	O	O
manifestation	NN	O	O
and	NN	O	O
transmissibility	NN	O	O
of	NN	O	O
retinoblastoma	NN	O	B-Disease
depend	NN	O	O
on	NN	O	O
the	NN	O	O
nature	NN	O	O
of	NN	O	O
the	NN	O	O
first	NN	O	O
mutation	NN	O	O
,	NN	O	O
its	NN	O	O
time	NN	O	O
in	NN	O	O
development	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
number	NN	O	O
and	NN	O	O
types	NN	O	O
of	NN	O	O
cells	NN	O	O
that	NN	O	O
are	NN	O	O
affected	NN	O	O
.	NN	O	O
.	NN	O	O

Hereditary	NN	O	B-Disease
deficiency	NN	O	I-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
fifth	NN	O	I-Disease
component	NN	O	I-Disease
of	NN	O	I-Disease
complement	NN	O	I-Disease
in	NN	O	O
man	NN	O	O
.	NN	O	O

I	NN	O	O
.	NN	O	O

Clinical	NN	O	O
,	NN	O	O
immunochemical	NN	O	O
,	NN	O	O
and	NN	O	O
family	NN	O	O
studies	NN	O	O
.	NN	O	O

The	NN	O	O
first	NN	O	O
recognized	NN	O	O
human	NN	O	O
kindred	NN	O	O
with	NN	O	O
hereditary	NN	O	B-Disease
deficiency	NN	O	I-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
fifth	NN	O	I-Disease
component	NN	O	I-Disease
of	NN	O	I-Disease
complement	NN	O	I-Disease
(	NN	O	O
C5	NN	O	O
)	NN	O	O
is	NN	O	O
described	NN	O	O
.	NN	O	O

The	NN	O	O
proband	NN	O	O
,	NN	O	O
a	NN	O	O
20	NN	O	O
-	NN	O	O
year	NN	O	O
-	NN	O	O
old	NN	O	O
black	NN	O	O
female	NN	O	O
with	NN	O	O
systemic	NN	O	B-Disease
lupus	NN	O	I-Disease
erythematosus	NN	O	I-Disease
since	NN	O	O
age	NN	O	O
11	NN	O	O
,	NN	O	O
lacked	NN	O	O
serum	NN	O	O
hemolytic	NN	O	O
complement	NN	O	O
activity	NN	O	O
,	NN	O	O
even	NN	O	O
during	NN	O	O
remission	NN	O	O
.	NN	O	O

C5	NN	O	O
was	NN	O	O
undetectable	NN	O	O
in	NN	O	O
her	NN	O	O
serum	NN	O	O
by	NN	O	O
both	NN	O	O
immunodiffusion	NN	O	O
and	NN	O	O
hemolytic	NN	O	O
assays	NN	O	O
.	NN	O	O

Other	NN	O	O
complement	NN	O	O
components	NN	O	O
were	NN	O	O
normal	NN	O	O
during	NN	O	O
remission	NN	O	O
of	NN	O	O
lupus	NN	O	O
,	NN	O	O
but	NN	O	O
C1	NN	O	O
,	NN	O	O
C4	NN	O	O
,	NN	O	O
C2	NN	O	O
,	NN	O	O
and	NN	O	O
C3	NN	O	O
levels	NN	O	O
fell	NN	O	O
during	NN	O	O
exacerbations	NN	O	O
.	NN	O	O

A	NN	O	O
younger	NN	O	O
half	NN	O	O
-	NN	O	O
sister	NN	O	O
,	NN	O	O
who	NN	O	O
had	NN	O	O
no	NN	O	O
underlying	NN	O	O
disease	NN	O	O
,	NN	O	O
was	NN	O	O
also	NN	O	O
found	NN	O	O
to	NN	O	O
lack	NN	O	O
immunochemically	NN	O	O
detectable	NN	O	O
C5	NN	O	O
.	NN	O	O

By	NN	O	O
hemolytic	NN	O	O
assay	NN	O	O
,	NN	O	O
she	NN	O	O
exhibited	NN	O	O
1	NN	O	O
-	NN	O	O
2	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
serum	NN	O	O
C5	NN	O	O
level	NN	O	O
and	NN	O	O
normal	NN	O	O
concentrations	NN	O	O
of	NN	O	O
other	NN	O	O
complement	NN	O	O
components	NN	O	O
.	NN	O	O

C5	NN	O	O
levels	NN	O	O
of	NN	O	O
other	NN	O	O
family	NN	O	O
members	NN	O	O
were	NN	O	O
either	NN	O	O
normal	NN	O	O
or	NN	O	O
approximately	NN	O	O
half	NN	O	O
-	NN	O	O
normal	NN	O	O
,	NN	O	O
consistent	NN	O	O
with	NN	O	O
autosomal	NN	O	O
codominant	NN	O	O
inheritance	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
determining	NN	O	O
C5	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

Normal	NN	O	O
hemolytic	NN	O	O
titers	NN	O	O
were	NN	O	O
restored	NN	O	O
to	NN	O	O
both	NN	O	O
homozygous	NN	O	O
C5	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
(	NN	O	O
C5D	NN	O	B-Disease
)	NN	O	O
sera	NN	O	O
by	NN	O	O
addition	NN	O	O
of	NN	O	O
highly	NN	O	O
purified	NN	O	O
human	NN	O	O
C5	NN	O	O
.	NN	O	O

In	NN	O	O
specific	NN	O	O
C5	NN	O	O
titrations	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
it	NN	O	O
was	NN	O	O
noted	NN	O	O
that	NN	O	O
when	NN	O	O
limited	NN	O	O
amounts	NN	O	O
of	NN	O	O
C5	NN	O	O
were	NN	O	O
assayed	NN	O	O
in	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
low	NN	O	O
dilutions	NN	O	O
of	NN	O	O
either	NN	O	O
C5D	NN	O	B-Disease
serum	NN	O	O
,	NN	O	O
curving	NN	O	O
rather	NN	O	O
than	NN	O	O
linear	NN	O	O
dose	NN	O	O
-	NN	O	O
response	NN	O	O
plots	NN	O	O
were	NN	O	O
consistently	NN	O	O
obtained	NN	O	O
,	NN	O	O
suggesting	NN	O	O
some	NN	O	O
inhibitory	NN	O	O
effect	NN	O	O
.	NN	O	O

Further	NN	O	O
studies	NN	O	O
suggested	NN	O	O
that	NN	O	O
low	NN	O	O
dilutions	NN	O	O
of	NN	O	O
C5D	NN	O	B-Disease
serum	NN	O	O
contain	NN	O	O
a	NN	O	O
factor	NN	O	O
(	NN	O	O
or	NN	O	O
factors	NN	O	O
)	NN	O	O
interfering	NN	O	O
at	NN	O	O
some	NN	O	O
step	NN	O	O
in	NN	O	O
the	NN	O	O
hemolytic	NN	O	O
assay	NN	O	O
of	NN	O	O
C5	NN	O	O
,	NN	O	O
rather	NN	O	O
than	NN	O	O
a	NN	O	O
true	NN	O	O
C5	NN	O	O
inhibitor	NN	O	O
or	NN	O	O
inactivator	NN	O	O
.	NN	O	O

Of	NN	O	O
clinical	NN	O	O
interest	NN	O	O
are	NN	O	O
(	NN	O	O
a	NN	O	O
)	NN	O	O
the	NN	O	O
documentation	NN	O	O
of	NN	O	O
membranous	NN	O	O
glomerulonephritis	NN	O	B-Disease
,	NN	O	O
vasculitis	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
arthritis	NN	O	B-Disease
in	NN	O	O
an	NN	O	O
individual	NN	O	O
lacking	NN	O	O
C5	NN	O	O
(	NN	O	O
and	NN	O	O
its	NN	O	O
biologic	NN	O	O
functions	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
(	NN	O	O
b	NN	O	O
)	NN	O	O
a	NN	O	O
remarkable	NN	O	O
propensity	NN	O	O
to	NN	O	O
bacterial	NN	O	B-Disease
infections	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
proband	NN	O	O
,	NN	O	O
even	NN	O	O
during	NN	O	O
periods	NN	O	O
of	NN	O	O
low	NN	O	O
-	NN	O	O
dose	NN	O	O
or	NN	O	O
alternate	NN	O	O
-	NN	O	O
day	NN	O	O
corticosteroid	NN	O	O
therapy	NN	O	O
.	NN	O	O

Other	NN	O	O
observations	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
C5D	NN	O	B-Disease
state	NN	O	O
is	NN	O	O
compatible	NN	O	O
with	NN	O	O
normal	NN	O	O
coagulation	NN	O	O
function	NN	O	O
and	NN	O	O
the	NN	O	O
capacity	NN	O	O
to	NN	O	O
mount	NN	O	O
a	NN	O	O
neutrophilic	NN	O	O
leukocytosis	NN	O	O
during	NN	O	O
pyogenic	NN	O	B-Disease
infection	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Susceptibility	NN	O	O
to	NN	O	O
ankylosing	NN	O	B-Disease
spondylitis	NN	O	I-Disease
in	NN	O	O
twins	NN	O	O
:	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
genes	NN	O	O
,	NN	O	O
HLA	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
environment	NN	O	O
.	NN	O	O

OBJECTIVE	NN	O	O
To	NN	O	O
determine	NN	O	O
the	NN	O	O
relative	NN	O	O
effects	NN	O	O
of	NN	O	O
genetic	NN	O	O
and	NN	O	O
environmental	NN	O	O
factors	NN	O	O
in	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
ankylosing	NN	O	B-Disease
spondylitis	NN	O	I-Disease
(	NN	O	O
AS	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

METHODS	NN	O	O
Twins	NN	O	O
with	NN	O	O
AS	NN	O	B-Disease
were	NN	O	O
identified	NN	O	O
from	NN	O	O
the	NN	O	O
Royal	NN	O	O
National	NN	O	O
Hospital	NN	O	O
for	NN	O	O
Rheumatic	NN	O	B-Disease
Diseases	NN	O	I-Disease
database	NN	O	O
.	NN	O	O

Clinical	NN	O	O
and	NN	O	O
radiographic	NN	O	O
examinations	NN	O	O
were	NN	O	O
performed	NN	O	O
to	NN	O	O
establish	NN	O	O
diagnoses	NN	O	O
,	NN	O	O
and	NN	O	O
disease	NN	O	O
severity	NN	O	O
was	NN	O	O
assessed	NN	O	O
using	NN	O	O
a	NN	O	O
combination	NN	O	O
of	NN	O	O
validated	NN	O	O
scoring	NN	O	O
systems	NN	O	O
.	NN	O	O

HLA	NN	O	O
typing	NN	O	O
for	NN	O	O
HLA	NN	O	O
-	NN	O	O
B27	NN	O	O
,	NN	O	O
HLA	NN	O	O
-	NN	O	O
B60	NN	O	O
,	NN	O	O
and	NN	O	O
HLA	NN	O	O
-	NN	O	O
DR1	NN	O	O
was	NN	O	O
performed	NN	O	O
by	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
with	NN	O	O
sequence	NN	O	O
-	NN	O	O
specific	NN	O	O
primers	NN	O	O
,	NN	O	O
and	NN	O	O
zygosity	NN	O	O
was	NN	O	O
assessed	NN	O	O
using	NN	O	O
microsatellite	NN	O	O
markers	NN	O	O
.	NN	O	O

Genetic	NN	O	O
and	NN	O	O
environmental	NN	O	O
variance	NN	O	O
components	NN	O	O
were	NN	O	O
assessed	NN	O	O
with	NN	O	O
the	NN	O	O
program	NN	O	O
Mx	NN	O	O
,	NN	O	O
using	NN	O	O
data	NN	O	O
from	NN	O	O
this	NN	O	O
and	NN	O	O
previous	NN	O	O
studies	NN	O	O
of	NN	O	O
twins	NN	O	O
with	NN	O	O
AS	NN	O	B-Disease
.	NN	O	O

RESULTS	NN	O	O
Six	NN	O	O
of	NN	O	O
8	NN	O	O
monozygotic	NN	O	O
(	NN	O	O
MZ	NN	O	O
)	NN	O	O
twin	NN	O	O
pairs	NN	O	O
were	NN	O	O
disease	NN	O	O
concordant	NN	O	O
,	NN	O	O
compared	NN	O	O
with	NN	O	O
4	NN	O	O
of	NN	O	O
15	NN	O	O
B27	NN	O	O
-	NN	O	O
positive	NN	O	O
dizygotic	NN	O	O
(	NN	O	O
DZ	NN	O	O
)	NN	O	O
twin	NN	O	O
pairs	NN	O	O
(	NN	O	O
27	NN	O	O
%	NN	O	O
)	NN	O	O
and	NN	O	O
4	NN	O	O
of	NN	O	O
32	NN	O	O
DZ	NN	O	O
twin	NN	O	O
pairs	NN	O	O
overall	NN	O	O
(	NN	O	O
12	NN	O	O
.	NN	O	O
5	NN	O	O
%	NN	O	O
)	NN	O	O
.	NN	O	O

Nonsignificant	NN	O	O
increases	NN	O	O
in	NN	O	O
similarity	NN	O	O
with	NN	O	O
regard	NN	O	O
to	NN	O	O
age	NN	O	O
at	NN	O	O
disease	NN	O	O
onset	NN	O	O
and	NN	O	O
all	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
severity	NN	O	O
scores	NN	O	O
assessed	NN	O	O
were	NN	O	O
noted	NN	O	O
in	NN	O	O
disease	NN	O	O
-	NN	O	O
concordant	NN	O	O
MZ	NN	O	O
twins	NN	O	O
compared	NN	O	O
with	NN	O	O
concordant	NN	O	O
DZ	NN	O	O
twins	NN	O	O
.	NN	O	O

HLA	NN	O	O
-	NN	O	O
B27	NN	O	O
and	NN	O	O
B60	NN	O	O
were	NN	O	O
associated	NN	O	O
with	NN	O	O
the	NN	O	O
disease	NN	O	O
in	NN	O	O
probands	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
rate	NN	O	O
of	NN	O	O
disease	NN	O	O
concordance	NN	O	O
was	NN	O	O
significantly	NN	O	O
increased	NN	O	O
among	NN	O	O
DZ	NN	O	O
twin	NN	O	O
pairs	NN	O	O
in	NN	O	O
which	NN	O	O
the	NN	O	O
co	NN	O	O
-	NN	O	O
twin	NN	O	O
was	NN	O	O
positive	NN	O	O
for	NN	O	O
both	NN	O	O
B27	NN	O	O
and	NN	O	O
DR1	NN	O	O
.	NN	O	O

Additive	NN	O	O
genetic	NN	O	O
effects	NN	O	O
were	NN	O	O
estimated	NN	O	O
to	NN	O	O
contribute	NN	O	O
97	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
population	NN	O	O
variance	NN	O	O
.	NN	O	O

CONCLUSION	NN	O	O
Susceptibility	NN	O	O
to	NN	O	O
AS	NN	O	B-Disease
is	NN	O	O
largely	NN	O	O
genetically	NN	O	O
determined	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
environmental	NN	O	O
trigger	NN	O	O
for	NN	O	O
the	NN	O	O
disease	NN	O	O
is	NN	O	O
probably	NN	O	O
ubiquitous	NN	O	O
.	NN	O	O

HLA	NN	O	O
-	NN	O	O
B27	NN	O	O
accounts	NN	O	O
for	NN	O	O
a	NN	O	O
minority	NN	O	O
of	NN	O	O
the	NN	O	O
overall	NN	O	O
genetic	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
AS	NN	O	B-Disease
.	NN	O	O

Cell	NN	O	O
cycle	NN	O	O
-	NN	O	O
dependent	NN	O	O
colocalization	NN	O	O
of	NN	O	O
BARD1	NN	O	O
and	NN	O	O
BRCA1	NN	O	O
proteins	NN	O	O
in	NN	O	O
discrete	NN	O	O
nuclear	NN	O	O
domains	NN	O	O
.	NN	O	O

Germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
predispose	NN	O	O
women	NN	O	O
to	NN	O	O
early	NN	O	O
-	NN	O	O
onset	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
by	NN	O	O
compromising	NN	O	O
the	NN	O	O
genes	NN	O	O
presumptive	NN	O	O
function	NN	O	O
as	NN	O	O
a	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
.	NN	O	O

Although	NN	O	O
the	NN	O	O
biochemical	NN	O	O
properties	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
polypeptides	NN	O	O
are	NN	O	O
not	NN	O	O
understood	NN	O	O
,	NN	O	O
their	NN	O	O
expression	NN	O	O
pattern	NN	O	O
and	NN	O	O
subcellular	NN	O	O
localization	NN	O	O
suggest	NN	O	O
a	NN	O	O
role	NN	O	O
in	NN	O	O
cell	NN	O	O
-	NN	O	O
cycle	NN	O	O
regulation	NN	O	O
.	NN	O	O

When	NN	O	O
resting	NN	O	O
cells	NN	O	O
are	NN	O	O
induced	NN	O	O
to	NN	O	O
proliferate	NN	O	O
,	NN	O	O
the	NN	O	O
steady	NN	O	O
-	NN	O	O
state	NN	O	O
levels	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
increase	NN	O	O
in	NN	O	O
late	NN	O	O
G1	NN	O	O
and	NN	O	O
reach	NN	O	O
a	NN	O	O
maximum	NN	O	O
during	NN	O	O
S	NN	O	O
phase	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
in	NN	O	O
S	NN	O	O
phase	NN	O	O
cells	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
polypeptides	NN	O	O
are	NN	O	O
hyperphosphorylated	NN	O	O
and	NN	O	O
accumulate	NN	O	O
into	NN	O	O
discrete	NN	O	O
subnuclear	NN	O	O
foci	NN	O	O
termed	NN	O	O
"	NN	O	O
BRCA1	NN	O	O
nuclear	NN	O	O
dots	NN	O	O
.	NN	O	O

"	NN	O	O
BRCA1	NN	O	O
associates	NN	O	O
in	NN	O	O
vivo	NN	O	O
with	NN	O	O
a	NN	O	O
structurally	NN	O	O
related	NN	O	O
protein	NN	O	O
termed	NN	O	O
BARD1	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
show	NN	O	O
that	NN	O	O
the	NN	O	O
steady	NN	O	O
-	NN	O	O
state	NN	O	O
levels	NN	O	O
of	NN	O	O
BARD1	NN	O	O
,	NN	O	O
unlike	NN	O	O
those	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
remain	NN	O	O
relatively	NN	O	O
constant	NN	O	O
during	NN	O	O
cell	NN	O	O
cycle	NN	O	O
progression	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
immunostaining	NN	O	O
revealed	NN	O	O
that	NN	O	O
BARD1	NN	O	O
resides	NN	O	O
within	NN	O	O
BRCA1	NN	O	O
nuclear	NN	O	O
dots	NN	O	O
during	NN	O	O
S	NN	O	O
phase	NN	O	O
of	NN	O	O
the	NN	O	O
cell	NN	O	O
cycle	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
during	NN	O	O
the	NN	O	O
G1	NN	O	O
phase	NN	O	O
.	NN	O	O

Nevertheless	NN	O	O
,	NN	O	O
BARD1	NN	O	O
polypeptides	NN	O	O
are	NN	O	O
found	NN	O	O
exclusively	NN	O	O
in	NN	O	O
the	NN	O	O
nuclear	NN	O	O
fractions	NN	O	O
of	NN	O	O
both	NN	O	O
G1	NN	O	O
-	NN	O	O
and	NN	O	O
S	NN	O	O
-	NN	O	O
phase	NN	O	O
cells	NN	O	O
.	NN	O	O

Therefore	NN	O	O
,	NN	O	O
progression	NN	O	O
to	NN	O	O
S	NN	O	O
phase	NN	O	O
is	NN	O	O
accompanied	NN	O	O
by	NN	O	O
the	NN	O	O
aggregation	NN	O	O
of	NN	O	O
nuclear	NN	O	O
BARD1	NN	O	O
polypeptides	NN	O	O
into	NN	O	O
BRCA1	NN	O	O
nuclear	NN	O	O
dots	NN	O	O
.	NN	O	O

This	NN	O	O
cell	NN	O	O
cycle	NN	O	O
-	NN	O	O
dependent	NN	O	O
colocalization	NN	O	O
of	NN	O	O
BARD1	NN	O	O
and	NN	O	O
BRCA1	NN	O	O
indicates	NN	O	O
a	NN	O	O
role	NN	O	O
for	NN	O	O
BARD1	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
-	NN	O	O
mediated	NN	O	O
tumor	NN	O	B-Disease
suppression	NN	O	O
.	NN	O	O

Ethnic	NN	O	O
differences	NN	O	O
in	NN	O	O
the	NN	O	O
HFE	NN	O	O
codon	NN	O	O
282	NN	O	O
(	NN	O	O
Cys	NN	O	O
/	NN	O	O
Tyr	NN	O	O
)	NN	O	O
polymorphism	NN	O	O
.	NN	O	O

Recent	NN	O	O
studies	NN	O	O
have	NN	O	O
shown	NN	O	O
that	NN	O	O
hereditary	NN	O	B-Disease
hemochromatosis	NN	O	I-Disease
(	NN	O	O
HH	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
likely	NN	O	O
to	NN	O	O
be	NN	O	O
caused	NN	O	O
by	NN	O	O
homozygosity	NN	O	O
for	NN	O	O
a	NN	O	O
Cys282Tyr	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
HFE	NN	O	O
gene	NN	O	O
located	NN	O	O
4	NN	O	O
.	NN	O	O

5	NN	O	O
Mb	NN	O	O
telomeric	NN	O	O
to	NN	O	O
HLA	NN	O	O
-	NN	O	O
A	NN	O	O
.	NN	O	O

Population	NN	O	O
studies	NN	O	O
of	NN	O	O
this	NN	O	O
polymorphism	NN	O	O
are	NN	O	O
facilitated	NN	O	O
by	NN	O	O
the	NN	O	O
fact	NN	O	O
that	NN	O	O
the	NN	O	O
Cys282Tyr	NN	O	O
mutation	NN	O	O
creates	NN	O	O
a	NN	O	O
Rsal	NN	O	O
restriction	NN	O	O
site	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
studied	NN	O	O
the	NN	O	O
codon	NN	O	O
282	NN	O	O
(	NN	O	O
Cys	NN	O	O
/	NN	O	O
Tyr	NN	O	O
)	NN	O	O
polymorphism	NN	O	O
in	NN	O	O
different	NN	O	O
ethnic	NN	O	O
groups	NN	O	O
.	NN	O	O

In	NN	O	O
agreement	NN	O	O
with	NN	O	O
previous	NN	O	O
observations	NN	O	O
the	NN	O	O
Tyr	NN	O	O
allele	NN	O	O
appeared	NN	O	O
to	NN	O	O
be	NN	O	O
rare	NN	O	O
or	NN	O	O
absent	NN	O	O
in	NN	O	O
Asiatic	NN	O	O
(	NN	O	O
Indian	NN	O	O
,	NN	O	O
Chinese	NN	O	O
)	NN	O	O
populations	NN	O	O
.	NN	O	O

The	NN	O	O
highest	NN	O	O
allele	NN	O	O
frequency	NN	O	O
(	NN	O	O
7	NN	O	O
.	NN	O	O
5	NN	O	O
%	NN	O	O
)	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
Swedes	NN	O	O
.	NN	O	O

Saamis	NN	O	O
(	NN	O	O
2	NN	O	O
%	NN	O	O
)	NN	O	O
and	NN	O	O
Mordvinians	NN	O	O
(	NN	O	O
1	NN	O	O
.	NN	O	O
8	NN	O	O
%	NN	O	O
)	NN	O	O
had	NN	O	O
significantly	NN	O	O
lower	NN	O	O
frequencies	NN	O	O
of	NN	O	O
the	NN	O	O
Tyr	NN	O	O
allele	NN	O	O
.	NN	O	O

Comparisons	NN	O	O
with	NN	O	O
allele	NN	O	O
frequencies	NN	O	O
based	NN	O	O
on	NN	O	O
prevalence	NN	O	O
estimates	NN	O	O
of	NN	O	O
HH	NN	O	B-Disease
showed	NN	O	O
some	NN	O	O
disagreements	NN	O	O
with	NN	O	O
the	NN	O	O
RFLP	NN	O	O
data	NN	O	O
,	NN	O	O
particularly	NN	O	O
in	NN	O	O
Finns	NN	O	O
.	NN	O	O

The	NN	O	O
newly	NN	O	O
described	NN	O	O
HFE	NN	O	O
marker	NN	O	O
provides	NN	O	O
a	NN	O	O
new	NN	O	O
approach	NN	O	O
to	NN	O	O
the	NN	O	O
screening	NN	O	O
of	NN	O	O
HH	NN	O	B-Disease
as	NN	O	O
well	NN	O	O
as	NN	O	O
studies	NN	O	O
of	NN	O	O
the	NN	O	O
relationship	NN	O	O
between	NN	O	O
the	NN	O	O
HFE	NN	O	O
Tyr	NN	O	O
allele	NN	O	O
and	NN	O	O
different	NN	O	O
disorders	NN	O	O
including	NN	O	O
cancer	NN	O	B-Disease

Autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
neurohypophyseal	NN	O	I-Disease
diabetes	NN	O	I-Disease
insipidus	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
encoding	NN	O	O
Gly23	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Val	NN	O	O
in	NN	O	O
neurophysin	NN	O	O
II	NN	O	O
.	NN	O	O

Autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
neurohypophyseal	NN	O	I-Disease
diabetes	NN	O	I-Disease
insipidus	NN	O	I-Disease
(	NN	O	O
ADNDI	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
inherited	NN	O	B-Disease
disease	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
progressive	NN	O	O
degeneration	NN	O	O
of	NN	O	O
the	NN	O	O
magnocellular	NN	O	O
neurons	NN	O	O
of	NN	O	O
the	NN	O	O
hypothalamus	NN	O	O
leading	NN	O	O
to	NN	O	O
decreased	NN	O	O
ability	NN	O	O
to	NN	O	O
produce	NN	O	O
the	NN	O	O
hormone	NN	O	O
arginine	NN	O	O
vasopressin	NN	O	O
(	NN	O	O
AVP	NN	O	O
)	NN	O	O
.	NN	O	O

Affected	NN	O	O
individuals	NN	O	O
are	NN	O	O
not	NN	O	O
symptomatic	NN	O	O
at	NN	O	O
birth	NN	O	O
,	NN	O	O
but	NN	O	O
usually	NN	O	O
develop	NN	O	O
diabetes	NN	O	B-Disease
insipidus	NN	O	I-Disease
at	NN	O	O
1	NN	O	O
-	NN	O	O
6	NN	O	O
yr	NN	O	O
of	NN	O	O
age	NN	O	O
.	NN	O	O

The	NN	O	O
genetic	NN	O	O
locus	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
is	NN	O	O
the	NN	O	O
AVP	NN	O	O
-	NN	O	O
neurophysin	NN	O	O
II	NN	O	O
(	NN	O	O
NPII	NN	O	O
)	NN	O	O
gene	NN	O	O
,	NN	O	O
and	NN	O	O
mutations	NN	O	O
that	NN	O	O
cause	NN	O	O
ADNDI	NN	O	B-Disease
have	NN	O	O
been	NN	O	O
found	NN	O	O
in	NN	O	O
both	NN	O	O
the	NN	O	O
signal	NN	O	O
peptide	NN	O	O
of	NN	O	O
the	NN	O	O
prepro	NN	O	O
-	NN	O	O
AVP	NN	O	O
-	NN	O	O
NPII	NN	O	O
precursor	NN	O	O
and	NN	O	O
within	NN	O	O
NPII	NN	O	O
itself	NN	O	O
.	NN	O	O

An	NN	O	O
affected	NN	O	O
girl	NN	O	O
who	NN	O	O
presented	NN	O	O
at	NN	O	O
9	NN	O	O
months	NN	O	O
of	NN	O	O
age	NN	O	O
and	NN	O	O
her	NN	O	O
similarly	NN	O	O
affected	NN	O	O
younger	NN	O	O
brother	NN	O	O
and	NN	O	O
father	NN	O	O
were	NN	O	O
all	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
a	NN	O	O
novel	NN	O	O
missense	NN	O	O
mutation	NN	O	O
(	NN	O	O
G1758	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
T	NN	O	O
)	NN	O	O
encoding	NN	O	O
the	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitution	NN	O	O
Gly23	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Val	NN	O	O
within	NN	O	O
NPII	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
was	NN	O	O
confirmed	NN	O	O
by	NN	O	O
restriction	NN	O	O
endonuclease	NN	O	O
analysis	NN	O	O
.	NN	O	O

A	NN	O	O
T1	NN	O	O
-	NN	O	O
weighted	NN	O	O
magnetic	NN	O	O
resonance	NN	O	O
imaging	NN	O	O
of	NN	O	O
the	NN	O	O
fathers	NN	O	O
pituitary	NN	O	O
gland	NN	O	O
demonstrates	NN	O	O
an	NN	O	O
attenuated	NN	O	O
posterior	NN	O	O
pituitary	NN	O	O
bright	NN	O	O
spot	NN	O	O
.	NN	O	O

This	NN	O	O
mutation	NN	O	O
may	NN	O	O
be	NN	O	O
valuable	NN	O	O
for	NN	O	O
developing	NN	O	O
models	NN	O	O
of	NN	O	O
dominantly	NN	O	B-Disease
inherited	NN	O	I-Disease
neurodegeneration	NN	O	I-Disease
,	NN	O	O
as	NN	O	O
the	NN	O	O
early	NN	O	O
age	NN	O	O
of	NN	O	O
onset	NN	O	O
of	NN	O	O
symptoms	NN	O	O
suggests	NN	O	O
that	NN	O	O
this	NN	O	O
mutation	NN	O	O
may	NN	O	O
be	NN	O	O
particularly	NN	O	O
deleterious	NN	O	O
to	NN	O	O
the	NN	O	O
magnocellular	NN	O	O
neuron	NN	O	O
.	NN	O	O
.	NN	O	O

Frequent	NN	O	O
inactivation	NN	O	O
of	NN	O	O
PTEN	NN	O	O
/	NN	O	O
MMAC1	NN	O	O
in	NN	O	O
primary	NN	O	O
prostate	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Sporadic	NN	O	B-Disease
prostate	NN	O	I-Disease
carcinoma	NN	O	I-Disease
is	NN	O	O
the	NN	O	O
most	NN	O	O
common	NN	O	O
male	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
Western	NN	O	O
world	NN	O	O
,	NN	O	O
yet	NN	O	O
many	NN	O	O
of	NN	O	O
the	NN	O	O
major	NN	O	O
genetic	NN	O	O
events	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
progression	NN	O	O
of	NN	O	O
this	NN	O	O
often	NN	O	O
fatal	NN	O	O
cancer	NN	O	B-Disease
remain	NN	O	O
to	NN	O	O
be	NN	O	O
elucidated	NN	O	O
.	NN	O	O

Numerous	NN	O	O
cytogenetic	NN	O	O
and	NN	O	O
allelotype	NN	O	O
studies	NN	O	O
have	NN	O	O
reported	NN	O	O
frequent	NN	O	O
loss	NN	O	O
of	NN	O	O
heterozygosity	NN	O	O
on	NN	O	O
chromosomal	NN	O	O
arm	NN	O	O
10q	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
prostate	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Deletion	NN	O	O
mapping	NN	O	O
studies	NN	O	O
have	NN	O	O
unambiguously	NN	O	O
identified	NN	O	O
a	NN	O	O
region	NN	O	O
of	NN	O	O
chromosome	NN	O	O
10q23	NN	O	O
to	NN	O	O
be	NN	O	O
the	NN	O	O
minimal	NN	O	O
area	NN	O	O
of	NN	O	O
loss	NN	O	O
.	NN	O	O

A	NN	O	O
new	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
,	NN	O	O
PTEN	NN	O	O
/	NN	O	O
MMAC1	NN	O	O
,	NN	O	O
was	NN	O	O
isolated	NN	O	O
recently	NN	O	O
at	NN	O	O
this	NN	O	O
region	NN	O	O
of	NN	O	O
chromosome	NN	O	O
10q23	NN	O	O
and	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
inactivated	NN	O	O
by	NN	O	O
mutation	NN	O	O
in	NN	O	O
three	NN	O	O
prostate	NN	O	B-Disease
cancer	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
.	NN	O	O

We	NN	O	O
screened	NN	O	O
80	NN	O	O
prostate	NN	O	B-Disease
tumors	NN	O	I-Disease
by	NN	O	O
microsatellite	NN	O	O
analysis	NN	O	O
and	NN	O	O
found	NN	O	O
chromosome	NN	O	O
10q23	NN	O	O
to	NN	O	O
be	NN	O	O
deleted	NN	O	O
in	NN	O	O
23	NN	O	O
cases	NN	O	O
.	NN	O	O

We	NN	O	O
then	NN	O	O
proceeded	NN	O	O
with	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
entire	NN	O	O
PTEN	NN	O	O
/	NN	O	O
MMAC1	NN	O	O
coding	NN	O	O
region	NN	O	O
and	NN	O	O
tested	NN	O	O
for	NN	O	O
homozygous	NN	O	O
deletion	NN	O	O
with	NN	O	O
new	NN	O	O
intragenic	NN	O	O
markers	NN	O	O
in	NN	O	O
these	NN	O	O
23	NN	O	O
cases	NN	O	O
with	NN	O	O
10q23	NN	O	O
loss	NN	O	O
of	NN	O	O
heterozygosity	NN	O	O
.	NN	O	O

The	NN	O	O
identification	NN	O	O
of	NN	O	O
the	NN	O	O
second	NN	O	O
mutational	NN	O	O
event	NN	O	O
in	NN	O	O
10	NN	O	O
(	NN	O	O
43	NN	O	O
%	NN	O	O
)	NN	O	O
tumors	NN	O	B-Disease
establishes	NN	O	O
PTEN	NN	O	O
/	NN	O	O
MMAC1	NN	O	O
as	NN	O	O
a	NN	O	O
main	NN	O	O
inactivation	NN	O	O
target	NN	O	O
of	NN	O	O
10q	NN	O	O
loss	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
prostate	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Risk	NN	O	O
reversals	NN	O	O
in	NN	O	O
predictive	NN	O	O
testing	NN	O	O
for	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
first	NN	O	O
predictive	NN	O	O
testing	NN	O	O
for	NN	O	O
Huntington	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
HD	NN	O	B-Disease
)	NN	O	O
was	NN	O	O
based	NN	O	O
on	NN	O	O
analysis	NN	O	O
of	NN	O	O
linked	NN	O	O
polymorphic	NN	O	O
DNA	NN	O	O
markers	NN	O	O
to	NN	O	O
estimate	NN	O	O
the	NN	O	O
likelihood	NN	O	O
of	NN	O	O
inheriting	NN	O	O
the	NN	O	O
mutation	NN	O	O
for	NN	O	O
HD	NN	O	B-Disease
.	NN	O	O

Limits	NN	O	O
to	NN	O	O
accuracy	NN	O	O
included	NN	O	O
recombination	NN	O	O
between	NN	O	O
the	NN	O	O
DNA	NN	O	O
markers	NN	O	O
and	NN	O	O
the	NN	O	O
mutation	NN	O	O
,	NN	O	O
pedigree	NN	O	O
structure	NN	O	O
,	NN	O	O
and	NN	O	O
whether	NN	O	O
DNA	NN	O	O
samples	NN	O	O
were	NN	O	O
available	NN	O	O
from	NN	O	O
family	NN	O	O
members	NN	O	O
.	NN	O	O

With	NN	O	O
direct	NN	O	O
tests	NN	O	O
for	NN	O	O
the	NN	O	O
HD	NN	O	B-Disease
mutation	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
assessed	NN	O	O
the	NN	O	O
accuracy	NN	O	O
of	NN	O	O
results	NN	O	O
obtained	NN	O	O
by	NN	O	O
linkage	NN	O	O
approaches	NN	O	O
when	NN	O	O
requested	NN	O	O
to	NN	O	O
do	NN	O	O
so	NN	O	O
by	NN	O	O
the	NN	O	O
test	NN	O	O
individuals	NN	O	O
.	NN	O	O

For	NN	O	O
six	NN	O	O
such	NN	O	O
individuals	NN	O	O
,	NN	O	O
there	NN	O	O
was	NN	O	O
significant	NN	O	O
disparity	NN	O	O
between	NN	O	O
the	NN	O	O
tests	NN	O	O
.	NN	O	O

Three	NN	O	O
went	NN	O	O
from	NN	O	O
a	NN	O	O
decreased	NN	O	O
risk	NN	O	O
to	NN	O	O
an	NN	O	O
increased	NN	O	O
risk	NN	O	O
,	NN	O	O
while	NN	O	O
in	NN	O	O
another	NN	O	O
three	NN	O	O
the	NN	O	O
risk	NN	O	O
was	NN	O	O
decreased	NN	O	O
.	NN	O	O

Knowledge	NN	O	O
of	NN	O	O
the	NN	O	O
potential	NN	O	O
reasons	NN	O	O
for	NN	O	O
these	NN	O	O
changes	NN	O	O
in	NN	O	O
results	NN	O	O
and	NN	O	O
impact	NN	O	O
of	NN	O	O
these	NN	O	O
risk	NN	O	O
reversals	NN	O	O
on	NN	O	O
both	NN	O	O
patients	NN	O	O
and	NN	O	O
the	NN	O	O
counseling	NN	O	O
team	NN	O	O
can	NN	O	O
assist	NN	O	O
in	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
strategies	NN	O	O
for	NN	O	O
the	NN	O	O
prevention	NN	O	O
and	NN	O	O
,	NN	O	O
where	NN	O	O
necessary	NN	O	O
,	NN	O	O
management	NN	O	O
of	NN	O	O
a	NN	O	O
risk	NN	O	O
reversal	NN	O	O
in	NN	O	O
any	NN	O	O
predictive	NN	O	O
testing	NN	O	O
program	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
novel	NN	O	O
common	NN	O	O
missense	NN	O	O
mutation	NN	O	O
G301C	NN	O	O
in	NN	O	O
the	NN	O	O
N	NN	O	O
-	NN	O	O
acetylgalactosamine	NN	O	O
-	NN	O	O
6	NN	O	O
-	NN	O	O
sulfate	NN	O	O
sulfatase	NN	O	O
gene	NN	O	O
in	NN	O	O
mucopolysaccharidosis	NN	O	B-Disease
IVA	NN	O	I-Disease
.	NN	O	O

Mucopolysaccharidosis	NN	O	B-Disease
IVA	NN	O	I-Disease
(	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
lysosomal	NN	O	I-Disease
storage	NN	O	I-Disease
disorder	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
a	NN	O	O
genetic	NN	O	B-Disease
defect	NN	O	I-Disease
in	NN	O	O
N	NN	O	O
-	NN	O	O
acetylgalactosamine	NN	O	O
-	NN	O	O
6	NN	O	O
-	NN	O	O
sulfate	NN	O	O
sulfatase	NN	O	O
(	NN	O	O
GALNS	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
previous	NN	O	O
studies	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
found	NN	O	O
two	NN	O	O
common	NN	O	O
mutations	NN	O	O
in	NN	O	O
Caucasians	NN	O	O
and	NN	O	O
Japanese	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

To	NN	O	O
characterize	NN	O	O
the	NN	O	O
mutational	NN	O	O
spectrum	NN	O	O
in	NN	O	O
various	NN	O	O
ethnic	NN	O	O
groups	NN	O	O
,	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
GALNS	NN	O	O
gene	NN	O	O
in	NN	O	O
Colombian	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
patients	NN	O	O
were	NN	O	O
investigated	NN	O	O
,	NN	O	O
and	NN	O	O
genetic	NN	O	O
backgrounds	NN	O	O
were	NN	O	O
extensively	NN	O	O
analyzed	NN	O	O
to	NN	O	O
identify	NN	O	O
racial	NN	O	O
origin	NN	O	O
,	NN	O	O
based	NN	O	O
on	NN	O	O
mitochondrial	NN	O	O
DNA	NN	O	O
(	NN	O	O
mtDNA	NN	O	O
)	NN	O	O
lineages	NN	O	O
.	NN	O	O

Three	NN	O	O
novel	NN	O	O
missense	NN	O	O
mutations	NN	O	O
never	NN	O	O
identified	NN	O	O
previously	NN	O	O
in	NN	O	O
other	NN	O	O
populations	NN	O	O
and	NN	O	O
found	NN	O	O
in	NN	O	O
16	NN	O	O
out	NN	O	O
of	NN	O	O
19	NN	O	O
Colombian	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
unrelated	NN	O	O
alleles	NN	O	O
account	NN	O	O
for	NN	O	O
84	NN	O	O
.	NN	O	O

2	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
alleles	NN	O	O
in	NN	O	O
this	NN	O	O
study	NN	O	O
.	NN	O	O

The	NN	O	O
G301C	NN	O	O
and	NN	O	O
S162F	NN	O	O
mutations	NN	O	O
account	NN	O	O
for	NN	O	O
68	NN	O	O
.	NN	O	O

4	NN	O	O
%	NN	O	O
and	NN	O	O
10	NN	O	O
.	NN	O	O

5	NN	O	O
%	NN	O	O
of	NN	O	O
mutations	NN	O	O
,	NN	O	O
respectively	NN	O	O
,	NN	O	O
whereas	NN	O	O
the	NN	O	O
remaining	NN	O	O
F69V	NN	O	O
is	NN	O	O
limited	NN	O	O
to	NN	O	O
a	NN	O	O
single	NN	O	O
allele	NN	O	O
.	NN	O	O

The	NN	O	O
skewed	NN	O	O
prevalence	NN	O	O
of	NN	O	O
G301C	NN	O	O
in	NN	O	O
only	NN	O	O
Colombian	NN	O	O
patients	NN	O	O
and	NN	O	O
haplotype	NN	O	O
analysis	NN	O	O
by	NN	O	O
restriction	NN	O	O
fragment	NN	O	O
length	NN	O	O
polymorphisms	NN	O	O
in	NN	O	O
the	NN	O	O
GALNS	NN	O	O
gene	NN	O	O
suggest	NN	O	O
that	NN	O	O
G301C	NN	O	O
originated	NN	O	O
from	NN	O	O
a	NN	O	O
common	NN	O	O
ancestor	NN	O	O
.	NN	O	O

Investigation	NN	O	O
of	NN	O	O
the	NN	O	O
genetic	NN	O	O
background	NN	O	O
by	NN	O	O
means	NN	O	O
of	NN	O	O
mtDNA	NN	O	O
lineages	NN	O	O
indicate	NN	O	O
that	NN	O	O
all	NN	O	O
our	NN	O	O
patients	NN	O	O
are	NN	O	O
probably	NN	O	O
of	NN	O	O
native	NN	O	O
American	NN	O	O
descent	NN	O	O

Low	NN	O	O
frequency	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
45	NN	O	O
German	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
.	NN	O	O

In	NN	O	O
this	NN	O	O
study	NN	O	O
we	NN	O	O
investigated	NN	O	O
45	NN	O	O
German	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
for	NN	O	O
germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
gene	NN	O	O
.	NN	O	O

We	NN	O	O
identified	NN	O	O
four	NN	O	O
germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
three	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
families	NN	O	O
and	NN	O	O
in	NN	O	O
one	NN	O	O
breast	NN	O	B-Disease
-	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
family	NN	O	O
.	NN	O	O
among	NN	O	O
these	NN	O	O
were	NN	O	O
one	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
,	NN	O	O
one	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
,	NN	O	O
one	NN	O	O
novel	NN	O	O
splice	NN	O	O
site	NN	O	O
mutation	NN	O	O
,	NN	O	O
and	NN	O	O
one	NN	O	O
missense	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
missense	NN	O	O
mutation	NN	O	O
was	NN	O	O
also	NN	O	O
found	NN	O	O
in	NN	O	O
2	NN	O	O
.	NN	O	O

8	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
general	NN	O	O
population	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
it	NN	O	O
is	NN	O	O
not	NN	O	O
disease	NN	O	O
associated	NN	O	O
.	NN	O	O

The	NN	O	O
average	NN	O	O
age	NN	O	O
of	NN	O	O
disease	NN	O	O
onset	NN	O	O
in	NN	O	O
those	NN	O	O
families	NN	O	O
harbouring	NN	O	O
causative	NN	O	O
mutations	NN	O	O
was	NN	O	O
between	NN	O	O
32	NN	O	O
.	NN	O	O

3	NN	O	O
and	NN	O	O
37	NN	O	O
.	NN	O	O

4	NN	O	O
years	NN	O	O
,	NN	O	O
whereas	NN	O	O
the	NN	O	O
family	NN	O	O
harbouring	NN	O	O
the	NN	O	O
missense	NN	O	O
mutation	NN	O	O
had	NN	O	O
an	NN	O	O
average	NN	O	O
age	NN	O	O
of	NN	O	O
onset	NN	O	O
of	NN	O	O
51	NN	O	O
.	NN	O	O

2	NN	O	O
years	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
show	NN	O	O
that	NN	O	O
BRCA1	NN	O	O
is	NN	O	O
implicated	NN	O	O
in	NN	O	O
a	NN	O	O
small	NN	O	O
fraction	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
suggesting	NN	O	O
the	NN	O	O
involvement	NN	O	O
of	NN	O	O
another	NN	O	O
susceptibility	NN	O	O
gene	NN	O	O
(	NN	O	O
s	NN	O	O
)	NN	O	O

Paternal	NN	O	O
transmission	NN	O	O
of	NN	O	O
congenital	NN	O	B-Disease
myotonic	NN	O	I-Disease
dystrophy	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
a	NN	O	O
rare	NN	O	O
case	NN	O	O
of	NN	O	O
paternally	NN	O	O
transmitted	NN	O	O
congenital	NN	O	B-Disease
myotonic	NN	O	I-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

The	NN	O	O
proband	NN	O	O
is	NN	O	O
a	NN	O	O
23	NN	O	O
year	NN	O	O
old	NN	O	O
,	NN	O	O
mentally	NN	O	B-Disease
retarded	NN	O	I-Disease
male	NN	O	O
who	NN	O	O
suffers	NN	O	O
severe	NN	O	O
muscular	NN	O	B-Disease
weakness	NN	O	I-Disease
.	NN	O	O

He	NN	O	O
presented	NN	O	O
with	NN	O	O
respiratory	NN	O	O
and	NN	O	O
feeding	NN	O	O
difficulties	NN	O	O
at	NN	O	O
birth	NN	O	O
.	NN	O	O

His	NN	O	O
two	NN	O	O
sibs	NN	O	O
suffer	NN	O	O
from	NN	O	O
childhood	NN	O	O
onset	NN	O	O
DM	NN	O	B-Disease
.	NN	O	O

Their	NN	O	O
late	NN	O	O
father	NN	O	O
had	NN	O	O
the	NN	O	O
adult	NN	O	O
type	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
,	NN	O	O
with	NN	O	O
onset	NN	O	O
around	NN	O	O
30	NN	O	O
years	NN	O	O
.	NN	O	O

Only	NN	O	O
six	NN	O	O
other	NN	O	O
cases	NN	O	O
of	NN	O	O
paternal	NN	O	O
transmission	NN	O	O
of	NN	O	O
congenital	NN	O	B-Disease
DM	NN	O	I-Disease
have	NN	O	O
been	NN	O	O
reported	NN	O	O
recently	NN	O	O
.	NN	O	O

We	NN	O	O
review	NN	O	O
the	NN	O	O
sex	NN	O	O
related	NN	O	O
effects	NN	O	O
on	NN	O	O
transmission	NN	O	O
of	NN	O	O
congenital	NN	O	B-Disease
DM	NN	O	I-Disease
.	NN	O	O

Decreased	NN	O	O
fertility	NN	O	O
of	NN	O	O
males	NN	O	O
with	NN	O	O
adult	NN	O	O
onset	NN	O	O
DM	NN	O	B-Disease
and	NN	O	O
contraction	NN	O	O
of	NN	O	O
the	NN	O	O
repeat	NN	O	O
upon	NN	O	O
male	NN	O	O
transmission	NN	O	O
contribute	NN	O	O
to	NN	O	O
the	NN	O	O
almost	NN	O	O
absent	NN	O	O
occurrence	NN	O	O
of	NN	O	O
paternal	NN	O	O
transmission	NN	O	O
of	NN	O	O
congenital	NN	O	B-Disease
DM	NN	O	I-Disease
.	NN	O	O

Also	NN	O	O
the	NN	O	O
fathers	NN	O	O
of	NN	O	O
the	NN	O	O
reported	NN	O	O
congenitally	NN	O	O
affected	NN	O	O
children	NN	O	O
showed	NN	O	O
,	NN	O	O
on	NN	O	O
average	NN	O	O
,	NN	O	O
shorter	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
lengths	NN	O	O
and	NN	O	O
hence	NN	O	O
less	NN	O	O
severe	NN	O	O
clinical	NN	O	O
symptoms	NN	O	O
than	NN	O	O
the	NN	O	O
mothers	NN	O	O
of	NN	O	O
children	NN	O	O
with	NN	O	O
congenital	NN	O	B-Disease
DM	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
paternal	NN	O	O
transmission	NN	O	O
of	NN	O	O
congenital	NN	O	B-Disease
DM	NN	O	I-Disease
is	NN	O	O
rare	NN	O	O
and	NN	O	O
preferentially	NN	O	O
occurs	NN	O	O
with	NN	O	O
onset	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
past	NN	O	O
30	NN	O	O
years	NN	O	O
in	NN	O	O
the	NN	O	O
father	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
RB1	NN	O	O
gene	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
child	NN	O	O
with	NN	O	O
ectopic	NN	O	B-Disease
intracranial	NN	O	I-Disease
retinoblastoma	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
RB1	NN	O	O
gene	NN	O	O
mutation	NN	O	O
was	NN	O	O
investigated	NN	O	O
in	NN	O	O
a	NN	O	O
child	NN	O	O
with	NN	O	O
ectopic	NN	O	B-Disease
intracranial	NN	O	I-Disease
retinoblastoma	NN	O	I-Disease
using	NN	O	O
DNA	NN	O	O
obtained	NN	O	O
from	NN	O	O
both	NN	O	O
the	NN	O	O
pineal	NN	O	B-Disease
and	NN	O	I-Disease
retinal	NN	O	I-Disease
tumours	NN	O	I-Disease
of	NN	O	O
the	NN	O	O
patient	NN	O	O
.	NN	O	O

A	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
17	NN	O	O
(	NN	O	O
codon	NN	O	O
556	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
present	NN	O	O
homozygously	NN	O	O
in	NN	O	O
both	NN	O	O
the	NN	O	O
retinal	NN	O	B-Disease
and	NN	O	I-Disease
the	NN	O	I-Disease
pineal	NN	O	I-Disease
tumours	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
same	NN	O	O
mutation	NN	O	O
was	NN	O	O
present	NN	O	O
heterozygously	NN	O	O
in	NN	O	O
the	NN	O	O
DNA	NN	O	O
from	NN	O	O
the	NN	O	O
constitutional	NN	O	O
cells	NN	O	O
of	NN	O	O
the	NN	O	O
patient	NN	O	O
,	NN	O	O
proving	NN	O	O
it	NN	O	O
to	NN	O	O
be	NN	O	O
of	NN	O	O
germline	NN	O	O
origin	NN	O	O
.	NN	O	O

The	NN	O	O
initial	NN	O	O
mutation	NN	O	O
was	NN	O	O
shown	NN	O	O
to	NN	O	O
have	NN	O	O
occurred	NN	O	O
in	NN	O	O
the	NN	O	O
paternally	NN	O	O
derived	NN	O	O
RB1	NN	O	O
allele	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
is	NN	O	O
in	NN	O	O
an	NN	O	O
area	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
that	NN	O	O
encodes	NN	O	O
the	NN	O	O
protein	NN	O	O
-	NN	O	O
binding	NN	O	O
region	NN	O	O
known	NN	O	O
as	NN	O	O
the	NN	O	O
pocket	NN	O	O
region	NN	O	O
and	NN	O	O
has	NN	O	O
been	NN	O	O
detected	NN	O	O
in	NN	O	O
other	NN	O	O
cases	NN	O	O
of	NN	O	O
retinoblastoma	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Low	NN	O	O
levels	NN	O	O
of	NN	O	O
beta	NN	O	O
hexosaminidase	NN	O	O
A	NN	O	O
in	NN	O	O
healthy	NN	O	O
individuals	NN	O	O
with	NN	O	O
apparent	NN	O	O
deficiency	NN	O	O
of	NN	O	O
this	NN	O	O
enzyme	NN	O	O
.	NN	O	O

Appreciable	NN	O	O
beta	NN	O	O
hexosaminidase	NN	O	O
A	NN	O	O
(	NN	O	O
hex	NN	O	O
A	NN	O	O
)	NN	O	O
activity	NN	O	O
has	NN	O	O
been	NN	O	O
detected	NN	O	O
in	NN	O	O
cultured	NN	O	O
skin	NN	O	O
fibroblasts	NN	O	O
and	NN	O	O
melanoma	NN	O	B-Disease
tissue	NN	O	O
from	NN	O	O
healthy	NN	O	O
individuals	NN	O	O
previously	NN	O	O
reported	NN	O	O
as	NN	O	O
having	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
hex	NN	O	I-Disease
A	NN	O	I-Disease
activity	NN	O	O
indistinguishable	NN	O	O
from	NN	O	O
that	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
Tay	NN	O	B-Disease
-	NN	O	I-Disease
Sachs	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
TSD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Identification	NN	O	O
and	NN	O	O
quantitation	NN	O	O
of	NN	O	O
hex	NN	O	O
A	NN	O	O
,	NN	O	O
amounting	NN	O	O
to	NN	O	O
3	NN	O	O
.	NN	O	O

5	NN	O	O
%	NN	O	O
-	NN	O	O
6	NN	O	O
.	NN	O	O

9	NN	O	O
%	NN	O	O
of	NN	O	O
total	NN	O	O
beta	NN	O	O
hexosaminidase	NN	O	O
activity	NN	O	O
,	NN	O	O
has	NN	O	O
been	NN	O	O
obtained	NN	O	O
by	NN	O	O
cellulose	NN	O	O
acetate	NN	O	O
gel	NN	O	O
electrophoresis	NN	O	O
,	NN	O	O
DEAE	NN	O	O
-	NN	O	O
cellulose	NN	O	O
ion	NN	O	O
-	NN	O	O
exchange	NN	O	O
chromatography	NN	O	O
,	NN	O	O
radial	NN	O	O
immunodiffusion	NN	O	O
,	NN	O	O
and	NN	O	O
radioimmunoassay	NN	O	O
.	NN	O	O

Previous	NN	O	O
family	NN	O	O
studies	NN	O	O
suggested	NN	O	O
that	NN	O	O
these	NN	O	O
individuals	NN	O	O
may	NN	O	O
be	NN	O	O
compound	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
the	NN	O	O
common	NN	O	O
mutant	NN	O	O
TSD	NN	O	B-Disease
gene	NN	O	O
and	NN	O	O
a	NN	O	O
rare	NN	O	O
(	NN	O	O
allelic	NN	O	O
)	NN	O	O
mutant	NN	O	O
gene	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
the	NN	O	O
postulated	NN	O	O
rate	NN	O	O
mutant	NN	O	O
gene	NN	O	O
appears	NN	O	O
to	NN	O	O
code	NN	O	O
for	NN	O	O
the	NN	O	O
expression	NN	O	O
of	NN	O	O
low	NN	O	O
amounts	NN	O	O
of	NN	O	O
hex	NN	O	O
A	NN	O	O
.	NN	O	O

Heterozygotes	NN	O	O
for	NN	O	O
the	NN	O	O
rare	NN	O	O
mutant	NN	O	O
may	NN	O	O
be	NN	O	O
indistinguishable	NN	O	O
from	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
the	NN	O	O
common	NN	O	O
TSD	NN	O	B-Disease
mutant	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
direct	NN	O	O
visualization	NN	O	O
and	NN	O	O
quantitation	NN	O	O
of	NN	O	O
hex	NN	O	O
A	NN	O	O
by	NN	O	O
the	NN	O	O
methods	NN	O	O
described	NN	O	O
may	NN	O	O
prevent	NN	O	O
false	NN	O	O
-	NN	O	O
positive	NN	O	O
prenatal	NN	O	O
diagnosis	NN	O	O
of	NN	O	O
TSD	NN	O	B-Disease
in	NN	O	O
fetuses	NN	O	O
having	NN	O	O
the	NN	O	O
incomplete	NN	O	O
hex	NN	O	B-Disease
A	NN	O	I-Disease
deficiency	NN	O	I-Disease
of	NN	O	O
the	NN	O	O
type	NN	O	O
described	NN	O	O
in	NN	O	O
the	NN	O	O
four	NN	O	O
healthy	NN	O	O
individuals	NN	O	O

The	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
Smad4	NN	O	O
/	NN	O	O
Dpc4	NN	O	O
is	NN	O	O
required	NN	O	O
for	NN	O	O
gastrulation	NN	O	O
and	NN	O	O
later	NN	O	O
for	NN	O	O
anterior	NN	O	O
development	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
embryo	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
SMAD4	NN	O	O
/	NN	O	O
DPC4	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
,	NN	O	O
a	NN	O	O
key	NN	O	O
signal	NN	O	O
transducer	NN	O	O
in	NN	O	O
most	NN	O	O
TGFbeta	NN	O	O
-	NN	O	O
related	NN	O	O
pathways	NN	O	O
,	NN	O	O
are	NN	O	O
involved	NN	O	O
in	NN	O	O
50	NN	O	O
%	NN	O	O
of	NN	O	O
pancreatic	NN	O	B-Disease
cancers	NN	O	I-Disease
.	NN	O	O

Homozygous	NN	O	O
Smad4	NN	O	O
mutant	NN	O	O
mice	NN	O	O
die	NN	O	O
before	NN	O	O
day	NN	O	O
7	NN	O	O
.	NN	O	O

5	NN	O	O
of	NN	O	O
embryogenesis	NN	O	O
.	NN	O	O

Mutant	NN	O	O
embryos	NN	O	O
have	NN	O	O
reduced	NN	O	O
size	NN	O	O
,	NN	O	O
fail	NN	O	O
to	NN	O	O
gastrulate	NN	O	O
or	NN	O	O
express	NN	O	O
a	NN	O	O
mesodermal	NN	O	O
marker	NN	O	O
,	NN	O	O
and	NN	O	O
show	NN	O	O
abnormal	NN	O	O
visceral	NN	O	O
endoderm	NN	O	O
development	NN	O	O
.	NN	O	O

Growth	NN	O	B-Disease
retardation	NN	O	I-Disease
of	NN	O	O
the	NN	O	O
Smad4	NN	O	O
-	NN	O	O
deficient	NN	O	O
embryos	NN	O	O
results	NN	O	O
from	NN	O	O
reduced	NN	O	O
cell	NN	O	O
proliferation	NN	O	O
rather	NN	O	O
than	NN	O	O
increased	NN	O	O
apoptosis	NN	O	O
.	NN	O	O

Aggregation	NN	O	O
of	NN	O	O
mutant	NN	O	O
Smad4	NN	O	O
ES	NN	O	O
cells	NN	O	O
with	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
tetraploid	NN	O	O
morulae	NN	O	O
rescues	NN	O	O
the	NN	O	O
gastrulation	NN	O	B-Disease
defect	NN	O	I-Disease
.	NN	O	O

These	NN	O	O
results	NN	O	O
indicate	NN	O	O
that	NN	O	O
Smad4	NN	O	O
is	NN	O	O
initially	NN	O	O
required	NN	O	O
for	NN	O	O
the	NN	O	O
differentiation	NN	O	O
of	NN	O	O
the	NN	O	O
visceral	NN	O	O
endoderm	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
gastrulation	NN	O	B-Disease
defect	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
epiblast	NN	O	O
is	NN	O	O
secondary	NN	O	O
and	NN	O	O
non	NN	O	O
-	NN	O	O
cell	NN	O	O
autonomous	NN	O	O
.	NN	O	O

Rescued	NN	O	O
embryos	NN	O	O
show	NN	O	O
severe	NN	O	O
anterior	NN	O	O
truncations	NN	O	O
,	NN	O	O
indicating	NN	O	O
a	NN	O	O
second	NN	O	O
important	NN	O	O
role	NN	O	O
for	NN	O	O
Smad4	NN	O	O
in	NN	O	O
anterior	NN	O	O
patterning	NN	O	O
during	NN	O	O
embryogenesis	NN	O	O
.	NN	O	O

Prevalence	NN	O	O
of	NN	O	O
p16	NN	O	O
and	NN	O	O
CDK4	NN	O	O
germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
48	NN	O	O
melanoma	NN	O	B-Disease
-	NN	O	O
prone	NN	O	O
families	NN	O	O
in	NN	O	O
France	NN	O	O
.	NN	O	O

The	NN	O	O
French	NN	O	O
Familial	NN	O	B-Disease
Melanoma	NN	O	I-Disease
Study	NN	O	O
Group	NN	O	O
.	NN	O	O

Germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
p16	NN	O	O
and	NN	O	O
CDK4	NN	O	O
genes	NN	O	O
have	NN	O	O
been	NN	O	O
reported	NN	O	O
in	NN	O	O
a	NN	O	O
subset	NN	O	O
of	NN	O	O
melanoma	NN	O	B-Disease
pedigrees	NN	O	O
,	NN	O	O
but	NN	O	O
their	NN	O	O
prevalence	NN	O	O
is	NN	O	O
not	NN	O	O
well	NN	O	O
known	NN	O	O
.	NN	O	O

We	NN	O	O
searched	NN	O	O
for	NN	O	O
such	NN	O	O
germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
48	NN	O	O
French	NN	O	O
melanoma	NN	O	B-Disease
-	NN	O	O
prone	NN	O	O
families	NN	O	O
selected	NN	O	O
according	NN	O	O
to	NN	O	O
two	NN	O	O
major	NN	O	O
criteria	NN	O	O
families	NN	O	O
with	NN	O	O
at	NN	O	O
least	NN	O	O
three	NN	O	O
affected	NN	O	O
members	NN	O	O
(	NN	O	O
n	NN	O	O
=	NN	O	O
20	NN	O	O
)	NN	O	O
or	NN	O	O
families	NN	O	O
with	NN	O	O
two	NN	O	O
affected	NN	O	O
members	NN	O	O
,	NN	O	O
one	NN	O	O
of	NN	O	O
them	NN	O	O
affected	NN	O	O
before	NN	O	O
the	NN	O	O
age	NN	O	O
of	NN	O	O
50	NN	O	O
(	NN	O	O
n	NN	O	O
=	NN	O	O
28	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
one	NN	O	O
additional	NN	O	O
minor	NN	O	O
criterion	NN	O	O
.	NN	O	O

Sixteen	NN	O	O
different	NN	O	O
p16	NN	O	O
germline	NN	O	O
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
21	NN	O	O
families	NN	O	O
,	NN	O	O
while	NN	O	O
one	NN	O	O
germline	NN	O	O
mutation	NN	O	O
,	NN	O	O
Arg24His	NN	O	O
,	NN	O	O
was	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
CDK4	NN	O	O
gene	NN	O	O
.	NN	O	O

The	NN	O	O
frequency	NN	O	O
of	NN	O	O
p16	NN	O	O
gene	NN	O	O
mutation	NN	O	O
in	NN	O	O
our	NN	O	O
sample	NN	O	O
(	NN	O	O
44	NN	O	O
%	NN	O	O
)	NN	O	O
is	NN	O	O
among	NN	O	O
the	NN	O	O
highest	NN	O	O
rates	NN	O	O
yet	NN	O	O
reported	NN	O	O
and	NN	O	O
the	NN	O	O
CDK4	NN	O	O
mutation	NN	O	O
is	NN	O	O
the	NN	O	O
second	NN	O	O
mutation	NN	O	O
detected	NN	O	O
in	NN	O	O
this	NN	O	O
gene	NN	O	O
worldwide	NN	O	O
.	NN	O	O

In	NN	O	O
summary	NN	O	O
,	NN	O	O
our	NN	O	O
results	NN	O	O
show	NN	O	O
frequent	NN	O	O
involvement	NN	O	O
of	NN	O	O
the	NN	O	O
p16	NN	O	O
gene	NN	O	O
in	NN	O	O
familial	NN	O	B-Disease
melanoma	NN	O	I-Disease
and	NN	O	O
confirm	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
the	NN	O	O
CDK4	NN	O	O
gene	NN	O	O
as	NN	O	O
a	NN	O	O
melanoma	NN	O	B-Disease
-	NN	O	O
predisposing	NN	O	O
gene	NN	O	O
.	NN	O	O
.	NN	O	O

Progression	NN	O	O
of	NN	O	O
somatic	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
length	NN	O	O
heterogeneity	NN	O	O
in	NN	O	O
the	NN	O	O
blood	NN	O	O
cells	NN	O	O
of	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

The	NN	O	O
genetic	NN	O	O
basis	NN	O	O
of	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
the	NN	O	O
expansion	NN	O	O
of	NN	O	O
an	NN	O	O
unstable	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
in	NN	O	O
the	NN	O	O
34	NN	O	O
UTR	NN	O	O
of	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
protein	NN	O	O
kinase	NN	O	O
gene	NN	O	O
on	NN	O	O
chromosome	NN	O	O
19	NN	O	O
.	NN	O	O

One	NN	O	O
of	NN	O	O
the	NN	O	O
principal	NN	O	O
features	NN	O	O
of	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
mutation	NN	O	O
is	NN	O	O
an	NN	O	O
extraordinarily	NN	O	O
high	NN	O	O
level	NN	O	O
of	NN	O	O
somatic	NN	O	O
mosaicism	NN	O	O
,	NN	O	O
due	NN	O	O
to	NN	O	O
an	NN	O	O
extremely	NN	O	O
high	NN	O	O
degree	NN	O	O
of	NN	O	O
somatic	NN	O	O
instability	NN	O	O
both	NN	O	O
within	NN	O	O
and	NN	O	O
between	NN	O	O
different	NN	O	O
tissues	NN	O	O
.	NN	O	O

This	NN	O	O
instability	NN	O	O
appears	NN	O	O
to	NN	O	O
be	NN	O	O
biased	NN	O	O
towards	NN	O	O
further	NN	O	O
expansion	NN	O	O
and	NN	O	O
continuous	NN	O	O
throughout	NN	O	O
the	NN	O	O
life	NN	O	O
of	NN	O	O
an	NN	O	O
individual	NN	O	O
,	NN	O	O
features	NN	O	O
that	NN	O	O
could	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
the	NN	O	O
progressive	NN	O	O
nature	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O

Although	NN	O	O
increasing	NN	O	O
measured	NN	O	O
allele	NN	O	O
size	NN	O	O
between	NN	O	O
patients	NN	O	O
clearly	NN	O	O
correlates	NN	O	O
with	NN	O	O
an	NN	O	O
increased	NN	O	O
severity	NN	O	O
of	NN	O	O
symptoms	NN	O	O
and	NN	O	O
an	NN	O	O
earlier	NN	O	O
age	NN	O	O
of	NN	O	O
onset	NN	O	O
,	NN	O	O
this	NN	O	O
correlation	NN	O	O
is	NN	O	O
not	NN	O	O
precise	NN	O	O
and	NN	O	O
measured	NN	O	O
allele	NN	O	O
length	NN	O	O
cannot	NN	O	O
be	NN	O	O
used	NN	O	O
as	NN	O	O
an	NN	O	O
accurate	NN	O	O
predictor	NN	O	O
of	NN	O	O
age	NN	O	O
of	NN	O	O
onset	NN	O	O
.	NN	O	O

In	NN	O	O
order	NN	O	O
to	NN	O	O
further	NN	O	O
characterize	NN	O	O
the	NN	O	O
dynamics	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
CTG	NN	O	O
repeat	NN	O	O
somatic	NN	O	O
instability	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
studied	NN	O	O
repeat	NN	O	O
length	NN	O	O
changes	NN	O	O
over	NN	O	O
time	NN	O	O
in	NN	O	O
111	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
patients	NN	O	O
with	NN	O	O
varying	NN	O	O
clinical	NN	O	O
severity	NN	O	O
and	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
size	NN	O	O
over	NN	O	O
time	NN	O	O
intervals	NN	O	O
of	NN	O	O
1	NN	O	O
-	NN	O	O
7	NN	O	O
years	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
found	NN	O	O
a	NN	O	O
direct	NN	O	O
progression	NN	O	O
of	NN	O	O
the	NN	O	O
size	NN	O	O
heterogeneity	NN	O	O
over	NN	O	O
time	NN	O	O
related	NN	O	O
to	NN	O	O
initial	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
size	NN	O	O
and	NN	O	O
the	NN	O	O
time	NN	O	O
interval	NN	O	O
and	NN	O	O
always	NN	O	O
biased	NN	O	O
towards	NN	O	O
further	NN	O	O
expansion	NN	O	O
.	NN	O	O

Attempts	NN	O	O
to	NN	O	O
mathematically	NN	O	O
model	NN	O	O
the	NN	O	O
dynamics	NN	O	O
have	NN	O	O
proved	NN	O	O
only	NN	O	O
partially	NN	O	O
successful	NN	O	O
suggesting	NN	O	O
that	NN	O	O
individual	NN	O	O
specific	NN	O	O
genetic	NN	O	O
and	NN	O	O
/	NN	O	O
or	NN	O	O
environmental	NN	O	O
factors	NN	O	O
also	NN	O	O
play	NN	O	O
a	NN	O	O
role	NN	O	O
in	NN	O	O
somatic	NN	O	O
mosaicism	NN	O	O
.	NN	O	O
.	NN	O	O

Aspartylglucosaminuria	NN	O	B-Disease
among	NN	O	O
Palestinian	NN	O	O
Arabs	NN	O	O
.	NN	O	O

Aspartylglucosaminuria	NN	O	B-Disease
(	NN	O	O
AGU	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
a	NN	O	O
rare	NN	O	O
disorder	NN	O	B-Disease
of	NN	O	I-Disease
glycoprotein	NN	O	I-Disease
metabolism	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
the	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
lysosomal	NN	O	I-Disease
enzyme	NN	O	I-Disease
aspartylglucosaminidase	NN	O	I-Disease
(	NN	O	O
AGA	NN	O	O
)	NN	O	O
.	NN	O	O

AGU	NN	O	B-Disease
is	NN	O	O
inherited	NN	O	O
as	NN	O	O
an	NN	O	O
autosomal	NN	O	O
recessive	NN	O	O
trait	NN	O	O
and	NN	O	O
occurs	NN	O	O
with	NN	O	O
a	NN	O	O
high	NN	O	O
frequency	NN	O	O
in	NN	O	O
Finland	NN	O	O
because	NN	O	O
of	NN	O	O
a	NN	O	O
founder	NN	O	O
effect	NN	O	O
.	NN	O	O

While	NN	O	O
very	NN	O	O
few	NN	O	O
patients	NN	O	O
with	NN	O	O
AGU	NN	O	B-Disease
have	NN	O	O
been	NN	O	O
reported	NN	O	O
from	NN	O	O
non	NN	O	O
-	NN	O	O
Finnish	NN	O	O
origin	NN	O	O
,	NN	O	O
we	NN	O	O
diagnosed	NN	O	O
the	NN	O	O
disorder	NN	O	O
in	NN	O	O
8	NN	O	O
patients	NN	O	O
originating	NN	O	O
from	NN	O	O
3	NN	O	O
unrelated	NN	O	O
families	NN	O	O
,	NN	O	O
all	NN	O	O
Palestinian	NN	O	O
Arabs	NN	O	O
from	NN	O	O
the	NN	O	O
region	NN	O	O
of	NN	O	O
Jerusalem	NN	O	O
.	NN	O	O

The	NN	O	O
clinical	NN	O	O
diagnosis	NN	O	O
of	NN	O	O
AGU	NN	O	B-Disease
is	NN	O	O
often	NN	O	O
difficult	NN	O	O
,	NN	O	O
in	NN	O	O
particular	NN	O	O
early	NN	O	O
in	NN	O	O
the	NN	O	O
course	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
,	NN	O	O
and	NN	O	O
most	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
are	NN	O	O
diagnosed	NN	O	O
after	NN	O	O
the	NN	O	O
age	NN	O	O
of	NN	O	O
5	NN	O	O
years	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
since	NN	O	O
these	NN	O	O
patients	NN	O	O
excrete	NN	O	O
early	NN	O	O
large	NN	O	O
amounts	NN	O	O
of	NN	O	O
aspartylglucosamine	NN	O	O
in	NN	O	O
urine	NN	O	O
,	NN	O	O
biochemical	NN	O	O
screening	NN	O	O
is	NN	O	O
easy	NN	O	O
by	NN	O	O
urine	NN	O	O
chromatography	NN	O	O
.	NN	O	O
.	NN	O	O

Detection	NN	O	O
of	NN	O	O
heterozygous	NN	O	O
carriers	NN	O	O
of	NN	O	O
the	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
ATM	NN	O	O
)	NN	O	O
gene	NN	O	O
by	NN	O	O
G2	NN	O	O
phase	NN	O	O
chromosomal	NN	O	O
radiosensitivity	NN	O	O
of	NN	O	O
peripheral	NN	O	O
blood	NN	O	O
lymphocytes	NN	O	O
.	NN	O	O

In	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
patients	NN	O	O
,	NN	O	O
mutations	NN	O	O
in	NN	O	O
a	NN	O	O
single	NN	O	O
gene	NN	O	O
,	NN	O	O
ATM	NN	O	O
,	NN	O	O
result	NN	O	O
in	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
syndrome	NN	O	I-Disease
that	NN	O	O
embraces	NN	O	O
a	NN	O	O
variety	NN	O	O
of	NN	O	O
clinical	NN	O	O
features	NN	O	O
and	NN	O	O
manifests	NN	O	O
extreme	NN	O	O
radiosensitivity	NN	O	O
and	NN	O	O
a	NN	O	O
strong	NN	O	O
pre	NN	O	O
-	NN	O	O
disposition	NN	O	O
to	NN	O	O
malignancy	NN	O	B-Disease
.	NN	O	O

Heterozygotes	NN	O	O
for	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
have	NN	O	O
no	NN	O	O
clinical	NN	O	O
expression	NN	O	O
of	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
but	NN	O	O
may	NN	O	O
be	NN	O	O
cancer	NN	O	B-Disease
prone	NN	O	O
with	NN	O	O
a	NN	O	O
moderate	NN	O	O
increase	NN	O	O
in	NN	O	O
in	NN	O	O
vitro	NN	O	O
radiosensitivity	NN	O	O
.	NN	O	O

We	NN	O	O
performed	NN	O	O
a	NN	O	O
blind	NN	O	O
chromosomal	NN	O	O
analysis	NN	O	O
on	NN	O	O
G2	NN	O	O
-	NN	O	O
phase	NN	O	O
lymphocytes	NN	O	O
from	NN	O	O
7	NN	O	O
unrelated	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
,	NN	O	O
13	NN	O	O
obligate	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
heterozygotes	NN	O	O
(	NN	O	O
parents	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
14	NN	O	O
normal	NN	O	O
controls	NN	O	O
following	NN	O	O
X	NN	O	O
-	NN	O	O
irradiation	NN	O	O
with	NN	O	O
1	NN	O	O
Gy	NN	O	O
in	NN	O	O
order	NN	O	O
to	NN	O	O
evaluate	NN	O	O
this	NN	O	O
cytogenetic	NN	O	O
method	NN	O	O
as	NN	O	O
a	NN	O	O
tool	NN	O	O
for	NN	O	O
detection	NN	O	O
of	NN	O	O
ATM	NN	O	O
carriers	NN	O	O
.	NN	O	O

Both	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
homozygotes	NN	O	O
and	NN	O	O
heterozygotes	NN	O	O
showed	NN	O	O
significantly	NN	O	O
increased	NN	O	O
levels	NN	O	O
of	NN	O	O
radiation	NN	O	O
-	NN	O	O
induced	NN	O	O
chromatid	NN	O	O
damage	NN	O	O
relative	NN	O	O
to	NN	O	O
that	NN	O	O
of	NN	O	O
normal	NN	O	O
controls	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
show	NN	O	O
that	NN	O	O
the	NN	O	O
G2	NN	O	O
-	NN	O	O
phase	NN	O	O
chromosomal	NN	O	O
radiosensitivity	NN	O	O
assay	NN	O	O
can	NN	O	O
be	NN	O	O
used	NN	O	O
for	NN	O	O
the	NN	O	O
detection	NN	O	O
of	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
heterozygotes	NN	O	O
.	NN	O	O

In	NN	O	O
combination	NN	O	O
with	NN	O	O
molecular	NN	O	O
genetic	NN	O	O
analyses	NN	O	O
,	NN	O	O
this	NN	O	O
test	NN	O	O
may	NN	O	O
be	NN	O	O
of	NN	O	O
value	NN	O	O
in	NN	O	O
studies	NN	O	O
of	NN	O	O
familial	NN	O	B-Disease
and	NN	O	I-Disease
sporadic	NN	O	I-Disease
cancers	NN	O	I-Disease
aimed	NN	O	O
at	NN	O	O
determination	NN	O	O
of	NN	O	O
the	NN	O	O
potential	NN	O	O
involvement	NN	O	O
of	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
in	NN	O	O
tumor	NN	O	B-Disease
risk	NN	O	O
or	NN	O	O
development	NN	O	O
.	NN	O	O
.	NN	O	O

Ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
:	NN	O	O
identification	NN	O	O
and	NN	O	O
detection	NN	O	O
of	NN	O	O
founder	NN	O	O
-	NN	O	O
effect	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
in	NN	O	O
ethnic	NN	O	O
populations	NN	O	O
.	NN	O	O

To	NN	O	O
facilitate	NN	O	O
the	NN	O	O
evaluation	NN	O	O
of	NN	O	O
ATM	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
other	NN	O	O
diseases	NN	O	O
,	NN	O	O
such	NN	O	O
as	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
have	NN	O	O
attempted	NN	O	O
to	NN	O	O
define	NN	O	O
the	NN	O	O
most	NN	O	O
common	NN	O	O
mutations	NN	O	O
and	NN	O	O
their	NN	O	O
frequencies	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
homozygotes	NN	O	O
from	NN	O	O
10	NN	O	O
ethnic	NN	O	O
populations	NN	O	O
.	NN	O	O

Both	NN	O	O
genomic	NN	O	O
mutations	NN	O	O
and	NN	O	O
their	NN	O	O
effects	NN	O	O
on	NN	O	O
cDNA	NN	O	O
were	NN	O	O
characterized	NN	O	O
.	NN	O	O

Protein	NN	O	O
-	NN	O	O
truncation	NN	O	O
testing	NN	O	O
of	NN	O	O
the	NN	O	O
entire	NN	O	O
ATM	NN	O	O
cDNA	NN	O	O
detected	NN	O	O
92	NN	O	O
(	NN	O	O
66	NN	O	O
%	NN	O	O
)	NN	O	O
truncating	NN	O	O
mutations	NN	O	O
in	NN	O	O
140	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
screened	NN	O	O
.	NN	O	O

The	NN	O	O
haplotyping	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
identical	NN	O	O
mutations	NN	O	O
indicates	NN	O	O
that	NN	O	O
almost	NN	O	O
all	NN	O	O
of	NN	O	O
these	NN	O	O
represent	NN	O	O
common	NN	O	O
ancestry	NN	O	O
and	NN	O	O
that	NN	O	O
very	NN	O	O
few	NN	O	O
spontaneously	NN	O	O
recurring	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
exist	NN	O	O
.	NN	O	O

Assays	NN	O	O
requiring	NN	O	O
minimal	NN	O	O
amounts	NN	O	O
of	NN	O	O
genomic	NN	O	O
DNA	NN	O	O
were	NN	O	O
designed	NN	O	O
to	NN	O	O
allow	NN	O	O
rapid	NN	O	O
screening	NN	O	O
for	NN	O	O
common	NN	O	O
ethnic	NN	O	O
mutations	NN	O	O
.	NN	O	O

These	NN	O	O
rapid	NN	O	O
assays	NN	O	O
detected	NN	O	O
mutations	NN	O	O
in	NN	O	O
76	NN	O	O
%	NN	O	O
of	NN	O	O
Costa	NN	O	O
Rican	NN	O	O
patients	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
,	NN	O	O
50	NN	O	O
%	NN	O	O
of	NN	O	O
Norwegian	NN	O	O
patients	NN	O	O
(	NN	O	O
1	NN	O	O
)	NN	O	O
,	NN	O	O
25	NN	O	O
%	NN	O	O
of	NN	O	O
Polish	NN	O	O
patients	NN	O	O
(	NN	O	O
4	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
14	NN	O	O
%	NN	O	O
of	NN	O	O
Italian	NN	O	O
patients	NN	O	O
(	NN	O	O
1	NN	O	O
)	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
in	NN	O	O
patients	NN	O	O
of	NN	O	O
Amish	NN	O	O
/	NN	O	O
Mennonite	NN	O	O
and	NN	O	O
Irish	NN	O	O
English	NN	O	O
backgrounds	NN	O	O
.	NN	O	O

Additional	NN	O	O
mutations	NN	O	O
were	NN	O	O
observed	NN	O	O
in	NN	O	O
Japanese	NN	O	O
,	NN	O	O
Utah	NN	O	O
Mormon	NN	O	O
,	NN	O	O
and	NN	O	O
African	NN	O	O
American	NN	O	O
patients	NN	O	O
.	NN	O	O

These	NN	O	O
assays	NN	O	O
should	NN	O	O
facilitate	NN	O	O
screening	NN	O	O
for	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
heterozygotes	NN	O	O
in	NN	O	O
the	NN	O	O
populations	NN	O	O
studied	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
tumor	NN	O	I-Disease
suppressor	NN	O	O
gene	NN	O	O
is	NN	O	O
required	NN	O	O
for	NN	O	O
cell	NN	O	O
cycle	NN	O	O
exit	NN	O	O
upon	NN	O	O
serum	NN	O	O
withdrawal	NN	O	O
.	NN	O	O

The	NN	O	O
inactivation	NN	O	O
of	NN	O	O
the	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
(	NN	O	I-Disease
VHL	NN	O	I-Disease
)	NN	O	I-Disease
tumor	NN	O	I-Disease
suppressor	NN	O	O
gene	NN	O	O
predisposes	NN	O	O
affected	NN	O	O
individuals	NN	O	O
to	NN	O	O
the	NN	O	O
human	NN	O	O
VHL	NN	O	B-Disease
cancer	NN	O	I-Disease
syndrome	NN	O	I-Disease
and	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
sporadic	NN	O	B-Disease
renal	NN	O	I-Disease
cell	NN	O	I-Disease
carcinomas	NN	O	I-Disease
(	NN	O	O
RCC	NN	O	B-Disease
)	NN	O	O
and	NN	O	O
brain	NN	O	B-Disease
hemangioblastomas	NN	O	I-Disease
.	NN	O	O

VHL	NN	O	O
-	NN	O	O
negative	NN	O	O
786	NN	O	O
-	NN	O	O
0	NN	O	O
RCC	NN	O	B-Disease
cells	NN	O	O
are	NN	O	O
tumorigenic	NN	O	O
in	NN	O	O
nude	NN	O	O
mice	NN	O	O
which	NN	O	O
is	NN	O	O
suppressed	NN	O	O
by	NN	O	O
the	NN	O	O
reintroduction	NN	O	O
of	NN	O	O
VHL	NN	O	B-Disease
.	NN	O	O

Remarkably	NN	O	O
,	NN	O	O
this	NN	O	O
occurs	NN	O	O
without	NN	O	O
affecting	NN	O	O
the	NN	O	O
growth	NN	O	O
rate	NN	O	O
and	NN	O	O
cell	NN	O	O
cycle	NN	O	O
profile	NN	O	O
of	NN	O	O
these	NN	O	O
cells	NN	O	O
in	NN	O	O
culture	NN	O	O
.	NN	O	O

The	NN	O	O
786	NN	O	O
-	NN	O	O
0	NN	O	O
cell	NN	O	O
line	NN	O	O
,	NN	O	O
like	NN	O	O
many	NN	O	O
cancer	NN	O	B-Disease
cells	NN	O	O
,	NN	O	O
fails	NN	O	O
to	NN	O	O
exit	NN	O	O
the	NN	O	O
cell	NN	O	O
cycle	NN	O	O
upon	NN	O	O
serum	NN	O	O
withdrawal	NN	O	O
.	NN	O	O

Here	NN	O	O
,	NN	O	O
it	NN	O	O
is	NN	O	O
shown	NN	O	O
that	NN	O	O
reintroduction	NN	O	O
of	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
restores	NN	O	O
the	NN	O	O
ability	NN	O	O
of	NN	O	O
VHL	NN	O	O
-	NN	O	O
negative	NN	O	O
RCC	NN	O	B-Disease
cancer	NN	O	I-Disease
cells	NN	O	O
to	NN	O	O
exit	NN	O	O
the	NN	O	O
cell	NN	O	O
cycle	NN	O	O
and	NN	O	O
enter	NN	O	O
G0	NN	O	O
/	NN	O	O
quiescence	NN	O	O
in	NN	O	O
low	NN	O	O
serum	NN	O	O
.	NN	O	O

Both	NN	O	O
VHL	NN	O	O
-	NN	O	O
positive	NN	O	O
and	NN	O	O
VHL	NN	O	O
-	NN	O	O
negative	NN	O	O
RCC	NN	O	B-Disease
cells	NN	O	O
exit	NN	O	O
the	NN	O	O
cell	NN	O	O
cycle	NN	O	O
by	NN	O	O
contact	NN	O	O
inhibition	NN	O	O
.	NN	O	O

The	NN	O	O
cyclin	NN	O	O
-	NN	O	O
dependent	NN	O	O
kinase	NN	O	O
inhibitor	NN	O	O
,	NN	O	O
p27	NN	O	O
,	NN	O	O
accumulates	NN	O	O
upon	NN	O	O
serum	NN	O	O
withdrawal	NN	O	O
,	NN	O	O
only	NN	O	O
in	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
VHL	NN	O	B-Disease
,	NN	O	O
as	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
the	NN	O	O
stabilization	NN	O	O
of	NN	O	O
the	NN	O	O
protein	NN	O	O
.	NN	O	O

We	NN	O	O
propose	NN	O	O
that	NN	O	O
the	NN	O	O
loss	NN	O	O
of	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
VHL	NN	O	B-Disease
gene	NN	O	O
results	NN	O	O
in	NN	O	O
a	NN	O	O
specific	NN	O	O
cellular	NN	O	O
defect	NN	O	O
in	NN	O	O
serum	NN	O	O
-	NN	O	O
dependent	NN	O	O
growth	NN	O	O
control	NN	O	O
,	NN	O	O
which	NN	O	O
may	NN	O	O
initiate	NN	O	O
tumor	NN	O	B-Disease
formation	NN	O	O
.	NN	O	O

This	NN	O	O
is	NN	O	O
corrected	NN	O	O
by	NN	O	O
the	NN	O	O
reintroduction	NN	O	O
of	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
VHL	NN	O	B-Disease
,	NN	O	O
implicating	NN	O	O
VHL	NN	O	B-Disease
as	NN	O	O
the	NN	O	O
first	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
regulation	NN	O	O
of	NN	O	O
cell	NN	O	O
cycle	NN	O	O
exit	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
consistent	NN	O	O
with	NN	O	O
its	NN	O	O
gatekeeper	NN	O	O
function	NN	O	O
in	NN	O	O
the	NN	O	O
kidney	NN	O	O
.	NN	O	O
.	NN	O	O

Piebaldism	NN	O	B-Disease
with	NN	O	O
deafness	NN	O	B-Disease
:	NN	O	O
molecular	NN	O	O
evidence	NN	O	O
for	NN	O	O
an	NN	O	O
expanded	NN	O	O
syndrome	NN	O	O
.	NN	O	O

In	NN	O	O
a	NN	O	O
South	NN	O	O
African	NN	O	O
girl	NN	O	O
of	NN	O	O
Xhosa	NN	O	O
stock	NN	O	O
with	NN	O	O
severe	NN	O	O
piebaldism	NN	O	B-Disease
and	NN	O	O
profound	NN	O	O
congenital	NN	O	O
sensorineural	NN	O	B-Disease
deafness	NN	O	I-Disease
we	NN	O	O
identified	NN	O	O
a	NN	O	O
novel	NN	O	O
missense	NN	O	O
substitution	NN	O	O
at	NN	O	O
a	NN	O	O
highly	NN	O	O
conserved	NN	O	O
residue	NN	O	O
in	NN	O	O
the	NN	O	O
intracellular	NN	O	O
kinase	NN	O	O
domain	NN	O	O
of	NN	O	O
the	NN	O	O
KIT	NN	O	O
proto	NN	O	O
-	NN	O	O
oncogene	NN	O	O
,	NN	O	O
R796G	NN	O	O
.	NN	O	O

Though	NN	O	O
auditory	NN	O	B-Disease
anomalies	NN	O	I-Disease
have	NN	O	O
been	NN	O	O
observed	NN	O	O
in	NN	O	O
mice	NN	O	O
with	NN	O	O
dominant	NN	O	O
white	NN	O	O
spotting	NN	O	O
(	NN	O	O
W	NN	O	O
)	NN	O	O
due	NN	O	O
to	NN	O	O
KIT	NN	O	O
mutations	NN	O	O
,	NN	O	O
deafness	NN	O	B-Disease
is	NN	O	O
not	NN	O	O
typical	NN	O	O
in	NN	O	O
human	NN	O	O
piebaldism	NN	O	B-Disease
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
the	NN	O	O
occurrence	NN	O	O
of	NN	O	O
sensorineural	NN	O	B-Disease
deafness	NN	O	I-Disease
in	NN	O	O
this	NN	O	O
patient	NN	O	O
extends	NN	O	O
considerably	NN	O	O
the	NN	O	O
phenotypic	NN	O	O
range	NN	O	O
of	NN	O	O
piebaldism	NN	O	B-Disease
due	NN	O	O
to	NN	O	O
KIT	NN	O	O
gene	NN	O	O
mutation	NN	O	O
in	NN	O	O
humans	NN	O	O
and	NN	O	O
tightens	NN	O	O
the	NN	O	O
clinical	NN	O	O
similarity	NN	O	O
between	NN	O	O
piebaldism	NN	O	B-Disease
and	NN	O	O
the	NN	O	O
various	NN	O	O
forms	NN	O	O
of	NN	O	O
Waardenburg	NN	O	B-Disease
syndrome	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Cycloheximide	NN	O	O
facilitates	NN	O	O
the	NN	O	O
identification	NN	O	O
of	NN	O	O
aberrant	NN	O	O
transcripts	NN	O	O
resulting	NN	O	O
from	NN	O	O
a	NN	O	O
novel	NN	O	O
splice	NN	O	O
-	NN	O	O
site	NN	O	O
mutation	NN	O	O
in	NN	O	O
COL17A1	NN	O	O
in	NN	O	O
a	NN	O	O
patient	NN	O	O
with	NN	O	O
generalized	NN	O	O
atrophic	NN	O	B-Disease
benign	NN	O	I-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
.	NN	O	O

Patients	NN	O	O
with	NN	O	O
generalized	NN	O	O
atrophic	NN	O	B-Disease
benign	NN	O	I-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
often	NN	O	O
show	NN	O	O
decreased	NN	O	O
expression	NN	O	O
of	NN	O	O
type	NN	O	O
XVII	NN	O	O
collagen	NN	O	O
,	NN	O	O
a	NN	O	O
transmembrane	NN	O	O
hemidesmosomal	NN	O	O
protein	NN	O	O
encoded	NN	O	O
by	NN	O	O
COL17A1	NN	O	O
.	NN	O	O

This	NN	O	O
report	NN	O	O
documents	NN	O	O
a	NN	O	O
novel	NN	O	O
splice	NN	O	O
-	NN	O	O
site	NN	O	O
mutation	NN	O	O
in	NN	O	O
COL17A1	NN	O	O
in	NN	O	O
a	NN	O	O
patient	NN	O	O
with	NN	O	O
generalized	NN	O	O
atrophic	NN	O	B-Disease
benign	NN	O	I-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
applies	NN	O	O
a	NN	O	O
new	NN	O	O
methodology	NN	O	O
to	NN	O	O
define	NN	O	O
and	NN	O	O
characterize	NN	O	O
the	NN	O	O
resulting	NN	O	O
mRNA	NN	O	O
splice	NN	O	O
variants	NN	O	O
.	NN	O	O

Mutational	NN	O	O
analysis	NN	O	O
of	NN	O	O
COL17A1	NN	O	O
identified	NN	O	O
a	NN	O	O
maternally	NN	O	O
inherited	NN	O	O
G	NN	O	O
-	NN	O	O
to	NN	O	O
-	NN	O	O
T	NN	O	O
transversion	NN	O	O
at	NN	O	O
the	NN	O	O
-	NN	O	O
1	NN	O	O
position	NN	O	O
of	NN	O	O
exon	NN	O	O
32	NN	O	O
.	NN	O	O

This	NN	O	O
acceptor	NN	O	O
splice	NN	O	O
-	NN	O	O
site	NN	O	O
mutation	NN	O	O
led	NN	O	O
to	NN	O	O
the	NN	O	O
formation	NN	O	O
of	NN	O	O
aberrant	NN	O	O
transcripts	NN	O	O
present	NN	O	O
at	NN	O	O
extremely	NN	O	O
low	NN	O	O
levels	NN	O	O
.	NN	O	O

Based	NN	O	O
on	NN	O	O
our	NN	O	O
recent	NN	O	O
finding	NN	O	O
that	NN	O	O
cycloheximide	NN	O	O
stabilized	NN	O	O
mutant	NN	O	O
COL17A1	NN	O	O
transcripts	NN	O	O
in	NN	O	O
keratinocytes	NN	O	O
homozygous	NN	O	O
for	NN	O	O
a	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
,	NN	O	O
the	NN	O	O
effects	NN	O	O
of	NN	O	O
the	NN	O	O
splice	NN	O	O
-	NN	O	O
site	NN	O	O
mutation	NN	O	O
on	NN	O	O
splicing	NN	O	O
of	NN	O	O
COL17A1	NN	O	O
transcripts	NN	O	O
were	NN	O	O
determined	NN	O	O
using	NN	O	O
reverse	NN	O	O
transcriptase	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
of	NN	O	O
total	NN	O	O
RNA	NN	O	O
from	NN	O	O
keratinocytes	NN	O	O
incubated	NN	O	O
for	NN	O	O
2	NN	O	O
.	NN	O	O

5	NN	O	O
h	NN	O	O
in	NN	O	O
the	NN	O	O
presence	NN	O	O
or	NN	O	O
absence	NN	O	O
of	NN	O	O
10	NN	O	O
microg	NN	O	O
cycloheximide	NN	O	O
per	NN	O	O
ml	NN	O	O
.	NN	O	O

Using	NN	O	O
this	NN	O	O
approach	NN	O	O
,	NN	O	O
an	NN	O	O
abnormally	NN	O	O
spliced	NN	O	O
transcript	NN	O	O
was	NN	O	O
identified	NN	O	O
that	NN	O	O
contains	NN	O	O
an	NN	O	O
extra	NN	O	O
264	NN	O	O
bases	NN	O	O
upstream	NN	O	O
from	NN	O	O
exon	NN	O	O
32	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
a	NN	O	O
premature	NN	O	O
termination	NN	O	O
codon	NN	O	O
27	NN	O	O
bp	NN	O	O
downstream	NN	O	O
from	NN	O	O
the	NN	O	O
cryptic	NN	O	O
splice	NN	O	O
site	NN	O	O
.	NN	O	O

Three	NN	O	O
other	NN	O	O
splice	NN	O	O
variants	NN	O	O
,	NN	O	O
including	NN	O	O
one	NN	O	O
derived	NN	O	O
from	NN	O	O
the	NN	O	O
skipping	NN	O	O
of	NN	O	O
exon	NN	O	O
32	NN	O	O
,	NN	O	O
were	NN	O	O
also	NN	O	O
identified	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
indicate	NN	O	O
the	NN	O	O
usefulness	NN	O	O
of	NN	O	O
cycloheximide	NN	O	O
treatment	NN	O	O
in	NN	O	O
evaluating	NN	O	O
the	NN	O	O
abnormal	NN	O	O
processing	NN	O	O
of	NN	O	O
mRNA	NN	O	O
due	NN	O	O
to	NN	O	O
splice	NN	O	O
-	NN	O	O
site	NN	O	O
mutations	NN	O	O
,	NN	O	O
because	NN	O	O
(	NN	O	O
i	NN	O	O
)	NN	O	O
aberrant	NN	O	O
splicing	NN	O	O
often	NN	O	O
generates	NN	O	O
a	NN	O	O
premature	NN	O	O
termination	NN	O	O
codon	NN	O	O
,	NN	O	O
(	NN	O	O
ii	NN	O	O
)	NN	O	O
transcripts	NN	O	O
with	NN	O	O
premature	NN	O	O
termination	NN	O	O
codons	NN	O	O
can	NN	O	O
occur	NN	O	O
at	NN	O	O
low	NN	O	O
or	NN	O	O
undetectable	NN	O	O
levels	NN	O	O
due	NN	O	O
to	NN	O	O
nonsense	NN	O	O
-	NN	O	O
mediated	NN	O	O
mRNA	NN	O	O
decay	NN	O	O
,	NN	O	O
and	NN	O	O
(	NN	O	O
iii	NN	O	O
)	NN	O	O
the	NN	O	O
levels	NN	O	O
of	NN	O	O
these	NN	O	O
transcripts	NN	O	O
can	NN	O	O
be	NN	O	O
increased	NN	O	O
by	NN	O	O
cycloheximide	NN	O	O
.	NN	O	O

A	NN	O	O
deletion	NN	O	O
mutation	NN	O	O
in	NN	O	O
COL17A1	NN	O	O
in	NN	O	O
five	NN	O	O
Austrian	NN	O	O
families	NN	O	O
with	NN	O	O
generalized	NN	O	O
atrophic	NN	O	B-Disease
benign	NN	O	I-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
represents	NN	O	O
propagation	NN	O	O
of	NN	O	O
an	NN	O	O
ancestral	NN	O	O
allele	NN	O	O
.	NN	O	O

Patients	NN	O	O
with	NN	O	O
generalized	NN	O	O
atrophic	NN	O	B-Disease
benign	NN	O	I-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
,	NN	O	O
a	NN	O	O
usually	NN	O	O
nonlethal	NN	O	O
form	NN	O	O
of	NN	O	O
junctional	NN	O	B-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
,	NN	O	O
have	NN	O	O
generalized	NN	O	O
blistering	NN	O	B-Disease
,	NN	O	O
nail	NN	O	B-Disease
dystrophy	NN	O	I-Disease
,	NN	O	O
patchy	NN	O	B-Disease
alopecia	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
dental	NN	O	B-Disease
abnormalities	NN	O	I-Disease
.	NN	O	O

Skin	NN	O	B-Disease
fragility	NN	O	I-Disease
in	NN	O	O
most	NN	O	O
cases	NN	O	O
is	NN	O	O
due	NN	O	O
to	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
gene	NN	O	O
encoding	NN	O	O
type	NN	O	O
XVII	NN	O	O
collagen	NN	O	O
(	NN	O	O
COL17A1	NN	O	O
)	NN	O	O
.	NN	O	O

Recently	NN	O	O
,	NN	O	O
we	NN	O	O
reported	NN	O	O
five	NN	O	O
Austrian	NN	O	O
families	NN	O	O
with	NN	O	O
generalized	NN	O	O
atrophic	NN	O	B-Disease
benign	NN	O	I-Disease
epidermolysis	NN	O	I-Disease
bullosa	NN	O	I-Disease
who	NN	O	O
share	NN	O	O
the	NN	O	O
same	NN	O	O
COL17A1	NN	O	O
mutation	NN	O	O
.	NN	O	O

Affected	NN	O	O
individuals	NN	O	O
in	NN	O	O
three	NN	O	O
families	NN	O	O
are	NN	O	O
homozygous	NN	O	O
for	NN	O	O
4003delTC	NN	O	O
,	NN	O	O
whereas	NN	O	O
those	NN	O	O
in	NN	O	O
two	NN	O	O
others	NN	O	O
are	NN	O	O
compound	NN	O	O
heterozygotes	NN	O	O
.	NN	O	O

To	NN	O	O
determine	NN	O	O
if	NN	O	O
the	NN	O	O
occurrence	NN	O	O
of	NN	O	O
4003delTC	NN	O	O
in	NN	O	O
these	NN	O	O
unrelated	NN	O	O
families	NN	O	O
signifies	NN	O	O
propagation	NN	O	O
of	NN	O	O
an	NN	O	O
ancestral	NN	O	O
allele	NN	O	O
or	NN	O	O
a	NN	O	O
mutational	NN	O	O
hot	NN	O	O
spot	NN	O	O
,	NN	O	O
haplotypes	NN	O	O
were	NN	O	O
determined	NN	O	O
for	NN	O	O
polymorphisms	NN	O	O
both	NN	O	O
within	NN	O	O
and	NN	O	O
flanking	NN	O	O
COL17A1	NN	O	O
.	NN	O	O

Five	NN	O	O
intragenic	NN	O	O
polymorphisms	NN	O	O
were	NN	O	O
chosen	NN	O	O
based	NN	O	O
on	NN	O	O
their	NN	O	O
informativeness	NN	O	O
.	NN	O	O

One	NN	O	O
of	NN	O	O
these	NN	O	O
,	NN	O	O
not	NN	O	O
previously	NN	O	O
reported	NN	O	O
,	NN	O	O
was	NN	O	O
2988	NN	O	O
A	NN	O	O
or	NN	O	O
C	NN	O	O
that	NN	O	O
introduces	NN	O	O
a	NN	O	O
new	NN	O	O
restriction	NN	O	O
site	NN	O	O
for	NN	O	O
Eco0109	NN	O	O
I	NN	O	O
.	NN	O	O

All	NN	O	O
the	NN	O	O
4003delTC	NN	O	O
alleles	NN	O	O
showed	NN	O	O
the	NN	O	O
same	NN	O	O
haplotype	NN	O	O
for	NN	O	O
these	NN	O	O
five	NN	O	O
polymorphic	NN	O	O
markers	NN	O	O
.	NN	O	O

Fourteen	NN	O	O
microsatellite	NN	O	O
polymorphisms	NN	O	O
were	NN	O	O
selected	NN	O	O
based	NN	O	O
on	NN	O	O
their	NN	O	O
high	NN	O	O
heterozygosity	NN	O	O
and	NN	O	O
their	NN	O	O
location	NN	O	O
within	NN	O	O
10q23	NN	O	O
-	NN	O	O
q25	NN	O	O
near	NN	O	O
COL17A1	NN	O	O
.	NN	O	O

Three	NN	O	O
families	NN	O	O
shared	NN	O	O
microsatellite	NN	O	O
polymorphisms	NN	O	O
covering	NN	O	O
at	NN	O	O
most	NN	O	O
19	NN	O	O
cM	NN	O	O
,	NN	O	O
whereas	NN	O	O
the	NN	O	O
others	NN	O	O
shared	NN	O	O
smaller	NN	O	O
regions	NN	O	O
consistent	NN	O	O
with	NN	O	O
cross	NN	O	O
-	NN	O	O
over	NN	O	O
events	NN	O	O
during	NN	O	O
passage	NN	O	O
of	NN	O	O
this	NN	O	O
mutation	NN	O	O
through	NN	O	O
several	NN	O	O
generations	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
indicate	NN	O	O
that	NN	O	O
4003delTC	NN	O	O
occurs	NN	O	O
on	NN	O	O
a	NN	O	O
single	NN	O	O
ancestral	NN	O	O
allele	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
haptoglobin	NN	O	O
-	NN	O	O
gene	NN	O	O
deletion	NN	O	O
responsible	NN	O	O
for	NN	O	O
anhaptoglobinemia	NN	O	B-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
found	NN	O	O
an	NN	O	O
allelic	NN	O	O
deletion	NN	O	O
of	NN	O	O
the	NN	O	O
haptoglobin	NN	O	O
(	NN	O	O
Hp	NN	O	O
)	NN	O	O
gene	NN	O	O
from	NN	O	O
an	NN	O	O
individual	NN	O	O
with	NN	O	O
anhaptoglobinemia	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
Hp	NN	O	O
gene	NN	O	O
cluster	NN	O	O
consists	NN	O	O
of	NN	O	O
coding	NN	O	O
regions	NN	O	O
of	NN	O	O
the	NN	O	O
alpha	NN	O	O
chain	NN	O	O
and	NN	O	O
beta	NN	O	O
chain	NN	O	O
of	NN	O	O
the	NN	O	O
haptoglobin	NN	O	O
gene	NN	O	O
(	NN	O	O
Hp	NN	O	O
)	NN	O	O
and	NN	O	O
of	NN	O	O
the	NN	O	O
alpha	NN	O	O
chain	NN	O	O
and	NN	O	O
beta	NN	O	O
chain	NN	O	O
of	NN	O	O
the	NN	O	O
haptoglobin	NN	O	O
-	NN	O	O
related	NN	O	O
gene	NN	O	O
(	NN	O	O
Hpr	NN	O	O
)	NN	O	O
,	NN	O	O
in	NN	O	O
tandem	NN	O	O
from	NN	O	O
the	NN	O	O
5	NN	O	O
side	NN	O	O
.	NN	O	O

Southern	NN	O	O
blot	NN	O	O
and	NN	O	O
PCR	NN	O	O
analyses	NN	O	O
have	NN	O	O
indicated	NN	O	O
that	NN	O	O
the	NN	O	O
individual	NN	O	O
with	NN	O	O
anhaptoglobinemia	NN	O	B-Disease
was	NN	O	O
homozygous	NN	O	O
for	NN	O	O
the	NN	O	O
gene	NN	O	O
deletion	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
gene	NN	O	O
deletion	NN	O	O
was	NN	O	O
included	NN	O	O
at	NN	O	O
least	NN	O	O
from	NN	O	O
the	NN	O	O
promoter	NN	O	O
region	NN	O	O
of	NN	O	O
Hp	NN	O	O
to	NN	O	O
Hpr	NN	O	O
alpha	NN	O	O
but	NN	O	O
not	NN	O	O
to	NN	O	O
Hpr	NN	O	O
beta	NN	O	O
(	NN	O	O
Hpdel	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
we	NN	O	O
found	NN	O	O
seven	NN	O	O
individuals	NN	O	O
with	NN	O	O
hypohaptoglobinemia	NN	O	B-Disease
in	NN	O	O
three	NN	O	O
families	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
genotypes	NN	O	O
of	NN	O	O
six	NN	O	O
of	NN	O	O
the	NN	O	O
seven	NN	O	O
individuals	NN	O	O
were	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
Hp2	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
.	NN	O	O

The	NN	O	O
phenotypes	NN	O	O
and	NN	O	O
genotypes	NN	O	O
in	NN	O	O
one	NN	O	O
of	NN	O	O
these	NN	O	O
three	NN	O	O
families	NN	O	O
showed	NN	O	O
the	NN	O	O
father	NN	O	O
to	NN	O	O
be	NN	O	O
hypohaptoglobinemic	NN	O	B-Disease
(	NN	O	O
Hp2	NN	O	O
)	NN	O	O
and	NN	O	O
Hp2	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
,	NN	O	O
the	NN	O	O
mother	NN	O	O
to	NN	O	O
be	NN	O	O
Hp2	NN	O	O
-	NN	O	O
1	NN	O	O
and	NN	O	O
Hp1	NN	O	O
/	NN	O	O
Hp2	NN	O	O
,	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
children	NN	O	O
to	NN	O	O
be	NN	O	O
hypohaptoglobinemic	NN	O	B-Disease
(	NN	O	O
Hp2	NN	O	O
)	NN	O	O
and	NN	O	O
Hp2	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
other	NN	O	O
child	NN	O	O
to	NN	O	O
be	NN	O	O
Hp1	NN	O	O
and	NN	O	O
Hp1	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
,	NN	O	O
showing	NN	O	O
an	NN	O	O
anomalous	NN	O	O
inheritance	NN	O	O
of	NN	O	O
Hp	NN	O	O
phenotypes	NN	O	O
in	NN	O	O
the	NN	O	O
child	NN	O	O
with	NN	O	O
Hp1	NN	O	O
.	NN	O	O

The	NN	O	O
Hp2	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
individuals	NN	O	O
had	NN	O	O
an	NN	O	O
extremely	NN	O	O
low	NN	O	O
level	NN	O	O
of	NN	O	O
Hp	NN	O	O
(	NN	O	O
mean	NN	O	O
+	NN	O	O
/	NN	O	O
-	NN	O	O
SD	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
049	NN	O	O
+	NN	O	O
/	NN	O	O
-	NN	O	O
0	NN	O	O
.	NN	O	O
043	NN	O	O
mg	NN	O	O
/	NN	O	O
ml	NN	O	O
;	NN	O	O
n	NN	O	O
=	NN	O	O
6	NN	O	O
)	NN	O	O
,	NN	O	O
compared	NN	O	O
with	NN	O	O
the	NN	O	O
level	NN	O	O
(	NN	O	O
1	NN	O	O
.	NN	O	O
64	NN	O	O
+	NN	O	O
/	NN	O	O
-	NN	O	O
1	NN	O	O
.	NN	O	O
07	NN	O	O
mg	NN	O	O
/	NN	O	O
ml	NN	O	O
)	NN	O	O
obtained	NN	O	O
from	NN	O	O
52	NN	O	O
healthy	NN	O	O
volunteers	NN	O	O
having	NN	O	O
phenotype	NN	O	O
Hp2	NN	O	O
,	NN	O	O
whereas	NN	O	O
the	NN	O	O
serum	NN	O	O
Hp	NN	O	O
level	NN	O	O
of	NN	O	O
an	NN	O	O
individual	NN	O	O
with	NN	O	O
Hp1	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
was	NN	O	O
0	NN	O	O
.	NN	O	O

50	NN	O	O
mg	NN	O	O
/	NN	O	O
ml	NN	O	O
,	NN	O	O
which	NN	O	O
was	NN	O	O
approximately	NN	O	O
half	NN	O	O
the	NN	O	O
level	NN	O	O
of	NN	O	O
Hp	NN	O	O
in	NN	O	O
control	NN	O	O
sera	NN	O	O
from	NN	O	O
the	NN	O	O
Hp1	NN	O	O
phenotype	NN	O	O
(	NN	O	O
1	NN	O	O
.	NN	O	O
26	NN	O	O
+	NN	O	O
/	NN	O	O
-	NN	O	O
0	NN	O	O
.	NN	O	O
33	NN	O	O
mg	NN	O	O
/	NN	O	O
ml	NN	O	O
;	NN	O	O
n	NN	O	O
=	NN	O	O
9	NN	O	O
)	NN	O	O
,	NN	O	O
showing	NN	O	O
a	NN	O	O
gene	NN	O	O
-	NN	O	O
dosage	NN	O	O
effect	NN	O	O
.	NN	O	O

The	NN	O	O
other	NN	O	O
allele	NN	O	O
(	NN	O	O
Hp2	NN	O	O
)	NN	O	O
of	NN	O	O
individuals	NN	O	O
with	NN	O	O
Hp2	NN	O	O
/	NN	O	O
Hpdel	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
,	NN	O	O
in	NN	O	O
all	NN	O	O
exons	NN	O	O
,	NN	O	O
no	NN	O	O
mutation	NN	O	O
,	NN	O	O
by	NN	O	O
DNA	NN	O	O
sequencing	NN	O	O
.	NN	O	O

On	NN	O	O
the	NN	O	O
basis	NN	O	O
of	NN	O	O
the	NN	O	O
present	NN	O	O
study	NN	O	O
,	NN	O	O
the	NN	O	O
mechanism	NN	O	O
of	NN	O	O
anhaptoglobinemia	NN	O	B-Disease
and	NN	O	O
the	NN	O	O
mechanism	NN	O	O
of	NN	O	O
anomalous	NN	O	O
inheritance	NN	O	O
of	NN	O	O
Hp	NN	O	O
phenotypes	NN	O	O
were	NN	O	O
well	NN	O	O
explained	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
the	NN	O	O
mechanism	NN	O	O
of	NN	O	O
hypohaptoglobinemia	NN	O	B-Disease
remains	NN	O	O
unknown	NN	O	O

ATM	NN	O	O
mutations	NN	O	O
and	NN	O	O
phenotypes	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
families	NN	O	O
in	NN	O	O
the	NN	O	O
British	NN	O	O
Isles	NN	O	O
:	NN	O	O
expression	NN	O	O
of	NN	O	O
mutant	NN	O	O
ATM	NN	O	O
and	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
leukemia	NN	O	B-Disease
,	NN	O	O
lymphoma	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
the	NN	O	O
spectrum	NN	O	O
of	NN	O	O
59	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
observed	NN	O	O
in	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
patients	NN	O	O
in	NN	O	O
the	NN	O	O
British	NN	O	O
Isles	NN	O	O
.	NN	O	O

Of	NN	O	O
51	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
identified	NN	O	O
in	NN	O	O
families	NN	O	O
native	NN	O	O
to	NN	O	O
the	NN	O	O
British	NN	O	O
Isles	NN	O	O
,	NN	O	O
11	NN	O	O
were	NN	O	O
founder	NN	O	O
mutations	NN	O	O
,	NN	O	O
and	NN	O	O
2	NN	O	O
of	NN	O	O
these	NN	O	O
11	NN	O	O
conferred	NN	O	O
a	NN	O	O
milder	NN	O	O
clinical	NN	O	O
phenotype	NN	O	O
with	NN	O	O
respect	NN	O	O
to	NN	O	O
both	NN	O	O
cerebellar	NN	O	B-Disease
degeneration	NN	O	I-Disease
and	NN	O	O
cellular	NN	O	O
features	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
,	NN	O	O
in	NN	O	O
two	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
families	NN	O	O
,	NN	O	O
an	NN	O	O
ATM	NN	O	O
mutation	NN	O	O
(	NN	O	O
7271T	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
)	NN	O	O
that	NN	O	O
may	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
an	NN	O	O
increased	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
both	NN	O	O
homozygotes	NN	O	O
and	NN	O	O
heterozygotes	NN	O	O
(	NN	O	O
relative	NN	O	O
risk	NN	O	O
12	NN	O	O
.	NN	O	O
7	NN	O	O
;	NN	O	O
P	NN	O	O
=	NN	O	O
.	NN	O	O
0025	NN	O	O
)	NN	O	O
,	NN	O	O
although	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
less	NN	O	O
severe	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
phenotype	NN	O	O
in	NN	O	O
terms	NN	O	O
of	NN	O	O
the	NN	O	O
degree	NN	O	O
of	NN	O	O
cerebellar	NN	O	B-Disease
degeneration	NN	O	I-Disease
.	NN	O	O

This	NN	O	O
mutation	NN	O	O
(	NN	O	O
7271T	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
)	NN	O	O
also	NN	O	O
allows	NN	O	O
expression	NN	O	O
of	NN	O	O
full	NN	O	O
-	NN	O	O
length	NN	O	O
ATM	NN	O	O
protein	NN	O	O
at	NN	O	O
a	NN	O	O
level	NN	O	O
comparable	NN	O	O
with	NN	O	O
that	NN	O	O
in	NN	O	O
unaffected	NN	O	O
individuals	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
studied	NN	O	O
18	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
,	NN	O	O
in	NN	O	O
15	NN	O	O
families	NN	O	O
,	NN	O	O
who	NN	O	O
developed	NN	O	O
leukemia	NN	O	B-Disease
,	NN	O	O
lymphoma	NN	O	B-Disease
,	NN	O	O
preleukemic	NN	O	O
T	NN	O	O
-	NN	O	O
cell	NN	O	O
proliferation	NN	O	O
,	NN	O	O
or	NN	O	O
Hodgkin	NN	O	B-Disease
lymphoma	NN	O	I-Disease
,	NN	O	O
mostly	NN	O	O
in	NN	O	O
childhood	NN	O	O
.	NN	O	O

A	NN	O	O
wide	NN	O	O
variety	NN	O	O
of	NN	O	O
ATM	NN	O	O
mutation	NN	O	O
types	NN	O	O
,	NN	O	O
including	NN	O	O
missense	NN	O	O
mutations	NN	O	O
and	NN	O	O
in	NN	O	O
-	NN	O	O
frame	NN	O	O
deletions	NN	O	O
,	NN	O	O
were	NN	O	O
seen	NN	O	O
in	NN	O	O
these	NN	O	O
patients	NN	O	O
.	NN	O	O

We	NN	O	O
also	NN	O	O
show	NN	O	O
that	NN	O	O
25	NN	O	O
%	NN	O	O
of	NN	O	O
all	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
carried	NN	O	O
in	NN	O	O
-	NN	O	O
frame	NN	O	O
deletions	NN	O	O
or	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
many	NN	O	O
of	NN	O	O
which	NN	O	O
were	NN	O	O
also	NN	O	O
associated	NN	O	O
with	NN	O	O
expression	NN	O	O
of	NN	O	O
mutant	NN	O	O
ATM	NN	O	O
protein	NN	O	O
.	NN	O	O

The	NN	O	O
DMPK	NN	O	O
gene	NN	O	O
of	NN	O	O
severely	NN	O	O
affected	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
patients	NN	O	O
is	NN	O	O
hypermethylated	NN	O	O
proximal	NN	O	O
to	NN	O	O
the	NN	O	O
largely	NN	O	O
expanded	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
.	NN	O	O

Using	NN	O	O
methylation	NN	O	O
-	NN	O	O
sensitive	NN	O	O
restriction	NN	O	O
enzymes	NN	O	O
,	NN	O	O
we	NN	O	O
characterized	NN	O	O
the	NN	O	O
methylation	NN	O	O
pattern	NN	O	O
on	NN	O	O
the	NN	O	O
5	NN	O	O
side	NN	O	O
of	NN	O	O
the	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
in	NN	O	O
the	NN	O	O
DMPK	NN	O	O
gene	NN	O	O
of	NN	O	O
normal	NN	O	O
individuals	NN	O	O
and	NN	O	O
of	NN	O	O
patients	NN	O	O
affected	NN	O	O
with	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
,	NN	O	O
showing	NN	O	O
expansions	NN	O	O
of	NN	O	O
the	NN	O	O
repetitive	NN	O	O
sequence	NN	O	O
.	NN	O	O

The	NN	O	O
gene	NN	O	O
segment	NN	O	O
analyzed	NN	O	O
corresponds	NN	O	O
to	NN	O	O
the	NN	O	O
genomic	NN	O	O
SacI	NN	O	O
-	NN	O	O
HindIII	NN	O	O
fragment	NN	O	O
carrying	NN	O	O
exons	NN	O	O
11	NN	O	O
-	NN	O	O
15	NN	O	O
.	NN	O	O

There	NN	O	O
is	NN	O	O
constitutive	NN	O	O
methylation	NN	O	O
in	NN	O	O
intron	NN	O	O
12	NN	O	O
at	NN	O	O
restriction	NN	O	O
sites	NN	O	O
of	NN	O	O
SacII	NN	O	O
and	NN	O	O
HhaI	NN	O	O
,	NN	O	O
localized	NN	O	O
1	NN	O	O
,	NN	O	O
159	NN	O	O
-	NN	O	O
1	NN	O	O
,	NN	O	O
232	NN	O	O
bp	NN	O	O
upstream	NN	O	O
of	NN	O	O
the	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
,	NN	O	O
whereas	NN	O	O
most	NN	O	O
,	NN	O	O
if	NN	O	O
not	NN	O	O
all	NN	O	O
,	NN	O	O
of	NN	O	O
the	NN	O	O
other	NN	O	O
sites	NN	O	O
of	NN	O	O
SacII	NN	O	O
,	NN	O	O
HhaI	NN	O	O
,	NN	O	O
and	NN	O	O
HpaII	NN	O	O
in	NN	O	O
this	NN	O	O
region	NN	O	O
are	NN	O	O
unmethylated	NN	O	O
,	NN	O	O
in	NN	O	O
normal	NN	O	O
individuals	NN	O	O
and	NN	O	O
most	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
.	NN	O	O

In	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
young	NN	O	O
and	NN	O	O
severely	NN	O	O
affected	NN	O	O
patients	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
complete	NN	O	O
methylation	NN	O	O
of	NN	O	O
these	NN	O	O
restriction	NN	O	O
sites	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
the	NN	O	O
mutated	NN	O	O
allele	NN	O	O
.	NN	O	O

In	NN	O	O
most	NN	O	O
of	NN	O	O
these	NN	O	O
patients	NN	O	O
,	NN	O	O
the	NN	O	O
onset	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
was	NN	O	O
congenital	NN	O	O
.	NN	O	O

Preliminary	NN	O	O
in	NN	O	O
vivo	NN	O	O
footprinting	NN	O	O
data	NN	O	O
gave	NN	O	O
evidence	NN	O	O
for	NN	O	O
protein	NN	O	O
-	NN	O	O
DNA	NN	O	O
contact	NN	O	O
in	NN	O	O
normal	NN	O	O
genes	NN	O	O
at	NN	O	O
an	NN	O	O
Sp1	NN	O	O
consensus	NN	O	O
binding	NN	O	O
site	NN	O	O
upstream	NN	O	O
of	NN	O	O
the	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
and	NN	O	O
for	NN	O	O
a	NN	O	O
significant	NN	O	O
reduction	NN	O	O
of	NN	O	O
this	NN	O	O
interaction	NN	O	O
in	NN	O	O
cells	NN	O	O
with	NN	O	O
a	NN	O	O
hypermethylated	NN	O	O
DMPK	NN	O	O
gene	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
hemochromatosis	NN	O	B-Disease
gene	NN	O	O
product	NN	O	O
complexes	NN	O	O
with	NN	O	O
the	NN	O	O
transferrin	NN	O	O
receptor	NN	O	O
and	NN	O	O
lowers	NN	O	O
its	NN	O	O
affinity	NN	O	O
for	NN	O	O
ligand	NN	O	O
binding	NN	O	O
.	NN	O	O

We	NN	O	O
recently	NN	O	O
reported	NN	O	O
the	NN	O	O
positional	NN	O	O
cloning	NN	O	O
of	NN	O	O
a	NN	O	O
candidate	NN	O	O
gene	NN	O	O
for	NN	O	O
hereditary	NN	O	B-Disease
hemochromatosis	NN	O	I-Disease
called	NN	O	O
HFE	NN	O	O
.	NN	O	O

The	NN	O	O
gene	NN	O	O
product	NN	O	O
,	NN	O	O
a	NN	O	O
member	NN	O	O
of	NN	O	O
the	NN	O	O
major	NN	O	O
histocompatibility	NN	O	O
complex	NN	O	O
class	NN	O	O
I	NN	O	O
-	NN	O	O
like	NN	O	O
family	NN	O	O
,	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
a	NN	O	O
mutation	NN	O	O
,	NN	O	O
Cys	NN	O	O
-	NN	O	O
282	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Tyr	NN	O	O
(	NN	O	O
C282Y	NN	O	O
)	NN	O	O
,	NN	O	O
in	NN	O	O
85	NN	O	O
%	NN	O	O
of	NN	O	O
patient	NN	O	O
chromosomes	NN	O	O
.	NN	O	O

This	NN	O	O
mutation	NN	O	O
eliminates	NN	O	O
the	NN	O	O
ability	NN	O	O
of	NN	O	O
HFE	NN	O	O
to	NN	O	O
associate	NN	O	O
with	NN	O	O
beta2	NN	O	O
-	NN	O	O
microglobulin	NN	O	O
(	NN	O	O
beta2m	NN	O	O
)	NN	O	O
and	NN	O	O
prevents	NN	O	O
cell	NN	O	O
-	NN	O	O
surface	NN	O	O
expression	NN	O	O
.	NN	O	O

A	NN	O	O
second	NN	O	O
mutation	NN	O	O
that	NN	O	O
has	NN	O	O
no	NN	O	O
effect	NN	O	O
on	NN	O	O
beta2m	NN	O	O
association	NN	O	O
,	NN	O	O
H63D	NN	O	O
,	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
eight	NN	O	O
out	NN	O	O
of	NN	O	O
nine	NN	O	O
patients	NN	O	O
heterozygous	NN	O	O
for	NN	O	O
the	NN	O	O
C282Y	NN	O	O
mutant	NN	O	O
.	NN	O	O

In	NN	O	O
this	NN	O	O
report	NN	O	O
,	NN	O	O
we	NN	O	O
demonstrate	NN	O	O
in	NN	O	O
cultured	NN	O	O
293	NN	O	O
cells	NN	O	O
overexpressing	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
or	NN	O	O
mutant	NN	O	O
HFE	NN	O	O
proteins	NN	O	O
that	NN	O	O
both	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
and	NN	O	O
H63D	NN	O	O
HFE	NN	O	O
proteins	NN	O	O
form	NN	O	O
stable	NN	O	O
complexes	NN	O	O
with	NN	O	O
the	NN	O	O
transferrin	NN	O	O
receptor	NN	O	O
(	NN	O	O
TfR	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
C282Y	NN	O	O
mutation	NN	O	O
nearly	NN	O	O
completely	NN	O	O
prevents	NN	O	O
the	NN	O	O
association	NN	O	O
of	NN	O	O
the	NN	O	O
mutant	NN	O	O
HFE	NN	O	O
protein	NN	O	O
with	NN	O	O
the	NN	O	O
TfR	NN	O	O
.	NN	O	O

Studies	NN	O	O
on	NN	O	O
cell	NN	O	O
-	NN	O	O
associated	NN	O	O
transferrin	NN	O	O
at	NN	O	O
37	NN	O	O
degrees	NN	O	O
C	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
overexpressed	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
HFE	NN	O	O
protein	NN	O	O
decreases	NN	O	O
the	NN	O	O
affinity	NN	O	O
of	NN	O	O
the	NN	O	O
TfR	NN	O	O
for	NN	O	O
transferrin	NN	O	O
.	NN	O	O

The	NN	O	O
overexpressed	NN	O	O
H63D	NN	O	O
protein	NN	O	O
does	NN	O	O
not	NN	O	O
have	NN	O	O
this	NN	O	O
effect	NN	O	O
,	NN	O	O
providing	NN	O	O
the	NN	O	O
first	NN	O	O
direct	NN	O	O
evidence	NN	O	O
for	NN	O	O
a	NN	O	O
functional	NN	O	O
consequence	NN	O	O
of	NN	O	O
the	NN	O	O
H63D	NN	O	O
mutation	NN	O	O
.	NN	O	O

Addition	NN	O	O
of	NN	O	O
soluble	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
HFE	NN	O	O
/	NN	O	O
beta2m	NN	O	O
heterodimers	NN	O	O
to	NN	O	O
cultured	NN	O	O
cells	NN	O	O
also	NN	O	O
decreased	NN	O	O
the	NN	O	O
apparent	NN	O	O
affinity	NN	O	O
of	NN	O	O
the	NN	O	O
TfR	NN	O	O
for	NN	O	O
its	NN	O	O
ligand	NN	O	O
under	NN	O	O
steady	NN	O	O
-	NN	O	O
state	NN	O	O
conditions	NN	O	O
,	NN	O	O
both	NN	O	O
in	NN	O	O
293	NN	O	O
cells	NN	O	O
and	NN	O	O
in	NN	O	O
HeLa	NN	O	O
cells	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
at	NN	O	O
4	NN	O	O
degrees	NN	O	O
C	NN	O	O
,	NN	O	O
the	NN	O	O
added	NN	O	O
soluble	NN	O	O
complex	NN	O	O
of	NN	O	O
HFE	NN	O	O
/	NN	O	O
beta2m	NN	O	O
inhibited	NN	O	O
binding	NN	O	O
of	NN	O	O
transferrin	NN	O	O
to	NN	O	O
HeLa	NN	O	O
cell	NN	O	O
TfR	NN	O	O
in	NN	O	O
a	NN	O	O
concentration	NN	O	O
-	NN	O	O
dependent	NN	O	O
manner	NN	O	O
.	NN	O	O

Scatchard	NN	O	O
plots	NN	O	O
of	NN	O	O
these	NN	O	O
data	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
added	NN	O	O
heterodimer	NN	O	O
substantially	NN	O	O
reduced	NN	O	O
the	NN	O	O
affinity	NN	O	O
of	NN	O	O
TfR	NN	O	O
for	NN	O	O
transferrin	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
establish	NN	O	O
a	NN	O	O
molecular	NN	O	O
link	NN	O	O
between	NN	O	O
HFE	NN	O	O
and	NN	O	O
a	NN	O	O
key	NN	O	O
protein	NN	O	O
involved	NN	O	O
in	NN	O	O
iron	NN	O	O
transport	NN	O	O
,	NN	O	O
the	NN	O	O
TfR	NN	O	O
,	NN	O	O
and	NN	O	O
raise	NN	O	O
the	NN	O	O
possibility	NN	O	O
that	NN	O	O
alterations	NN	O	O
in	NN	O	O
this	NN	O	O
regulatory	NN	O	O
mechanism	NN	O	O
may	NN	O	O
play	NN	O	O
a	NN	O	O
role	NN	O	O
in	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
hemochromatosis	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Genomic	NN	O	O
organization	NN	O	O
of	NN	O	O
the	NN	O	O
UBE3A	NN	O	O
/	NN	O	O
E6	NN	O	O
-	NN	O	O
AP	NN	O	O
gene	NN	O	O
and	NN	O	O
related	NN	O	O
pseudogenes	NN	O	O
.	NN	O	O

The	NN	O	O
UBE3A	NN	O	O
gene	NN	O	O
encodes	NN	O	O
the	NN	O	O
E6	NN	O	O
-	NN	O	O
AP	NN	O	O
ubiquitin	NN	O	O
-	NN	O	O
protein	NN	O	O
ligase	NN	O	O
and	NN	O	O
has	NN	O	O
recently	NN	O	O
been	NN	O	O
shown	NN	O	O
to	NN	O	O
be	NN	O	O
mutated	NN	O	O
in	NN	O	O
Angelman	NN	O	B-Disease
syndrome	NN	O	I-Disease
patients	NN	O	O
who	NN	O	O
lack	NN	O	O
15q11	NN	O	O
-	NN	O	O
q13	NN	O	O
deletions	NN	O	O
or	NN	O	O
chromosome	NN	O	O
15	NN	O	O
paternal	NN	O	O
uniparental	NN	O	B-Disease
disomy	NN	O	I-Disease
.	NN	O	O

Previous	NN	O	O
UBE3A	NN	O	O
cDNA	NN	O	O
analysis	NN	O	O
has	NN	O	O
shown	NN	O	O
a	NN	O	O
coding	NN	O	O
region	NN	O	O
of	NN	O	O
approximately	NN	O	O
2	NN	O	O
.	NN	O	O

6	NN	O	O
kb	NN	O	O
and	NN	O	O
a	NN	O	O
3	NN	O	O
-	NN	O	O
untranslated	NN	O	O
region	NN	O	O
(	NN	O	O
UTR	NN	O	O
)	NN	O	O
of	NN	O	O
<	NN	O	O
50	NN	O	O
bp	NN	O	O
,	NN	O	O
whereas	NN	O	O
Northern	NN	O	O
analysis	NN	O	O
has	NN	O	O
indicated	NN	O	O
mRNA	NN	O	O
sizes	NN	O	O
of	NN	O	O
5	NN	O	O
-	NN	O	O
8	NN	O	O
kb	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
analyzed	NN	O	O
additional	NN	O	O
cDNA	NN	O	O
clones	NN	O	O
and	NN	O	O
provide	NN	O	O
evidence	NN	O	O
for	NN	O	O
an	NN	O	O
additional	NN	O	O
0	NN	O	O
.	NN	O	O

5	NN	O	O
kb	NN	O	O
of	NN	O	O
5	NN	O	O
-	NN	O	O
UTR	NN	O	O
and	NN	O	O
>	NN	O	O
2	NN	O	O
kb	NN	O	O
of	NN	O	O
3	NN	O	O
-	NN	O	O
UTR	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
established	NN	O	O
the	NN	O	O
genomic	NN	O	O
organization	NN	O	O
of	NN	O	O
UBE3A	NN	O	O
and	NN	O	O
the	NN	O	O
sequence	NN	O	O
of	NN	O	O
intron	NN	O	O
-	NN	O	O
exon	NN	O	O
borders	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
also	NN	O	O
mapped	NN	O	O
two	NN	O	O
highly	NN	O	O
homologous	NN	O	O
processed	NN	O	O
pseudogenes	NN	O	O
,	NN	O	O
UBE3AP1	NN	O	O
and	NN	O	O
UBE3AP2	NN	O	O
,	NN	O	O
to	NN	O	O
chromosomes	NN	O	O
2	NN	O	O
and	NN	O	O
21	NN	O	O
,	NN	O	O
respectively	NN	O	O
,	NN	O	O
and	NN	O	O
determined	NN	O	O
their	NN	O	O
genomic	NN	O	O
organization	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
will	NN	O	O
form	NN	O	O
the	NN	O	O
basis	NN	O	O
for	NN	O	O
studies	NN	O	O
of	NN	O	O
mutation	NN	O	O
and	NN	O	O
imprinting	NN	O	O
of	NN	O	O
UBE3A	NN	O	O
.	NN	O	O

Mutation	NN	O	O
spectrum	NN	O	O
and	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
analyses	NN	O	O
in	NN	O	O
Cowden	NN	O	B-Disease
disease	NN	O	I-Disease
and	NN	O	O
Bannayan	NN	O	B-Disease
-	NN	O	I-Disease
Zonana	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
two	NN	O	O
hamartoma	NN	O	B-Disease
syndromes	NN	O	I-Disease
with	NN	O	O
germline	NN	O	O
PTEN	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
tumour	NN	O	B-Disease
suppressor	NN	O	O
gene	NN	O	O
PTEN	NN	O	O
,	NN	O	O
which	NN	O	O
maps	NN	O	O
to	NN	O	O
10q23	NN	O	O
.	NN	O	O

3	NN	O	O
and	NN	O	O
encodes	NN	O	O
a	NN	O	O
403	NN	O	O
amino	NN	O	O
acid	NN	O	O
dual	NN	O	O
specificity	NN	O	O
phosphatase	NN	O	O
(	NN	O	O
protein	NN	O	O
tyrosine	NN	O	O
phosphatase	NN	O	O
;	NN	O	O
PTPase	NN	O	O
)	NN	O	O
,	NN	O	O
was	NN	O	O
shown	NN	O	O
recently	NN	O	O
to	NN	O	O
play	NN	O	O
a	NN	O	O
broad	NN	O	O
role	NN	O	O
in	NN	O	O
human	NN	O	O
malignancy	NN	O	B-Disease
.	NN	O	O

Somatic	NN	O	O
PTEN	NN	O	O
deletions	NN	O	O
and	NN	O	O
mutations	NN	O	O
were	NN	O	O
observed	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
breast	NN	O	I-Disease
,	NN	O	I-Disease
brain	NN	O	I-Disease
,	NN	O	I-Disease
prostate	NN	O	I-Disease
and	NN	O	I-Disease
kidney	NN	O	I-Disease
cancer	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
and	NN	O	O
in	NN	O	O
several	NN	O	O
primary	NN	O	O
tumours	NN	O	B-Disease
such	NN	O	O
as	NN	O	O
endometrial	NN	O	B-Disease
carcinomas	NN	O	I-Disease
,	NN	O	O
malignant	NN	O	B-Disease
melanoma	NN	O	I-Disease
and	NN	O	O
thyroid	NN	O	B-Disease
tumours	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
PTEN	NN	O	O
was	NN	O	O
identified	NN	O	O
as	NN	O	O
the	NN	O	O
susceptibility	NN	O	O
gene	NN	O	O
for	NN	O	O
two	NN	O	O
hamartoma	NN	O	B-Disease
syndromes	NN	O	I-Disease
Cowden	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
CD	NN	O	B-Disease
;	NN	O	O
MIM	NN	O	O
158350	NN	O	O
)	NN	O	O
and	NN	O	O
Bannayan	NN	O	B-Disease
-	NN	O	I-Disease
Zonana	NN	O	I-Disease
(	NN	O	I-Disease
BZS	NN	O	I-Disease
)	NN	O	I-Disease
or	NN	O	I-Disease
Ruvalcaba	NN	O	I-Disease
-	NN	O	I-Disease
Riley	NN	O	I-Disease
-	NN	O	I-Disease
Smith	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
MIM	NN	O	O
153480	NN	O	O
)	NN	O	O
.	NN	O	O

Constitutive	NN	O	O
DNA	NN	O	O
from	NN	O	O
37	NN	O	O
CD	NN	O	B-Disease
families	NN	O	O
and	NN	O	O
seven	NN	O	O
BZS	NN	O	B-Disease
families	NN	O	O
was	NN	O	O
screened	NN	O	O
for	NN	O	O
germline	NN	O	O
PTEN	NN	O	O
mutations	NN	O	O
.	NN	O	O

PTEN	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
30	NN	O	O
of	NN	O	O
37	NN	O	O
(	NN	O	O
81	NN	O	O
%	NN	O	O
)	NN	O	O
CD	NN	O	B-Disease
families	NN	O	O
,	NN	O	O
including	NN	O	O
missense	NN	O	O
and	NN	O	O
nonsense	NN	O	O
point	NN	O	O
mutations	NN	O	O
,	NN	O	O
deletions	NN	O	O
,	NN	O	O
insertions	NN	O	O
,	NN	O	O
a	NN	O	O
deletion	NN	O	O
/	NN	O	O
insertion	NN	O	O
and	NN	O	O
splice	NN	O	O
site	NN	O	O
mutations	NN	O	O
.	NN	O	O

These	NN	O	O
mutations	NN	O	O
were	NN	O	O
scattered	NN	O	O
over	NN	O	O
the	NN	O	O
entire	NN	O	O
length	NN	O	O
of	NN	O	O
PTEN	NN	O	O
,	NN	O	O
with	NN	O	O
the	NN	O	O
exception	NN	O	O
of	NN	O	O
the	NN	O	O
first	NN	O	O
,	NN	O	O
fourth	NN	O	O
and	NN	O	O
last	NN	O	O
exons	NN	O	O
.	NN	O	O

A	NN	O	O
hot	NN	O	O
spot	NN	O	O
for	NN	O	O
PTEN	NN	O	O
mutation	NN	O	O
in	NN	O	O
CD	NN	O	B-Disease
was	NN	O	O
identified	NN	O	O
in	NN	O	O
exon	NN	O	O
5	NN	O	O
that	NN	O	O
contains	NN	O	O
the	NN	O	O
PTPase	NN	O	O
core	NN	O	O
motif	NN	O	O
,	NN	O	O
with	NN	O	O
13	NN	O	O
of	NN	O	O
30	NN	O	O
(	NN	O	O
43	NN	O	O
%	NN	O	O
)	NN	O	O
CD	NN	O	B-Disease
mutations	NN	O	O
identified	NN	O	O
in	NN	O	O
this	NN	O	O
exon	NN	O	O
.	NN	O	O

Seven	NN	O	O
of	NN	O	O
30	NN	O	O
(	NN	O	O
23	NN	O	O
%	NN	O	O
)	NN	O	O
were	NN	O	O
within	NN	O	O
the	NN	O	O
core	NN	O	O
motif	NN	O	O
,	NN	O	O
the	NN	O	O
majority	NN	O	O
(	NN	O	O
five	NN	O	O
of	NN	O	O
seven	NN	O	O
)	NN	O	O
of	NN	O	O
which	NN	O	O
were	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
possibly	NN	O	O
pointing	NN	O	O
to	NN	O	O
the	NN	O	O
functional	NN	O	O
significance	NN	O	O
of	NN	O	O
this	NN	O	O
region	NN	O	O
.	NN	O	O

Germline	NN	O	O
PTEN	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
four	NN	O	O
of	NN	O	O
seven	NN	O	O
(	NN	O	O
57	NN	O	O
%	NN	O	O
)	NN	O	O
BZS	NN	O	B-Disease
families	NN	O	O
studied	NN	O	O
.	NN	O	O

Interestingly	NN	O	O
,	NN	O	O
none	NN	O	O
of	NN	O	O
these	NN	O	O
mutations	NN	O	O
was	NN	O	O
observed	NN	O	O
in	NN	O	O
the	NN	O	O
PTPase	NN	O	O
core	NN	O	O
motif	NN	O	O
.	NN	O	O

It	NN	O	O
is	NN	O	O
also	NN	O	O
worthy	NN	O	O
of	NN	O	O
note	NN	O	O
that	NN	O	O
a	NN	O	O
single	NN	O	O
nonsense	NN	O	O
point	NN	O	O
mutation	NN	O	O
,	NN	O	O
R233X	NN	O	O
,	NN	O	O
was	NN	O	O
observed	NN	O	O
in	NN	O	O
the	NN	O	O
germline	NN	O	O
DNA	NN	O	O
from	NN	O	O
two	NN	O	O
unrelated	NN	O	O
CD	NN	O	B-Disease
families	NN	O	O
and	NN	O	O
one	NN	O	O
BZS	NN	O	B-Disease
family	NN	O	O
.	NN	O	O

Genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
studies	NN	O	O
were	NN	O	O
not	NN	O	O
performed	NN	O	O
on	NN	O	O
this	NN	O	O
small	NN	O	O
group	NN	O	O
of	NN	O	O
BZS	NN	O	B-Disease
families	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
analysis	NN	O	O
inthe	NN	O	O
group	NN	O	O
of	NN	O	O
CD	NN	O	B-Disease
families	NN	O	O
revealed	NN	O	O
two	NN	O	O
possible	NN	O	O
associations	NN	O	O
worthy	NN	O	O
of	NN	O	O
follow	NN	O	O
-	NN	O	O
up	NN	O	O
in	NN	O	O
independent	NN	O	O
analyses	NN	O	O
.	NN	O	O

The	NN	O	O
first	NN	O	O
was	NN	O	O
an	NN	O	O
association	NN	O	O
noted	NN	O	O
in	NN	O	O
the	NN	O	O
group	NN	O	O
of	NN	O	O
CD	NN	O	B-Disease
families	NN	O	O
with	NN	O	O
breast	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

A	NN	O	O
correlation	NN	O	O
was	NN	O	O
observed	NN	O	O
between	NN	O	O
the	NN	O	O
presence	NN	O	O
/	NN	O	O
absence	NN	O	O
of	NN	O	O
a	NN	O	O
PTEN	NN	O	O
mutation	NN	O	O
and	NN	O	O
the	NN	O	O
type	NN	O	O
of	NN	O	O
breast	NN	O	O
involvement	NN	O	O
(	NN	O	O
unaffected	NN	O	O
versus	NN	O	O
benign	NN	O	O
versus	NN	O	O
malignant	NN	O	O
)	NN	O	O
.	NN	O	O

Specifically	NN	O	O
and	NN	O	O
more	NN	O	O
directly	NN	O	O
,	NN	O	O
an	NN	O	O
association	NN	O	O
was	NN	O	O
also	NN	O	O
observed	NN	O	O
between	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
PTEN	NN	O	O
mutation	NN	O	O
and	NN	O	O
malignant	NN	O	B-Disease
breast	NN	O	I-Disease
disease	NN	O	I-Disease
.	NN	O	O

Secondly	NN	O	O
,	NN	O	O
there	NN	O	O
appeared	NN	O	O
to	NN	O	O
be	NN	O	O
an	NN	O	O
interdependent	NN	O	O
association	NN	O	O
between	NN	O	O
mutations	NN	O	O
upstream	NN	O	O
and	NN	O	O
within	NN	O	O
the	NN	O	O
PTPase	NN	O	O
core	NN	O	O
motif	NN	O	O
,	NN	O	O
the	NN	O	O
core	NN	O	O
motif	NN	O	O
containing	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
involvement	NN	O	O
of	NN	O	O
all	NN	O	O
major	NN	O	O
organ	NN	O	O
systems	NN	O	O
(	NN	O	O
central	NN	O	O
nervous	NN	O	O
system	NN	O	O
,	NN	O	O
thyroid	NN	O	O
,	NN	O	O
breast	NN	O	O
,	NN	O	O
skin	NN	O	O
and	NN	O	O
gastrointestinal	NN	O	O
tract	NN	O	O
)	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
these	NN	O	O
observations	NN	O	O
would	NN	O	O
need	NN	O	O
to	NN	O	O
be	NN	O	O
confirmed	NN	O	O
by	NN	O	O
studying	NN	O	O
a	NN	O	O
larger	NN	O	O
number	NN	O	O
of	NN	O	O
CD	NN	O	B-Disease
families	NN	O	O
.	NN	O	O

Molecular	NN	O	O
defects	NN	O	O
leading	NN	O	O
to	NN	O	O
human	NN	O	O
complement	NN	O	B-Disease
component	NN	O	I-Disease
C6	NN	O	I-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
an	NN	O	O
African	NN	O	O
-	NN	O	O
American	NN	O	O
family	NN	O	O
.	NN	O	O

Complement	NN	O	B-Disease
component	NN	O	I-Disease
C6	NN	O	I-Disease
deficiency	NN	O	I-Disease
(	NN	O	O
C6D	NN	O	B-Disease
)	NN	O	O
was	NN	O	O
diagnosed	NN	O	O
in	NN	O	O
a	NN	O	O
16	NN	O	O
-	NN	O	O
year	NN	O	O
-	NN	O	O
old	NN	O	O
African	NN	O	O
-	NN	O	O
American	NN	O	O
male	NN	O	O
with	NN	O	O
meningococcal	NN	O	B-Disease
meningitis	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
patients	NN	O	O
father	NN	O	O
and	NN	O	O
two	NN	O	O
brothers	NN	O	O
also	NN	O	O
had	NN	O	O
C6D	NN	O	B-Disease
,	NN	O	O
but	NN	O	O
gave	NN	O	O
no	NN	O	O
history	NN	O	O
of	NN	O	O
meningitis	NN	O	B-Disease
or	NN	O	O
other	NN	O	O
neisserial	NN	O	B-Disease
infection	NN	O	I-Disease
.	NN	O	O

By	NN	O	O
using	NN	O	O
exon	NN	O	O
-	NN	O	O
specific	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
(	NN	O	O
PCR	NN	O	O
)	NN	O	O
/	NN	O	O
single	NN	O	O
-	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
as	NN	O	O
a	NN	O	O
screening	NN	O	O
step	NN	O	O
and	NN	O	O
nucleotide	NN	O	O
sequencing	NN	O	O
of	NN	O	O
target	NN	O	O
exons	NN	O	O
,	NN	O	O
we	NN	O	O
determined	NN	O	O
that	NN	O	O
the	NN	O	O
proband	NN	O	O
was	NN	O	O
a	NN	O	O
compound	NN	O	O
heterozygote	NN	O	O
for	NN	O	O
two	NN	O	O
C6	NN	O	O
gene	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
first	NN	O	O
,	NN	O	O
1195delC	NN	O	O
located	NN	O	O
in	NN	O	O
exon	NN	O	O
7	NN	O	O
,	NN	O	O
is	NN	O	O
a	NN	O	O
novel	NN	O	O
mutation	NN	O	O
,	NN	O	O
while	NN	O	O
the	NN	O	O
second	NN	O	O
,	NN	O	O
1936delG	NN	O	O
in	NN	O	O
exon	NN	O	O
12	NN	O	O
,	NN	O	O
has	NN	O	O
been	NN	O	O
described	NN	O	O
before	NN	O	O
to	NN	O	O
cause	NN	O	O
C6D	NN	O	B-Disease
in	NN	O	O
an	NN	O	O
unrelated	NN	O	O
African	NN	O	O
-	NN	O	O
American	NN	O	O
individual	NN	O	O
.	NN	O	O

Both	NN	O	O
mutations	NN	O	O
result	NN	O	O
in	NN	O	O
premature	NN	O	O
termination	NN	O	O
codons	NN	O	O
and	NN	O	O
C6	NN	O	O
null	NN	O	O
alleles	NN	O	O
.	NN	O	O

Allele	NN	O	O
-	NN	O	O
specific	NN	O	O
PCR	NN	O	O
indicated	NN	O	O
that	NN	O	O
the	NN	O	O
probands	NN	O	O
two	NN	O	O
brothers	NN	O	O
also	NN	O	O
inherited	NN	O	O
the	NN	O	O
1195delC	NN	O	O
mutation	NN	O	O
from	NN	O	O
their	NN	O	O
heterozygous	NN	O	O
mother	NN	O	O
and	NN	O	O
the	NN	O	O
1936delG	NN	O	O
mutation	NN	O	O
from	NN	O	O
their	NN	O	O
homozygous	NN	O	O
father	NN	O	O
.	NN	O	O
.	NN	O	O

PAX6	NN	O	O
mutations	NN	O	O
reviewed	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
PAX6	NN	O	O
are	NN	O	O
responsible	NN	O	O
for	NN	O	O
human	NN	O	O
aniridia	NN	O	B-Disease
and	NN	O	O
have	NN	O	O
also	NN	O	O
been	NN	O	O
found	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
Peters	NN	O	B-Disease
anomaly	NN	O	I-Disease
,	NN	O	O
with	NN	O	O
congenital	NN	O	B-Disease
cataracts	NN	O	I-Disease
,	NN	O	O
with	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
keratitis	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
with	NN	O	O
isolated	NN	O	B-Disease
foveal	NN	O	I-Disease
hypoplasia	NN	O	I-Disease
.	NN	O	O

No	NN	O	O
locus	NN	O	O
other	NN	O	O
than	NN	O	O
chromosome	NN	O	O
11p13	NN	O	O
has	NN	O	O
been	NN	O	O
implicated	NN	O	O
in	NN	O	O
aniridia	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
PAX6	NN	O	O
is	NN	O	O
clearly	NN	O	O
the	NN	O	O
major	NN	O	O
,	NN	O	O
if	NN	O	O
not	NN	O	O
only	NN	O	O
,	NN	O	O
gene	NN	O	O
responsible	NN	O	O
.	NN	O	O

Twenty	NN	O	O
-	NN	O	O
eight	NN	O	O
percent	NN	O	O
of	NN	O	O
identified	NN	O	O
PAX6	NN	O	O
mutations	NN	O	O
are	NN	O	O
C	NN	O	O
-	NN	O	O
T	NN	O	O
changes	NN	O	O
at	NN	O	O
CpG	NN	O	O
dinucleotides	NN	O	O
,	NN	O	O
20	NN	O	O
%	NN	O	O
are	NN	O	O
splicing	NN	O	O
errors	NN	O	O
,	NN	O	O
and	NN	O	O
more	NN	O	O
than	NN	O	O
30	NN	O	O
%	NN	O	O
are	NN	O	O
deletion	NN	O	O
or	NN	O	O
insertion	NN	O	O
events	NN	O	O
.	NN	O	O

There	NN	O	O
is	NN	O	O
a	NN	O	O
noticeably	NN	O	O
elevated	NN	O	O
level	NN	O	O
of	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
paired	NN	O	O
domain	NN	O	O
compared	NN	O	O
with	NN	O	O
the	NN	O	O
rest	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
.	NN	O	O

Increased	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
homeodomain	NN	O	O
is	NN	O	O
accounted	NN	O	O
for	NN	O	O
by	NN	O	O
the	NN	O	O
hypermutable	NN	O	O
CpG	NN	O	O
dinucleotide	NN	O	O
in	NN	O	O
codon	NN	O	O
240	NN	O	O
.	NN	O	O

Very	NN	O	O
nearly	NN	O	O
all	NN	O	O
mutations	NN	O	O
appear	NN	O	O
to	NN	O	O
cause	NN	O	O
loss	NN	O	O
of	NN	O	O
function	NN	O	O
of	NN	O	O
the	NN	O	O
mutant	NN	O	O
allele	NN	O	O
,	NN	O	O
and	NN	O	O
more	NN	O	O
than	NN	O	O
80	NN	O	O
%	NN	O	O
of	NN	O	O
exonic	NN	O	O
substitutions	NN	O	O
result	NN	O	O
in	NN	O	O
nonsense	NN	O	O
codons	NN	O	O
.	NN	O	O

In	NN	O	O
a	NN	O	O
gene	NN	O	O
with	NN	O	O
such	NN	O	O
extraordinarily	NN	O	O
high	NN	O	O
sequence	NN	O	O
conservation	NN	O	O
throughout	NN	O	O
evolution	NN	O	O
,	NN	O	O
there	NN	O	O
are	NN	O	O
presumed	NN	O	O
undiscovered	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
these	NN	O	O
are	NN	O	O
hypothesized	NN	O	O
to	NN	O	O
exist	NN	O	O
in	NN	O	O
as	NN	O	O
-	NN	O	O
yet	NN	O	O
unidentified	NN	O	O
phenotypes	NN	O	O
.	NN	O	O
.	NN	O	O

Genetic	NN	O	O
heterogeneity	NN	O	O
and	NN	O	O
penetrance	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
genes	NN	O	O
in	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
families	NN	O	O
.	NN	O	O

The	NN	O	O
Breast	NN	O	B-Disease
Cancer	NN	O	I-Disease
Linkage	NN	O	O
Consortium	NN	O	O
.	NN	O	O

The	NN	O	O
contribution	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
to	NN	O	O
inherited	NN	O	B-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
was	NN	O	O
assessed	NN	O	O
by	NN	O	O
linkage	NN	O	O
and	NN	O	O
mutation	NN	O	O
analysis	NN	O	O
in	NN	O	O
237	NN	O	O
families	NN	O	O
,	NN	O	O
each	NN	O	O
with	NN	O	O
at	NN	O	O
least	NN	O	O
four	NN	O	O
cases	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
collected	NN	O	O
by	NN	O	O
the	NN	O	O
Breast	NN	O	B-Disease
Cancer	NN	O	I-Disease
Linkage	NN	O	O
Consortium	NN	O	O
.	NN	O	O

Families	NN	O	O
were	NN	O	O
included	NN	O	O
without	NN	O	O
regard	NN	O	O
to	NN	O	O
the	NN	O	O
occurrence	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
or	NN	O	I-Disease
other	NN	O	I-Disease
cancers	NN	O	I-Disease
.	NN	O	O

Overall	NN	O	O
,	NN	O	O
disease	NN	O	O
was	NN	O	O
linked	NN	O	O
to	NN	O	O
BRCA1	NN	O	O
in	NN	O	O
an	NN	O	O
estimated	NN	O	O
52	NN	O	O
%	NN	O	O
of	NN	O	O
families	NN	O	O
,	NN	O	O
to	NN	O	O
BRCA2	NN	O	O
in	NN	O	O
32	NN	O	O
%	NN	O	O
of	NN	O	O
families	NN	O	O
,	NN	O	O
and	NN	O	O
to	NN	O	O
neither	NN	O	O
gene	NN	O	O
in	NN	O	O
16	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
confidence	NN	O	O
interval	NN	O	O
[	NN	O	O
CI	NN	O	O
]	NN	O	O
6	NN	O	O
%	NN	O	O
-	NN	O	O
28	NN	O	O
%	NN	O	O
)	NN	O	O
,	NN	O	O
suggesting	NN	O	O
other	NN	O	O
predisposition	NN	O	O
genes	NN	O	O
.	NN	O	O

The	NN	O	O
majority	NN	O	O
(	NN	O	O
81	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
breast	NN	O	B-Disease
-	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
were	NN	O	O
due	NN	O	O
to	NN	O	O
BRCA1	NN	O	O
,	NN	O	O
with	NN	O	O
most	NN	O	O
others	NN	O	O
(	NN	O	O
14	NN	O	O
%	NN	O	O
)	NN	O	O
due	NN	O	O
to	NN	O	O
BRCA2	NN	O	O
.	NN	O	O

Conversely	NN	O	O
,	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
families	NN	O	O
with	NN	O	O
male	NN	O	B-Disease
and	NN	O	I-Disease
female	NN	O	I-Disease
breast	NN	O	I-Disease
cancer	NN	O	I-Disease
were	NN	O	O
due	NN	O	O
to	NN	O	O
BRCA2	NN	O	O
(	NN	O	O
76	NN	O	O
%	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
largest	NN	O	O
proportion	NN	O	O
(	NN	O	O
67	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
families	NN	O	O
due	NN	O	O
to	NN	O	O
other	NN	O	O
genes	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
families	NN	O	O
with	NN	O	O
four	NN	O	O
or	NN	O	O
five	NN	O	O
cases	NN	O	O
of	NN	O	O
female	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
only	NN	O	O
.	NN	O	O

These	NN	O	O
estimates	NN	O	O
were	NN	O	O
not	NN	O	O
substantially	NN	O	O
affected	NN	O	O
either	NN	O	O
by	NN	O	O
changing	NN	O	O
the	NN	O	O
assumed	NN	O	O
penetrance	NN	O	O
model	NN	O	O
for	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
by	NN	O	O
including	NN	O	O
or	NN	O	O
excluding	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
data	NN	O	O
.	NN	O	O

Among	NN	O	O
those	NN	O	O
families	NN	O	O
with	NN	O	O
disease	NN	O	O
due	NN	O	O
to	NN	O	O
BRCA1	NN	O	O
that	NN	O	O
were	NN	O	O
tested	NN	O	O
by	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
standard	NN	O	O
screening	NN	O	O
methods	NN	O	O
,	NN	O	O
mutations	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
coding	NN	O	O
sequence	NN	O	O
or	NN	O	O
splice	NN	O	O
sites	NN	O	O
in	NN	O	O
an	NN	O	O
estimated	NN	O	O
63	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
CI	NN	O	O
51	NN	O	O
%	NN	O	O
-	NN	O	O
77	NN	O	O
%	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
estimated	NN	O	O
sensitivity	NN	O	O
was	NN	O	O
identical	NN	O	O
for	NN	O	O
direct	NN	O	O
sequencing	NN	O	O
and	NN	O	O
other	NN	O	O
techniques	NN	O	O
.	NN	O	O

The	NN	O	O
penetrance	NN	O	O
of	NN	O	O
BRCA2	NN	O	O
was	NN	O	O
estimated	NN	O	O
by	NN	O	O
maximizing	NN	O	O
the	NN	O	O
LOD	NN	O	O
score	NN	O	O
in	NN	O	O
BRCA2	NN	O	O
-	NN	O	O
mutation	NN	O	O
families	NN	O	O
,	NN	O	O
over	NN	O	O
all	NN	O	O
possible	NN	O	O
penetrance	NN	O	O
functions	NN	O	O
.	NN	O	O

The	NN	O	O
estimated	NN	O	O
cumulative	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
reached	NN	O	O
28	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
CI	NN	O	O
9	NN	O	O
%	NN	O	O
-	NN	O	O
44	NN	O	O
%	NN	O	O
)	NN	O	O
by	NN	O	O
age	NN	O	O
50	NN	O	O
years	NN	O	O
and	NN	O	O
84	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
CI	NN	O	O
43	NN	O	O
%	NN	O	O
-	NN	O	O
95	NN	O	O
%	NN	O	O
)	NN	O	O
by	NN	O	O
age	NN	O	O
70	NN	O	O
years	NN	O	O
.	NN	O	O

The	NN	O	O
corresponding	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
risks	NN	O	O
were	NN	O	O
0	NN	O	O
.	NN	O	O

4	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
CI	NN	O	O
0	NN	O	O
%	NN	O	O
-	NN	O	O
1	NN	O	O
%	NN	O	O
)	NN	O	O
by	NN	O	O
age	NN	O	O
50	NN	O	O
years	NN	O	O
and	NN	O	O
27	NN	O	O
%	NN	O	O
(	NN	O	O
95	NN	O	O
%	NN	O	O
CI	NN	O	O
0	NN	O	O
%	NN	O	O
-	NN	O	O
47	NN	O	O
%	NN	O	O
)	NN	O	O
by	NN	O	O
age	NN	O	O
70	NN	O	O
years	NN	O	O
.	NN	O	O

The	NN	O	O
lifetime	NN	O	O
risk	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
appears	NN	O	O
similar	NN	O	O
to	NN	O	O
the	NN	O	O
risk	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
carriers	NN	O	O
,	NN	O	O
but	NN	O	O
there	NN	O	O
was	NN	O	O
some	NN	O	O
suggestion	NN	O	O
of	NN	O	O
a	NN	O	O
lower	NN	O	O
risk	NN	O	O
in	NN	O	O
BRCA2	NN	O	O
carriers	NN	O	O
<	NN	O	O
50	NN	O	O
years	NN	O	O
of	NN	O	O
age	NN	O	O
.	NN	O	O

Eye	NN	O	B-Disease
movement	NN	O	I-Disease
abnormalities	NN	O	I-Disease
correlate	NN	O	O
with	NN	O	O
genotype	NN	O	O
in	NN	O	O
autosomal	NN	O	O
dominant	NN	O	O
cerebellar	NN	O	B-Disease
ataxia	NN	O	I-Disease
type	NN	O	I-Disease
I	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
compared	NN	O	O
horizontal	NN	O	O
eye	NN	O	O
movements	NN	O	O
(	NN	O	O
visually	NN	O	O
guided	NN	O	O
saccades	NN	O	O
,	NN	O	O
antisaccades	NN	O	O
,	NN	O	O
and	NN	O	O
smooth	NN	O	O
pursuit	NN	O	O
)	NN	O	O
in	NN	O	O
control	NN	O	O
subjects	NN	O	O
(	NN	O	O
n	NN	O	O
=	NN	O	O
14	NN	O	O
)	NN	O	O
and	NN	O	O
patients	NN	O	O
with	NN	O	O
three	NN	O	O
forms	NN	O	O
of	NN	O	O
autosomal	NN	O	O
dominant	NN	O	O
cerebellar	NN	O	B-Disease
ataxias	NN	O	I-Disease
type	NN	O	I-Disease
I	NN	O	I-Disease
spinocerebellar	NN	O	B-Disease
ataxias	NN	O	I-Disease
1	NN	O	I-Disease
and	NN	O	I-Disease
2	NN	O	I-Disease
(	NN	O	O
SCA1	NN	O	B-Disease
,	NN	O	O
n	NN	O	O
=	NN	O	O
11	NN	O	O
;	NN	O	O
SCA2	NN	O	B-Disease
,	NN	O	O
n	NN	O	O
=	NN	O	O
10	NN	O	O
)	NN	O	O
and	NN	O	O
SCA3	NN	O	B-Disease
/	NN	O	O
Machado	NN	O	B-Disease
-	NN	O	I-Disease
Joseph	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
MJD	NN	O	B-Disease
)	NN	O	O
(	NN	O	O
n	NN	O	O
=	NN	O	O
16	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
SCA1	NN	O	B-Disease
,	NN	O	O
saccade	NN	O	O
amplitude	NN	O	O
was	NN	O	O
significantly	NN	O	O
increased	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
hypermetria	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
smooth	NN	O	O
pursuit	NN	O	O
gain	NN	O	O
was	NN	O	O
decreased	NN	O	O
.	NN	O	O

In	NN	O	O
SCA2	NN	O	B-Disease
,	NN	O	O
saccade	NN	O	O
velocity	NN	O	O
was	NN	O	O
markedly	NN	O	O
decreased	NN	O	O
.	NN	O	O

The	NN	O	O
percentage	NN	O	O
of	NN	O	O
errors	NN	O	O
in	NN	O	O
antisaccades	NN	O	O
was	NN	O	O
greatly	NN	O	O
increased	NN	O	O
and	NN	O	O
was	NN	O	O
significantly	NN	O	O
correlated	NN	O	O
with	NN	O	O
age	NN	O	O
at	NN	O	O
disease	NN	O	O
onset	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
a	NN	O	O
correlation	NN	O	O
between	NN	O	O
smooth	NN	O	O
pursuit	NN	O	O
gain	NN	O	O
and	NN	O	O
the	NN	O	O
number	NN	O	O
of	NN	O	O
trinucleotide	NN	O	O
repeats	NN	O	O
was	NN	O	O
found	NN	O	O
.	NN	O	O

In	NN	O	O
SCA3	NN	O	B-Disease
,	NN	O	O
gaze	NN	O	B-Disease
-	NN	O	I-Disease
evoked	NN	O	I-Disease
nystagmus	NN	O	I-Disease
was	NN	O	O
often	NN	O	O
present	NN	O	O
as	NN	O	O
was	NN	O	O
saccade	NN	O	O
hypometria	NN	O	O
and	NN	O	O
smooth	NN	O	O
pursuit	NN	O	O
gain	NN	O	O
was	NN	O	O
markedly	NN	O	O
decreased	NN	O	O
.	NN	O	O

Three	NN	O	O
major	NN	O	O
criteria	NN	O	O
,	NN	O	O
saccade	NN	O	O
amplitude	NN	O	O
,	NN	O	O
saccade	NN	O	O
velocity	NN	O	O
,	NN	O	O
and	NN	O	O
presence	NN	O	O
of	NN	O	O
gaze	NN	O	B-Disease
-	NN	O	I-Disease
evoked	NN	O	I-Disease
nystagmus	NN	O	I-Disease
,	NN	O	O
permitted	NN	O	O
the	NN	O	O
correct	NN	O	O
assignment	NN	O	O
of	NN	O	O
90	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
SCA1	NN	O	B-Disease
,	NN	O	O
90	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
SCA2	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
93	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
patients	NN	O	O
with	NN	O	O
SCA3	NN	O	B-Disease
to	NN	O	O
their	NN	O	O
genetically	NN	O	O
confirmed	NN	O	O
patient	NN	O	O
group	NN	O	O
and	NN	O	O
,	NN	O	O
therefore	NN	O	O
,	NN	O	O
may	NN	O	O
help	NN	O	O
orient	NN	O	O
diagnoses	NN	O	O
of	NN	O	O
SCA1	NN	O	B-Disease
,	NN	O	O
SCA2	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
SCA3	NN	O	B-Disease
at	NN	O	O
early	NN	O	O
clinical	NN	O	O
stages	NN	O	O
of	NN	O	O
the	NN	O	O
diseases	NN	O	O
.	NN	O	O
.	NN	O	O

Genetic	NN	O	O
basis	NN	O	O
and	NN	O	O
molecular	NN	O	O
mechanism	NN	O	O
for	NN	O	O
idiopathic	NN	O	B-Disease
ventricular	NN	O	I-Disease
fibrillation	NN	O	I-Disease
.	NN	O	O

Ventricular	NN	O	B-Disease
fibrillation	NN	O	I-Disease
causes	NN	O	O
more	NN	O	O
than	NN	O	O
300	NN	O	O
,	NN	O	O
000	NN	O	O
sudden	NN	O	O
deaths	NN	O	O
each	NN	O	O
year	NN	O	O
in	NN	O	O
the	NN	O	O
USA	NN	O	O
alone	NN	O	O
.	NN	O	O

In	NN	O	O
approximately	NN	O	O
5	NN	O	O
-	NN	O	O
12	NN	O	O
%	NN	O	O
of	NN	O	O
these	NN	O	O
cases	NN	O	O
,	NN	O	O
there	NN	O	O
are	NN	O	O
no	NN	O	O
demonstrable	NN	O	O
cardiac	NN	O	O
or	NN	O	O
non	NN	O	O
-	NN	O	O
cardiac	NN	O	O
causes	NN	O	O
to	NN	O	O
account	NN	O	O
for	NN	O	O
the	NN	O	O
episode	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
therefore	NN	O	O
classified	NN	O	O
as	NN	O	O
idiopathic	NN	O	B-Disease
ventricular	NN	O	I-Disease
fibrillation	NN	O	I-Disease
(	NN	O	O
IVF	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

A	NN	O	O
distinct	NN	O	O
group	NN	O	O
of	NN	O	O
IVF	NN	O	B-Disease
patients	NN	O	O
has	NN	O	O
been	NN	O	O
found	NN	O	O
to	NN	O	O
present	NN	O	O
with	NN	O	O
a	NN	O	O
characteristic	NN	O	O
electrocardiographic	NN	O	O
pattern	NN	O	O
.	NN	O	O

Because	NN	O	O
of	NN	O	O
the	NN	O	O
small	NN	O	O
size	NN	O	O
of	NN	O	O
most	NN	O	O
pedigrees	NN	O	O
and	NN	O	O
the	NN	O	O
high	NN	O	O
incidence	NN	O	O
of	NN	O	O
sudden	NN	O	B-Disease
death	NN	O	I-Disease
,	NN	O	O
however	NN	O	O
,	NN	O	O
molecular	NN	O	O
genetic	NN	O	O
studies	NN	O	O
of	NN	O	O
IVF	NN	O	B-Disease
have	NN	O	O
not	NN	O	O
yet	NN	O	O
been	NN	O	O
done	NN	O	O
.	NN	O	O

Because	NN	O	O
IVF	NN	O	B-Disease
causes	NN	O	O
cardiac	NN	O	O
rhythm	NN	O	O
disturbance	NN	O	O
,	NN	O	O
we	NN	O	O
investigated	NN	O	O
whether	NN	O	O
malfunction	NN	O	O
of	NN	O	O
ion	NN	O	O
channels	NN	O	O
could	NN	O	O
cause	NN	O	O
the	NN	O	O
disorder	NN	O	O
by	NN	O	O
studying	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
cardiac	NN	O	O
sodium	NN	O	O
channel	NN	O	O
gene	NN	O	O
SCN5A	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
now	NN	O	O
identified	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
,	NN	O	O
a	NN	O	O
splice	NN	O	O
-	NN	O	O
donor	NN	O	O
mutation	NN	O	O
,	NN	O	O
and	NN	O	O
a	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
coding	NN	O	O
region	NN	O	O
of	NN	O	O
SCN5A	NN	O	O
in	NN	O	O
three	NN	O	O
IVF	NN	O	B-Disease
families	NN	O	O
.	NN	O	O

We	NN	O	O
show	NN	O	O
that	NN	O	O
sodium	NN	O	O
channels	NN	O	O
with	NN	O	O
the	NN	O	O
missense	NN	O	O
mutation	NN	O	O
recover	NN	O	O
from	NN	O	O
inactivation	NN	O	O
more	NN	O	O
rapidly	NN	O	O
than	NN	O	O
normal	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
frameshift	NN	O	O
mutation	NN	O	O
causes	NN	O	O
the	NN	O	O
sodium	NN	O	O
channel	NN	O	O
to	NN	O	O
be	NN	O	O
non	NN	O	O
-	NN	O	O
functional	NN	O	O
.	NN	O	O

Our	NN	O	O
results	NN	O	O
indicate	NN	O	O
that	NN	O	O
mutations	NN	O	O
in	NN	O	O
cardiac	NN	O	O
ion	NN	O	O
-	NN	O	O
channel	NN	O	O
genes	NN	O	O
contribute	NN	O	O
to	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
developing	NN	O	O
IVF	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Molecular	NN	O	O
heterogeneity	NN	O	O
in	NN	O	O
mucopolysaccharidosis	NN	O	B-Disease
IVA	NN	O	I-Disease
in	NN	O	O
Australia	NN	O	O
and	NN	O	O
Northern	NN	O	O
Ireland	NN	O	O
:	NN	O	O
nine	NN	O	O
novel	NN	O	O
mutations	NN	O	O
including	NN	O	O
T312S	NN	O	O
,	NN	O	O
a	NN	O	O
common	NN	O	O
allele	NN	O	O
that	NN	O	O
confers	NN	O	O
a	NN	O	O
mild	NN	O	O
phenotype	NN	O	O
.	NN	O	O

Mucopolysaccharidosis	NN	O	B-Disease
IVA	NN	O	I-Disease
(	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
lysosomal	NN	O	I-Disease
storage	NN	O	I-Disease
disorder	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
a	NN	O	O
genetic	NN	O	B-Disease
defect	NN	O	I-Disease
in	NN	O	O
N	NN	O	O
-	NN	O	O
acetylgalactosamine	NN	O	O
-	NN	O	O
6	NN	O	O
-	NN	O	O
sulfate	NN	O	O
sulfatase	NN	O	O
(	NN	O	O
GALNS	NN	O	O
)	NN	O	O
.	NN	O	O

Previous	NN	O	O
studies	NN	O	O
of	NN	O	O
patients	NN	O	O
from	NN	O	O
a	NN	O	O
British	NN	O	O
-	NN	O	O
Irish	NN	O	O
population	NN	O	O
showed	NN	O	O
that	NN	O	O
the	NN	O	O
I113F	NN	O	O
mutation	NN	O	O
is	NN	O	O
the	NN	O	O
most	NN	O	O
common	NN	O	O
single	NN	O	O
mutation	NN	O	O
among	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
patients	NN	O	O
and	NN	O	O
produces	NN	O	O
a	NN	O	O
severe	NN	O	O
clinical	NN	O	O
phenotype	NN	O	O
.	NN	O	O

We	NN	O	O
studied	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
GALNS	NN	O	O
gene	NN	O	O
from	NN	O	O
23	NN	O	O
additional	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
patients	NN	O	O
(	NN	O	O
15	NN	O	O
from	NN	O	O
Australia	NN	O	O
,	NN	O	O
8	NN	O	O
from	NN	O	O
Northern	NN	O	O
Ireland	NN	O	O
)	NN	O	O
,	NN	O	O
with	NN	O	O
various	NN	O	O
clinical	NN	O	O
phenotypes	NN	O	O
(	NN	O	O
severe	NN	O	O
,	NN	O	O
16	NN	O	O
cases	NN	O	O
;	NN	O	O
intermediate	NN	O	O
,	NN	O	O
4	NN	O	O
cases	NN	O	O
;	NN	O	O
mild	NN	O	O
,	NN	O	O
3	NN	O	O
cases	NN	O	O
)	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
two	NN	O	O
common	NN	O	O
mutations	NN	O	O
that	NN	O	O
together	NN	O	O
accounted	NN	O	O
for	NN	O	O
32	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
44	NN	O	O
unrelated	NN	O	O
alleles	NN	O	O
in	NN	O	O
these	NN	O	O
patients	NN	O	O
.	NN	O	O

One	NN	O	O
is	NN	O	O
the	NN	O	O
T312S	NN	O	O
mutation	NN	O	O
,	NN	O	O
a	NN	O	O
novel	NN	O	O
mutation	NN	O	O
found	NN	O	O
exclusively	NN	O	O
in	NN	O	O
milder	NN	O	O
patients	NN	O	O
.	NN	O	O

The	NN	O	O
other	NN	O	O
is	NN	O	O
the	NN	O	O
previously	NN	O	O
described	NN	O	O
I113F	NN	O	O
that	NN	O	O
produces	NN	O	O
a	NN	O	O
severe	NN	O	O
phenotype	NN	O	O
.	NN	O	O

The	NN	O	O
I113F	NN	O	O
and	NN	O	O
T312S	NN	O	O
mutations	NN	O	O
accounted	NN	O	O
for	NN	O	O
8	NN	O	O
(	NN	O	O
18	NN	O	O
%	NN	O	O
)	NN	O	O
and	NN	O	O
6	NN	O	O
(	NN	O	O
14	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
44	NN	O	O
unrelated	NN	O	O
alleles	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

The	NN	O	O
relatively	NN	O	O
high	NN	O	O
residual	NN	O	O
GALNS	NN	O	O
activity	NN	O	O
seen	NN	O	O
when	NN	O	O
the	NN	O	O
T312S	NN	O	O
mutant	NN	O	O
cDNA	NN	O	O
is	NN	O	O
overexpressed	NN	O	O
in	NN	O	O
mutant	NN	O	O
cells	NN	O	O
provides	NN	O	O
an	NN	O	O
explanation	NN	O	O
for	NN	O	O
the	NN	O	O
mild	NN	O	O
phenotype	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
this	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
distribution	NN	O	O
and	NN	O	O
relative	NN	O	O
frequencies	NN	O	O
of	NN	O	O
the	NN	O	O
I113F	NN	O	O
and	NN	O	O
T312S	NN	O	O
mutations	NN	O	O
in	NN	O	O
Australia	NN	O	O
corresponded	NN	O	O
to	NN	O	O
those	NN	O	O
observed	NN	O	O
in	NN	O	O
Northern	NN	O	O
Ireland	NN	O	O
and	NN	O	O
are	NN	O	O
unique	NN	O	O
to	NN	O	O
these	NN	O	O
two	NN	O	O
populations	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
both	NN	O	O
mutations	NN	O	O
were	NN	O	O
probably	NN	O	O
introduced	NN	O	O
to	NN	O	O
Australia	NN	O	O
by	NN	O	O
Irish	NN	O	O
migrants	NN	O	O
during	NN	O	O
the	NN	O	O
19th	NN	O	O
century	NN	O	O
.	NN	O	O

Haplotype	NN	O	O
analysis	NN	O	O
using	NN	O	O
6	NN	O	O
RFLPs	NN	O	O
provides	NN	O	O
additional	NN	O	O
data	NN	O	O
that	NN	O	O
the	NN	O	O
I113F	NN	O	O
mutation	NN	O	O
originated	NN	O	O
from	NN	O	O
a	NN	O	O
common	NN	O	O
ancestor	NN	O	O
.	NN	O	O

The	NN	O	O
other	NN	O	O
9	NN	O	O
novel	NN	O	O
mutations	NN	O	O
identified	NN	O	O
in	NN	O	O
these	NN	O	O
23	NN	O	O
patients	NN	O	O
were	NN	O	O
each	NN	O	O
limited	NN	O	O
to	NN	O	O
a	NN	O	O
single	NN	O	O
family	NN	O	O
.	NN	O	O

These	NN	O	O
data	NN	O	O
provide	NN	O	O
further	NN	O	O
evidence	NN	O	O
for	NN	O	O
extensive	NN	O	O
allelic	NN	O	O
heterogeneity	NN	O	O
in	NN	O	O
MPS	NN	O	B-Disease
IVA	NN	O	I-Disease
in	NN	O	O
British	NN	O	O
-	NN	O	O
Irish	NN	O	O
patients	NN	O	O
and	NN	O	O
provide	NN	O	O
evidence	NN	O	O
for	NN	O	O
their	NN	O	O
transmission	NN	O	O
to	NN	O	O
Australia	NN	O	O
by	NN	O	O
British	NN	O	O
-	NN	O	O
Irish	NN	O	O
migrants	NN	O	O
.	NN	O	O
.	NN	O	O

Identification	NN	O	O
of	NN	O	O
constitutional	NN	O	O
WT1	NN	O	O
mutations	NN	O	O
,	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
isolated	NN	O	O
diffuse	NN	O	B-Disease
mesangial	NN	O	I-Disease
sclerosis	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
analysis	NN	O	O
of	NN	O	O
genotype	NN	O	O
/	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
by	NN	O	O
use	NN	O	O
of	NN	O	O
a	NN	O	O
computerized	NN	O	O
mutation	NN	O	O
database	NN	O	O
.	NN	O	O

Constitutional	NN	O	O
mutations	NN	O	O
of	NN	O	O
the	NN	O	O
WT1	NN	O	O
gene	NN	O	O
,	NN	O	O
encoding	NN	O	O
a	NN	O	O
zinc	NN	O	O
-	NN	O	O
finger	NN	O	O
transcription	NN	O	O
factor	NN	O	O
involved	NN	O	O
in	NN	O	O
renal	NN	O	O
and	NN	O	O
gonadal	NN	O	O
development	NN	O	O
,	NN	O	O
are	NN	O	O
found	NN	O	O
in	NN	O	O
most	NN	O	O
patients	NN	O	O
with	NN	O	O
Denys	NN	O	B-Disease
-	NN	O	I-Disease
Drash	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
DDS	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
or	NN	O	O
diffuse	NN	O	B-Disease
mesangial	NN	O	I-Disease
sclerosis	NN	O	I-Disease
(	NN	O	O
DMS	NN	O	B-Disease
)	NN	O	O
associated	NN	O	O
with	NN	O	O
pseudohermaphroditism	NN	O	B-Disease
and	NN	O	O
/	NN	O	O
or	NN	O	O
Wilms	NN	O	B-Disease
tumor	NN	O	I-Disease
(	NN	O	O
WT	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Most	NN	O	O
mutations	NN	O	O
in	NN	O	O
DDS	NN	O	B-Disease
patients	NN	O	O
lie	NN	O	O
in	NN	O	O
exon	NN	O	O
8	NN	O	O
or	NN	O	O
exon	NN	O	O
9	NN	O	O
,	NN	O	O
encoding	NN	O	O
zinc	NN	O	O
finger	NN	O	O
2	NN	O	O
or	NN	O	O
zinc	NN	O	O
finger	NN	O	O
3	NN	O	O
,	NN	O	O
respectively	NN	O	O
,	NN	O	O
with	NN	O	O
a	NN	O	O
hot	NN	O	O
spot	NN	O	O
(	NN	O	O
R394W	NN	O	O
)	NN	O	O
in	NN	O	O
exon	NN	O	O
9	NN	O	O
.	NN	O	O

We	NN	O	O
analyzed	NN	O	O
a	NN	O	O
series	NN	O	O
of	NN	O	O
24	NN	O	O
patients	NN	O	O
,	NN	O	O
10	NN	O	O
with	NN	O	O
isolated	NN	O	B-Disease
DMS	NN	O	I-Disease
(	NN	O	O
IDMS	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
10	NN	O	O
with	NN	O	O
DDS	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
4	NN	O	O
with	NN	O	O
urogenital	NN	O	B-Disease
abnormalities	NN	O	I-Disease
and	NN	O	O
/	NN	O	O
or	NN	O	O
WT	NN	O	B-Disease
.	NN	O	O

We	NN	O	O
report	NN	O	O
WT1	NN	O	O
heterozygous	NN	O	O
mutations	NN	O	O
in	NN	O	O
16	NN	O	O
patients	NN	O	O
,	NN	O	O
4	NN	O	O
of	NN	O	O
whom	NN	O	O
presented	NN	O	O
with	NN	O	O
IDMS	NN	O	B-Disease
.	NN	O	O

One	NN	O	O
male	NN	O	O
and	NN	O	O
two	NN	O	O
female	NN	O	O
IDMS	NN	O	B-Disease
patients	NN	O	O
with	NN	O	O
WT1	NN	O	O
mutations	NN	O	O
underwent	NN	O	O
normal	NN	O	O
puberty	NN	O	O
.	NN	O	O

Two	NN	O	O
mutations	NN	O	O
associated	NN	O	O
with	NN	O	O
IDMS	NN	O	B-Disease
are	NN	O	O
different	NN	O	O
from	NN	O	O
those	NN	O	O
described	NN	O	O
in	NN	O	O
DDS	NN	O	B-Disease
patients	NN	O	O
.	NN	O	O

No	NN	O	O
WT1	NN	O	O
mutations	NN	O	O
were	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
six	NN	O	O
other	NN	O	O
IDMS	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
suggesting	NN	O	O
genetic	NN	O	O
heterogeneity	NN	O	O
of	NN	O	O
this	NN	O	O
disease	NN	O	O
.	NN	O	O

We	NN	O	O
analyzed	NN	O	O
genotype	NN	O	O
/	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
,	NN	O	O
on	NN	O	O
the	NN	O	O
basis	NN	O	O
of	NN	O	O
the	NN	O	O
constitution	NN	O	O
of	NN	O	O
a	NN	O	O
WT1	NN	O	O
mutation	NN	O	O
database	NN	O	O
of	NN	O	O
84	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
,	NN	O	O
to	NN	O	O
compare	NN	O	O
the	NN	O	O
distribution	NN	O	O
and	NN	O	O
type	NN	O	O
of	NN	O	O
mutations	NN	O	O
,	NN	O	O
according	NN	O	O
to	NN	O	O
the	NN	O	O
different	NN	O	O
symptoms	NN	O	O
.	NN	O	O

This	NN	O	O
demonstrated	NN	O	O
(	NN	O	O
1	NN	O	O
)	NN	O	O
the	NN	O	O
association	NN	O	O
between	NN	O	O
mutations	NN	O	O
in	NN	O	O
exons	NN	O	O
8	NN	O	O
and	NN	O	O
9	NN	O	O
and	NN	O	O
DMS	NN	O	B-Disease
;	NN	O	O
(	NN	O	O
2	NN	O	O
)	NN	O	O
among	NN	O	O
patients	NN	O	O
with	NN	O	O
DMS	NN	O	B-Disease
,	NN	O	O
a	NN	O	O
higher	NN	O	O
frequency	NN	O	O
of	NN	O	O
exon	NN	O	O
8	NN	O	O
mutations	NN	O	O
among	NN	O	O
46	NN	O	O
,	NN	O	O
XY	NN	O	O
patients	NN	O	O
with	NN	O	O
female	NN	O	O
phenotype	NN	O	O
than	NN	O	O
among	NN	O	O
46	NN	O	O
,	NN	O	O
XY	NN	O	O
patients	NN	O	O
with	NN	O	O
sexual	NN	O	O
ambiguity	NN	O	O
or	NN	O	O
male	NN	O	O
phenotype	NN	O	O
;	NN	O	O
and	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
statistically	NN	O	O
significant	NN	O	O
evidence	NN	O	O
that	NN	O	O
mutations	NN	O	O
in	NN	O	O
exons	NN	O	O
8	NN	O	O
and	NN	O	O
9	NN	O	O
preferentially	NN	O	O
affect	NN	O	O
amino	NN	O	O
acids	NN	O	O
with	NN	O	O
different	NN	O	O
functions	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
185delAG	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
originated	NN	O	O
before	NN	O	O
the	NN	O	O
dispersion	NN	O	O
of	NN	O	O
Jews	NN	O	O
in	NN	O	O
the	NN	O	O
diaspora	NN	O	O
and	NN	O	O
is	NN	O	O
not	NN	O	O
limited	NN	O	O
to	NN	O	O
Ashkenazim	NN	O	O
.	NN	O	O

The	NN	O	O
185delAG	NN	O	O
mutation	NN	O	O
in	NN	O	O
BRCA1	NN	O	O
is	NN	O	O
detected	NN	O	O
in	NN	O	O
Ashkenazi	NN	O	O
Jews	NN	O	O
both	NN	O	O
in	NN	O	O
familial	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
and	NN	O	O
in	NN	O	O
the	NN	O	O
general	NN	O	O
population	NN	O	O
.	NN	O	O

All	NN	O	O
tested	NN	O	O
Ashkenazi	NN	O	O
mutation	NN	O	O
carriers	NN	O	O
share	NN	O	O
the	NN	O	O
same	NN	O	O
allelic	NN	O	O
pattern	NN	O	O
at	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
locus	NN	O	O
.	NN	O	O

Our	NN	O	O
previous	NN	O	O
study	NN	O	O
showed	NN	O	O
that	NN	O	O
this	NN	O	O
Ashkenazi	NN	O	O
mutation	NN	O	O
also	NN	O	O
occurs	NN	O	O
in	NN	O	O
Iraqi	NN	O	O
Jews	NN	O	O
with	NN	O	O
a	NN	O	O
similar	NN	O	O
allelic	NN	O	O
pattern	NN	O	O
.	NN	O	O

We	NN	O	O
extended	NN	O	O
our	NN	O	O
analysis	NN	O	O
to	NN	O	O
other	NN	O	O
non	NN	O	O
-	NN	O	O
Ashkenazi	NN	O	O
subsets	NN	O	O
354	NN	O	O
of	NN	O	O
Moroccan	NN	O	O
origin	NN	O	O
,	NN	O	O
200	NN	O	O
Yemenites	NN	O	O
and	NN	O	O
150	NN	O	O
Iranian	NN	O	O
Jews	NN	O	O
.	NN	O	O

Heteroduplex	NN	O	O
analysis	NN	O	O
complemented	NN	O	O
by	NN	O	O
direct	NN	O	O
DNA	NN	O	O
sequencing	NN	O	O
of	NN	O	O
abnormally	NN	O	O
migrating	NN	O	O
bands	NN	O	O
were	NN	O	O
employed	NN	O	O
.	NN	O	O

Four	NN	O	O
of	NN	O	O
Moroccan	NN	O	O
origin	NN	O	O
(	NN	O	O
1	NN	O	O
.	NN	O	O
1	NN	O	O
%	NN	O	O
)	NN	O	O
and	NN	O	O
none	NN	O	O
of	NN	O	O
the	NN	O	O
Yemenites	NN	O	O
or	NN	O	O
Iranians	NN	O	O
was	NN	O	O
a	NN	O	O
carrier	NN	O	O
of	NN	O	O
the	NN	O	O
185delAG	NN	O	O
mutation	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
allelic	NN	O	O
patterns	NN	O	O
were	NN	O	O
determined	NN	O	O
for	NN	O	O
four	NN	O	O
of	NN	O	O
these	NN	O	O
individuals	NN	O	O
and	NN	O	O
for	NN	O	O
12	NN	O	O
additional	NN	O	O
non	NN	O	O
-	NN	O	O
Ashkenazi	NN	O	O
185delAG	NN	O	O
mutation	NN	O	O
carriers	NN	O	O
who	NN	O	O
had	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Six	NN	O	O
non	NN	O	O
-	NN	O	O
Ashkenazi	NN	O	O
individuals	NN	O	O
shared	NN	O	O
the	NN	O	O
common	NN	O	O
Ashkenazi	NN	O	O
haplotype	NN	O	O
,	NN	O	O
four	NN	O	O
had	NN	O	O
a	NN	O	O
closely	NN	O	O
related	NN	O	O
pattern	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
rest	NN	O	O
(	NN	O	O
n	NN	O	O
=	NN	O	O
6	NN	O	O
)	NN	O	O
displayed	NN	O	O
a	NN	O	O
distinct	NN	O	O
BRCA1	NN	O	O
allelic	NN	O	O
pattern	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
the	NN	O	O
185delAG	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
occurs	NN	O	O
in	NN	O	O
some	NN	O	O
non	NN	O	O
-	NN	O	O
Ashkenazi	NN	O	O
populations	NN	O	O
at	NN	O	O
rates	NN	O	O
comparable	NN	O	O
with	NN	O	O
that	NN	O	O
of	NN	O	O
Ashkenazim	NN	O	O
.	NN	O	O

The	NN	O	O
majority	NN	O	O
of	NN	O	O
Jewish	NN	O	O
185delAG	NN	O	O
mutation	NN	O	O
carriers	NN	O	O
have	NN	O	O
a	NN	O	O
common	NN	O	O
allelic	NN	O	O
pattern	NN	O	O
,	NN	O	O
supporting	NN	O	O
the	NN	O	O
founder	NN	O	O
effect	NN	O	O
notion	NN	O	O
,	NN	O	O
but	NN	O	O
dating	NN	O	O
the	NN	O	O
mutations	NN	O	O
origin	NN	O	O
to	NN	O	O
an	NN	O	O
earlier	NN	O	O
date	NN	O	O
than	NN	O	O
currently	NN	O	O
estimated	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
the	NN	O	O
different	NN	O	O
allelic	NN	O	O
pattern	NN	O	O
at	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
locus	NN	O	O
even	NN	O	O
in	NN	O	O
some	NN	O	O
Jewish	NN	O	O
mutation	NN	O	O
carriers	NN	O	O
,	NN	O	O
might	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
mutation	NN	O	O
arose	NN	O	O
independently	NN	O	O
.	NN	O	O
.	NN	O	O

Crystal	NN	O	O
structure	NN	O	O
of	NN	O	O
the	NN	O	O
hemochromatosis	NN	O	B-Disease
protein	NN	O	O
HFE	NN	O	O
and	NN	O	O
characterization	NN	O	O
of	NN	O	O
its	NN	O	O
interaction	NN	O	O
with	NN	O	O
transferrin	NN	O	O
receptor	NN	O	O
.	NN	O	O

HFE	NN	O	O
is	NN	O	O
an	NN	O	O
MHC	NN	O	O
-	NN	O	O
related	NN	O	O
protein	NN	O	O
that	NN	O	O
is	NN	O	O
mutated	NN	O	O
in	NN	O	O
the	NN	O	O
iron	NN	O	B-Disease
-	NN	O	I-Disease
overload	NN	O	I-Disease
disease	NN	O	I-Disease
hereditary	NN	O	B-Disease
hemochromatosis	NN	O	I-Disease
.	NN	O	O

HFE	NN	O	O
binds	NN	O	O
to	NN	O	O
transferrin	NN	O	O
receptor	NN	O	O
(	NN	O	O
TfR	NN	O	O
)	NN	O	O
and	NN	O	O
reduces	NN	O	O
its	NN	O	O
affinity	NN	O	O
for	NN	O	O
iron	NN	O	O
-	NN	O	O
loaded	NN	O	O
transferrin	NN	O	O
,	NN	O	O
implicating	NN	O	O
HFE	NN	O	O
in	NN	O	O
iron	NN	O	O
metabolism	NN	O	O
.	NN	O	O

The	NN	O	O
2	NN	O	O
.	NN	O	O

6	NN	O	O
A	NN	O	O
crystal	NN	O	O
structure	NN	O	O
of	NN	O	O
HFE	NN	O	O
reveals	NN	O	O
the	NN	O	O
locations	NN	O	O
of	NN	O	O
hemochromatosis	NN	O	B-Disease
mutations	NN	O	O
and	NN	O	O
a	NN	O	O
patch	NN	O	O
of	NN	O	O
histidines	NN	O	O
that	NN	O	O
could	NN	O	O
be	NN	O	O
involved	NN	O	O
in	NN	O	O
pH	NN	O	O
-	NN	O	O
dependent	NN	O	O
interactions	NN	O	O
.	NN	O	O

We	NN	O	O
also	NN	O	O
demonstrate	NN	O	O
that	NN	O	O
soluble	NN	O	O
TfR	NN	O	O
and	NN	O	O
HFE	NN	O	O
bind	NN	O	O
tightly	NN	O	O
at	NN	O	O
the	NN	O	O
basic	NN	O	O
pH	NN	O	O
of	NN	O	O
the	NN	O	O
cell	NN	O	O
surface	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
at	NN	O	O
the	NN	O	O
acidic	NN	O	O
pH	NN	O	O
of	NN	O	O
intracellular	NN	O	O
vesicles	NN	O	O
.	NN	O	O

TfR	NN	O	O
HFE	NN	O	O
stoichiometry	NN	O	O
(	NN	O	O
2	NN	O	O
1	NN	O	O
)	NN	O	O
differs	NN	O	O
from	NN	O	O
TfR	NN	O	O
transferrin	NN	O	O
stoichiometry	NN	O	O
(	NN	O	O
2	NN	O	O
2	NN	O	O
)	NN	O	O
,	NN	O	O
implying	NN	O	O
a	NN	O	O
different	NN	O	O
mode	NN	O	O
of	NN	O	O
binding	NN	O	O
for	NN	O	O
HFE	NN	O	O
and	NN	O	O
transferrin	NN	O	O
to	NN	O	O
TfR	NN	O	O
,	NN	O	O
consistent	NN	O	O
with	NN	O	O
our	NN	O	O
demonstration	NN	O	O
that	NN	O	O
HFE	NN	O	O
,	NN	O	O
transferrin	NN	O	O
,	NN	O	O
and	NN	O	O
TfR	NN	O	O
form	NN	O	O
a	NN	O	O
ternary	NN	O	O
complex	NN	O	O
.	NN	O	O

Identification	NN	O	O
of	NN	O	O
three	NN	O	O
novel	NN	O	O
mutations	NN	O	O
and	NN	O	O
a	NN	O	O
high	NN	O	O
frequency	NN	O	O
of	NN	O	O
the	NN	O	O
Arg778Leu	NN	O	O
mutation	NN	O	O
in	NN	O	O
Korean	NN	O	O
patients	NN	O	O
with	NN	O	O
Wilson	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

Four	NN	O	O
mutations	NN	O	O
-	NN	O	O
-	NN	O	O
R778L	NN	O	O
,	NN	O	O
A874V	NN	O	O
,	NN	O	O
L1083F	NN	O	O
,	NN	O	O
and	NN	O	O
2304delC	NN	O	O
-	NN	O	O
-	NN	O	O
in	NN	O	O
the	NN	O	O
copper	NN	O	O
-	NN	O	O
transporting	NN	O	O
enzyme	NN	O	O
,	NN	O	O
P	NN	O	O
-	NN	O	O
type	NN	O	O
ATPase	NN	O	O
(	NN	O	O
ATP7B	NN	O	O
)	NN	O	O
,	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
Korean	NN	O	O
Patients	NN	O	O
with	NN	O	O
Wilson	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

Arg778Leu	NN	O	O
,	NN	O	O
the	NN	O	O
most	NN	O	O
frequently	NN	O	O
reported	NN	O	O
mutation	NN	O	O
of	NN	O	O
this	NN	O	O
enzyme	NN	O	O
,	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
six	NN	O	O
of	NN	O	O
eight	NN	O	O
unrelated	NN	O	O
patients	NN	O	O
studied	NN	O	O
,	NN	O	O
an	NN	O	O
allele	NN	O	O
frequency	NN	O	O
of	NN	O	O
37	NN	O	O
.	NN	O	O

5	NN	O	O
%	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
considerably	NN	O	O
higher	NN	O	O
than	NN	O	O
those	NN	O	O
in	NN	O	O
other	NN	O	O
Asian	NN	O	O
populations	NN	O	O
.	NN	O	O

The	NN	O	O
novel	NN	O	O
single	NN	O	O
nucleotide	NN	O	O
deletion	NN	O	O
,	NN	O	O
2304delC	NN	O	O
,	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
one	NN	O	O
patient	NN	O	O
.	NN	O	O

Since	NN	O	O
a	NN	O	O
mutation	NN	O	O
at	NN	O	O
cDNA	NN	O	O
nucleotide	NN	O	O
2302	NN	O	O
(	NN	O	O
2302insC	NN	O	O
)	NN	O	O
had	NN	O	O
been	NN	O	O
previously	NN	O	O
described	NN	O	O
,	NN	O	O
this	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
ATP7B	NN	O	O
gene	NN	O	O
may	NN	O	O
be	NN	O	O
susceptible	NN	O	O
to	NN	O	O
gene	NN	O	O
rearrangements	NN	O	O
causing	NN	O	O
Wilson	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

Disruption	NN	O	O
of	NN	O	O
splicing	NN	O	O
regulated	NN	O	O
by	NN	O	O
a	NN	O	O
CUG	NN	O	O
-	NN	O	O
binding	NN	O	O
protein	NN	O	O
in	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
a	NN	O	O
CTG	NN	O	O
expansion	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
untranslated	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

One	NN	O	O
model	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
pathogenesis	NN	O	O
suggests	NN	O	O
that	NN	O	O
RNAs	NN	O	O
from	NN	O	O
the	NN	O	O
expanded	NN	O	O
allele	NN	O	O
create	NN	O	O
a	NN	O	O
gain	NN	O	O
-	NN	O	O
of	NN	O	O
-	NN	O	O
function	NN	O	O
mutation	NN	O	O
by	NN	O	O
the	NN	O	O
inappropriate	NN	O	O
binding	NN	O	O
of	NN	O	O
proteins	NN	O	O
to	NN	O	O
the	NN	O	O
CUG	NN	O	O
repeats	NN	O	O
.	NN	O	O

Data	NN	O	O
presented	NN	O	O
here	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
conserved	NN	O	O
heterogeneous	NN	O	O
nuclear	NN	O	O
ribonucleoprotein	NN	O	O
,	NN	O	O
CUG	NN	O	O
-	NN	O	O
binding	NN	O	O
protein	NN	O	O
(	NN	O	O
CUG	NN	O	O
-	NN	O	O
BP	NN	O	O
)	NN	O	O
,	NN	O	O
may	NN	O	O
mediate	NN	O	O
the	NN	O	O
trans	NN	O	O
-	NN	O	O
dominant	NN	O	O
effect	NN	O	O
of	NN	O	O
the	NN	O	O
RNA	NN	O	O
.	NN	O	O

CUG	NN	O	O
-	NN	O	O
BP	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
bind	NN	O	O
to	NN	O	O
the	NN	O	O
human	NN	O	O
cardiac	NN	O	O
troponin	NN	O	O
T	NN	O	O
(	NN	O	O
cTNT	NN	O	O
)	NN	O	O
pre	NN	O	O
-	NN	O	O
messenger	NN	O	O
RNA	NN	O	O
and	NN	O	O
regulate	NN	O	O
its	NN	O	O
alternative	NN	O	O
splicing	NN	O	O
.	NN	O	O

Splicing	NN	O	O
of	NN	O	O
cTNT	NN	O	O
was	NN	O	O
disrupted	NN	O	O
in	NN	O	O
DM	NN	O	B-Disease
striated	NN	O	O
muscle	NN	O	O
and	NN	O	O
in	NN	O	O
normal	NN	O	O
cells	NN	O	O
expressing	NN	O	O
transcripts	NN	O	O
that	NN	O	O
contain	NN	O	O
CUG	NN	O	O
repeats	NN	O	O
.	NN	O	O

Altered	NN	O	O
expression	NN	O	O
of	NN	O	O
genes	NN	O	O
regulated	NN	O	O
posttranscriptionally	NN	O	O
by	NN	O	O
CUG	NN	O	O
-	NN	O	O
BP	NN	O	O
therefore	NN	O	O
may	NN	O	O
contribute	NN	O	O
to	NN	O	O
DM	NN	O	B-Disease
pathogenesis	NN	O	O
.	NN	O	O
.	NN	O	O

Identification	NN	O	O
of	NN	O	O
a	NN	O	O
novel	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
and	NN	O	O
a	NN	O	O
missense	NN	O	O
substitution	NN	O	O
in	NN	O	O
the	NN	O	O
vasopressin	NN	O	O
-	NN	O	O
neurophysin	NN	O	O
II	NN	O	O
gene	NN	O	O
in	NN	O	O
two	NN	O	O
Spanish	NN	O	O
kindreds	NN	O	O
with	NN	O	O
familial	NN	O	B-Disease
neurohypophyseal	NN	O	I-Disease
diabetes	NN	O	I-Disease
insipidus	NN	O	I-Disease
.	NN	O	O

Familial	NN	O	B-Disease
neurohypophyseal	NN	O	I-Disease
diabetes	NN	O	I-Disease
insipidus	NN	O	I-Disease
(	NN	O	O
FNDI	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disease	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
deficiency	NN	O	O
in	NN	O	O
the	NN	O	O
antidiuretic	NN	O	O
hormone	NN	O	O
arginine	NN	O	O
vasopressin	NN	O	O
(	NN	O	O
AVP	NN	O	O
)	NN	O	O
encoded	NN	O	O
by	NN	O	O
the	NN	O	O
AVP	NN	O	O
-	NN	O	O
neurophysin	NN	O	O
II	NN	O	O
(	NN	O	O
AVP	NN	O	O
-	NN	O	O
NPII	NN	O	O
)	NN	O	O
gene	NN	O	O
on	NN	O	O
chromosome	NN	O	O
20p13	NN	O	O
.	NN	O	O

In	NN	O	O
this	NN	O	O
study	NN	O	O
,	NN	O	O
we	NN	O	O
analyzed	NN	O	O
two	NN	O	O
families	NN	O	O
with	NN	O	O
FNDI	NN	O	B-Disease
using	NN	O	O
direct	NN	O	O
automated	NN	O	O
fluorescent	NN	O	O
,	NN	O	O
solid	NN	O	O
phase	NN	O	O
,	NN	O	O
single	NN	O	O
-	NN	O	O
stranded	NN	O	O
DNA	NN	O	O
sequencing	NN	O	O
of	NN	O	O
PCR	NN	O	O
-	NN	O	O
amplified	NN	O	O
AVP	NN	O	O
-	NN	O	O
NPII	NN	O	O
DNA	NN	O	O
.	NN	O	O

In	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
families	NN	O	O
,	NN	O	O
affected	NN	O	O
individuals	NN	O	O
presented	NN	O	O
a	NN	O	O
novel	NN	O	O
nonsense	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
3	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
,	NN	O	O
consisting	NN	O	O
in	NN	O	O
a	NN	O	O
G	NN	O	O
to	NN	O	O
T	NN	O	O
transition	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
2101	NN	O	O
,	NN	O	O
which	NN	O	O
produces	NN	O	O
a	NN	O	O
stop	NN	O	O
signal	NN	O	O
in	NN	O	O
codon	NN	O	O
82	NN	O	O
(	NN	O	O
Glu	NN	O	O
)	NN	O	O
of	NN	O	O
NPII	NN	O	O
.	NN	O	O

The	NN	O	O
premature	NN	O	O
termination	NN	O	O
eliminates	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
C	NN	O	O
-	NN	O	O
terminal	NN	O	O
domain	NN	O	O
of	NN	O	O
NPII	NN	O	O
,	NN	O	O
including	NN	O	O
a	NN	O	O
cysteine	NN	O	O
residue	NN	O	O
in	NN	O	O
position	NN	O	O
85	NN	O	O
,	NN	O	O
which	NN	O	O
could	NN	O	O
be	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
correct	NN	O	O
folding	NN	O	O
of	NN	O	O
the	NN	O	O
prohormone	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
second	NN	O	O
family	NN	O	O
,	NN	O	O
a	NN	O	O
G279A	NN	O	O
substitution	NN	O	O
at	NN	O	O
position	NN	O	O
-	NN	O	O
1	NN	O	O
of	NN	O	O
the	NN	O	O
signal	NN	O	O
peptide	NN	O	O
was	NN	O	O
observed	NN	O	O
in	NN	O	O
all	NN	O	O
affected	NN	O	O
individuals	NN	O	O
.	NN	O	O

This	NN	O	O
missense	NN	O	O
mutation	NN	O	O
,	NN	O	O
which	NN	O	O
replaces	NN	O	O
Ala	NN	O	O
with	NN	O	O
Thr	NN	O	O
,	NN	O	O
is	NN	O	O
frequent	NN	O	O
among	NN	O	O
FNDI	NN	O	B-Disease
patients	NN	O	O
and	NN	O	O
is	NN	O	O
thought	NN	O	O
to	NN	O	O
reduce	NN	O	O
the	NN	O	O
efficiency	NN	O	O
of	NN	O	O
cleavage	NN	O	O
by	NN	O	O
signal	NN	O	O
peptidases	NN	O	O
.	NN	O	O
.	NN	O	O

Genetic	NN	O	O
heterogeneity	NN	O	O
of	NN	O	O
Saethre	NN	O	B-Disease
-	NN	O	I-Disease
Chotzen	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
due	NN	O	O
to	NN	O	O
TWIST	NN	O	O
and	NN	O	O
FGFR	NN	O	O
mutations	NN	O	O
.	NN	O	O

Thirty	NN	O	O
-	NN	O	O
two	NN	O	O
unrelated	NN	O	O
patients	NN	O	O
with	NN	O	O
features	NN	O	O
of	NN	O	O
Saethre	NN	O	B-Disease
-	NN	O	I-Disease
Chotzen	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
a	NN	O	O
common	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
condition	NN	O	I-Disease
of	NN	O	O
craniosynostosis	NN	O	B-Disease
and	NN	O	O
limb	NN	O	B-Disease
anomalies	NN	O	I-Disease
,	NN	O	O
were	NN	O	O
screened	NN	O	O
for	NN	O	O
mutations	NN	O	O
in	NN	O	O
TWIST	NN	O	O
,	NN	O	O
FGFR2	NN	O	O
,	NN	O	O
and	NN	O	O
FGFR3	NN	O	O
.	NN	O	O

Nine	NN	O	O
novel	NN	O	O
and	NN	O	O
three	NN	O	O
recurrent	NN	O	O
TWIST	NN	O	O
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
12	NN	O	O
families	NN	O	O
.	NN	O	O

Seven	NN	O	O
families	NN	O	O
were	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
the	NN	O	O
FGFR3	NN	O	O
P250R	NN	O	O
mutation	NN	O	O
,	NN	O	O
and	NN	O	O
one	NN	O	O
individual	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
an	NN	O	O
FGFR2	NN	O	O
VV269	NN	O	O
-	NN	O	O
270	NN	O	O
deletion	NN	O	O
.	NN	O	O

To	NN	O	O
date	NN	O	O
,	NN	O	O
our	NN	O	O
detection	NN	O	O
rate	NN	O	O
for	NN	O	O
TWIST	NN	O	O
or	NN	O	O
FGFR	NN	O	O
mutations	NN	O	O
is	NN	O	O
68	NN	O	O
%	NN	O	O
in	NN	O	O
our	NN	O	O
Saethre	NN	O	B-Disease
-	NN	O	I-Disease
Chotzen	NN	O	I-Disease
syndrome	NN	O	I-Disease
patients	NN	O	O
,	NN	O	O
including	NN	O	O
our	NN	O	O
five	NN	O	O
patients	NN	O	O
elsewhere	NN	O	O
reported	NN	O	O
with	NN	O	O
TWIST	NN	O	O
mutations	NN	O	O
.	NN	O	O

More	NN	O	O
than	NN	O	O
35	NN	O	O
different	NN	O	O
TWIST	NN	O	O
mutations	NN	O	O
are	NN	O	O
now	NN	O	O
known	NN	O	O
in	NN	O	O
the	NN	O	O
literature	NN	O	O
.	NN	O	O

The	NN	O	O
most	NN	O	O
common	NN	O	O
phenotypic	NN	O	O
features	NN	O	O
,	NN	O	O
present	NN	O	O
in	NN	O	O
more	NN	O	O
than	NN	O	O
a	NN	O	O
third	NN	O	O
of	NN	O	O
our	NN	O	O
patients	NN	O	O
with	NN	O	O
TWIST	NN	O	O
mutations	NN	O	O
,	NN	O	O
are	NN	O	O
coronal	NN	O	B-Disease
synostosis	NN	O	I-Disease
,	NN	O	O
brachycephaly	NN	O	B-Disease
,	NN	O	O
low	NN	O	B-Disease
frontal	NN	O	I-Disease
hairline	NN	O	I-Disease
,	NN	O	O
facial	NN	O	B-Disease
asymmetry	NN	O	I-Disease
,	NN	O	O
ptosis	NN	O	B-Disease
,	NN	O	O
hypertelorism	NN	O	B-Disease
,	NN	O	O
broad	NN	O	B-Disease
great	NN	O	I-Disease
toes	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
clinodactyly	NN	O	B-Disease
.	NN	O	O

Significant	NN	O	O
intra	NN	O	O
-	NN	O	O
and	NN	O	O
interfamilial	NN	O	O
phenotypic	NN	O	O
variability	NN	O	O
is	NN	O	O
present	NN	O	O
for	NN	O	O
either	NN	O	O
TWIST	NN	O	O
mutations	NN	O	O
or	NN	O	O
FGFR	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
overlap	NN	O	O
in	NN	O	O
clinical	NN	O	O
features	NN	O	O
and	NN	O	O
the	NN	O	O
presence	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
same	NN	O	O
genes	NN	O	O
,	NN	O	O
of	NN	O	O
mutations	NN	O	O
for	NN	O	O
more	NN	O	O
than	NN	O	O
one	NN	O	O
craniosynostotic	NN	O	B-Disease
condition	NN	O	I-Disease
-	NN	O	O
such	NN	O	O
as	NN	O	O
Saethre	NN	O	B-Disease
-	NN	O	I-Disease
Chotzen	NN	O	I-Disease
,	NN	O	I-Disease
Crouzon	NN	O	I-Disease
,	NN	O	I-Disease
and	NN	O	I-Disease
Pfeiffer	NN	O	I-Disease
syndromes	NN	O	I-Disease
-	NN	O	O
support	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
TWIST	NN	O	O
and	NN	O	O
FGFRs	NN	O	O
are	NN	O	O
components	NN	O	O
of	NN	O	O
the	NN	O	O
same	NN	O	O
molecular	NN	O	O
pathway	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
modulation	NN	O	O
of	NN	O	O
craniofacial	NN	O	O
and	NN	O	O
limb	NN	O	O
development	NN	O	O
in	NN	O	O
humans	NN	O	O
.	NN	O	O
.	NN	O	O

Mutation	NN	O	O
analysis	NN	O	O
of	NN	O	O
UBE3A	NN	O	O
in	NN	O	O
Angelman	NN	O	B-Disease
syndrome	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

Angelman	NN	O	B-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
AS	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
chromosome	NN	O	O
15q11	NN	O	O
-	NN	O	O
q13	NN	O	O
deletions	NN	O	O
of	NN	O	O
maternal	NN	O	O
origin	NN	O	O
,	NN	O	O
by	NN	O	O
paternal	NN	O	O
uniparental	NN	O	B-Disease
disomy	NN	O	I-Disease
(	NN	O	O
UPD	NN	O	B-Disease
)	NN	O	O
15	NN	O	O
,	NN	O	O
by	NN	O	O
imprinting	NN	O	O
defects	NN	O	O
,	NN	O	O
and	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
UBE3A	NN	O	O
gene	NN	O	O
.	NN	O	O

UBE3A	NN	O	O
encodes	NN	O	O
a	NN	O	O
ubiquitin	NN	O	O
-	NN	O	O
protein	NN	O	O
ligase	NN	O	O
and	NN	O	O
shows	NN	O	O
brain	NN	O	O
-	NN	O	O
specific	NN	O	O
imprinting	NN	O	O
.	NN	O	O

Here	NN	O	O
we	NN	O	O
describe	NN	O	O
UBE3A	NN	O	O
coding	NN	O	O
-	NN	O	O
region	NN	O	O
mutations	NN	O	O
detected	NN	O	O
by	NN	O	O
SSCP	NN	O	O
analysis	NN	O	O
in	NN	O	O
13	NN	O	O
AS	NN	O	B-Disease
individuals	NN	O	O
or	NN	O	O
families	NN	O	O
.	NN	O	O

Two	NN	O	O
identical	NN	O	O
de	NN	O	O
novo	NN	O	O
5	NN	O	O
-	NN	O	O
bp	NN	O	O
duplications	NN	O	O
in	NN	O	O
exon	NN	O	O
16	NN	O	O
were	NN	O	O
found	NN	O	O
.	NN	O	O

Among	NN	O	O
the	NN	O	O
other	NN	O	O
11	NN	O	O
unique	NN	O	O
mutations	NN	O	O
,	NN	O	O
8	NN	O	O
were	NN	O	O
small	NN	O	O
deletions	NN	O	O
or	NN	O	O
insertions	NN	O	O
predicted	NN	O	O
to	NN	O	O
cause	NN	O	O
frameshifts	NN	O	O
,	NN	O	O
1	NN	O	O
was	NN	O	O
a	NN	O	O
mutation	NN	O	O
to	NN	O	O
a	NN	O	O
stop	NN	O	O
codon	NN	O	O
,	NN	O	O
1	NN	O	O
was	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
,	NN	O	O
and	NN	O	O
1	NN	O	O
was	NN	O	O
predicted	NN	O	O
to	NN	O	O
cause	NN	O	O
insertion	NN	O	O
of	NN	O	O
an	NN	O	O
isoleucine	NN	O	O
in	NN	O	O
the	NN	O	O
hect	NN	O	O
domain	NN	O	O
of	NN	O	O
the	NN	O	O
UBE3A	NN	O	O
protein	NN	O	O
,	NN	O	O
which	NN	O	O
functions	NN	O	O
in	NN	O	O
E2	NN	O	O
binding	NN	O	O
and	NN	O	O
ubiquitin	NN	O	O
transfer	NN	O	O
.	NN	O	O

Eight	NN	O	O
of	NN	O	O
the	NN	O	O
cases	NN	O	O
were	NN	O	O
familial	NN	O	O
,	NN	O	O
and	NN	O	O
five	NN	O	O
were	NN	O	O
sporadic	NN	O	O
.	NN	O	O

In	NN	O	O
two	NN	O	O
familial	NN	O	O
cases	NN	O	O
and	NN	O	O
one	NN	O	O
sporadic	NN	O	O
case	NN	O	O
,	NN	O	O
mosaicism	NN	O	O
for	NN	O	O
UBE3A	NN	O	O
mutations	NN	O	O
was	NN	O	O
detected	NN	O	O
in	NN	O	O
the	NN	O	O
mother	NN	O	O
of	NN	O	O
three	NN	O	O
AS	NN	O	B-Disease
sons	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
maternal	NN	O	O
grandfather	NN	O	O
of	NN	O	O
two	NN	O	O
AS	NN	O	B-Disease
first	NN	O	O
cousins	NN	O	O
,	NN	O	O
and	NN	O	O
in	NN	O	O
the	NN	O	O
mother	NN	O	O
of	NN	O	O
an	NN	O	O
AS	NN	O	B-Disease
daughter	NN	O	O
.	NN	O	O

The	NN	O	O
frequencies	NN	O	O
with	NN	O	O
which	NN	O	O
we	NN	O	O
detected	NN	O	O
mutations	NN	O	O
were	NN	O	O
5	NN	O	O
(	NN	O	O
14	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
35	NN	O	O
in	NN	O	O
sporadic	NN	O	O
cases	NN	O	O
and	NN	O	O
8	NN	O	O
(	NN	O	O
80	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
10	NN	O	O
in	NN	O	O
familial	NN	O	O
cases	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
hemochromatosis	NN	O	B-Disease
845	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
and	NN	O	O
187	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
mutations	NN	O	O
:	NN	O	O
prevalence	NN	O	O
in	NN	O	O
non	NN	O	O
-	NN	O	O
Caucasian	NN	O	O
populations	NN	O	O
.	NN	O	O

Hemochromatosis	NN	O	B-Disease
,	NN	O	O
the	NN	O	O
inherited	NN	O	B-Disease
disorder	NN	O	I-Disease
of	NN	O	I-Disease
iron	NN	O	I-Disease
metabolism	NN	O	I-Disease
,	NN	O	O
leads	NN	O	O
,	NN	O	O
if	NN	O	O
untreated	NN	O	O
,	NN	O	O
to	NN	O	O
progressive	NN	O	O
iron	NN	O	B-Disease
overload	NN	O	I-Disease
and	NN	O	O
premature	NN	O	B-Disease
death	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
hemochromatosis	NN	O	B-Disease
gene	NN	O	O
,	NN	O	O
HFE	NN	O	O
,	NN	O	O
recently	NN	O	O
has	NN	O	O
been	NN	O	O
identified	NN	O	O
,	NN	O	O
and	NN	O	O
characterization	NN	O	O
of	NN	O	O
this	NN	O	O
gene	NN	O	O
has	NN	O	O
shown	NN	O	O
that	NN	O	O
it	NN	O	O
contains	NN	O	O
two	NN	O	O
mutations	NN	O	O
that	NN	O	O
result	NN	O	O
in	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitutions	NN	O	O
-	NN	O	O
cDNA	NN	O	O
nucleotides	NN	O	O
845	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
(	NN	O	O
C282Y	NN	O	O
)	NN	O	O
and	NN	O	O
187	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
(	NN	O	O
H63D	NN	O	O
)	NN	O	O
.	NN	O	O

Although	NN	O	O
hemochromatosis	NN	O	B-Disease
is	NN	O	O
common	NN	O	O
in	NN	O	O
Caucasians	NN	O	O
,	NN	O	O
affecting	NN	O	O
>	NN	O	O
=	NN	O	O
1	NN	O	O
/	NN	O	O
300	NN	O	O
individuals	NN	O	O
of	NN	O	O
northern	NN	O	O
European	NN	O	O
origin	NN	O	O
,	NN	O	O
it	NN	O	O
has	NN	O	O
not	NN	O	O
been	NN	O	O
recognized	NN	O	O
in	NN	O	O
other	NN	O	O
populations	NN	O	O
.	NN	O	O

The	NN	O	O
present	NN	O	O
study	NN	O	O
used	NN	O	O
PCR	NN	O	O
and	NN	O	O
restriction	NN	O	O
-	NN	O	O
enzyme	NN	O	O
digestion	NN	O	O
to	NN	O	O
analyze	NN	O	O
the	NN	O	O
frequency	NN	O	O
of	NN	O	O
the	NN	O	O
845	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
and	NN	O	O
187	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
mutations	NN	O	O
in	NN	O	O
HLA	NN	O	O
-	NN	O	O
typed	NN	O	O
samples	NN	O	O
from	NN	O	O
non	NN	O	O
-	NN	O	O
Caucasian	NN	O	O
populations	NN	O	O
,	NN	O	O
comprising	NN	O	O
Australian	NN	O	O
Aboriginal	NN	O	O
,	NN	O	O
Chinese	NN	O	O
,	NN	O	O
and	NN	O	O
Pacific	NN	O	O
Islanders	NN	O	O
.	NN	O	O

Results	NN	O	O
showed	NN	O	O
that	NN	O	O
the	NN	O	O
845	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
mutation	NN	O	O
was	NN	O	O
present	NN	O	O
in	NN	O	O
these	NN	O	O
populations	NN	O	O
(	NN	O	O
allele	NN	O	O
frequency	NN	O	O
0	NN	O	O
.	NN	O	O
32	NN	O	O
%	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
,	NN	O	O
furthermore	NN	O	O
,	NN	O	O
it	NN	O	O
was	NN	O	O
always	NN	O	O
seen	NN	O	O
in	NN	O	O
conjunction	NN	O	O
with	NN	O	O
HLA	NN	O	O
haplotypes	NN	O	O
common	NN	O	O
in	NN	O	O
Caucasians	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
845	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
may	NN	O	O
have	NN	O	O
been	NN	O	O
introduced	NN	O	O
into	NN	O	O
these	NN	O	O
populations	NN	O	O
by	NN	O	O
Caucasian	NN	O	O
admixture	NN	O	O
.	NN	O	O

187	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
was	NN	O	O
present	NN	O	O
at	NN	O	O
an	NN	O	O
allele	NN	O	O
frequency	NN	O	O
of	NN	O	O
2	NN	O	O
.	NN	O	O

68	NN	O	O
%	NN	O	O
in	NN	O	O
the	NN	O	O
two	NN	O	O
populations	NN	O	O
analyzed	NN	O	O
(	NN	O	O
Australian	NN	O	O
Aboriginal	NN	O	O
and	NN	O	O
Chinese	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
Australian	NN	O	O
Aboriginal	NN	O	O
samples	NN	O	O
,	NN	O	O
187	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
associated	NN	O	O
with	NN	O	O
HLA	NN	O	O
haplotypes	NN	O	O
common	NN	O	O
in	NN	O	O
Caucasians	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
it	NN	O	O
was	NN	O	O
introduced	NN	O	O
by	NN	O	O
recent	NN	O	O
admixture	NN	O	O
.	NN	O	O

In	NN	O	O
the	NN	O	O
Chinese	NN	O	O
samples	NN	O	O
analyzed	NN	O	O
,	NN	O	O
187	NN	O	O
C	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
G	NN	O	O
was	NN	O	O
present	NN	O	O
in	NN	O	O
association	NN	O	O
with	NN	O	O
a	NN	O	O
wide	NN	O	O
variety	NN	O	O
of	NN	O	O
HLA	NN	O	O
haplotypes	NN	O	O
,	NN	O	O
showing	NN	O	O
this	NN	O	O
mutation	NN	O	O
to	NN	O	O
be	NN	O	O
widespread	NN	O	O
and	NN	O	O
likely	NN	O	O
to	NN	O	O
predate	NN	O	O
the	NN	O	O
more	NN	O	O
genetically	NN	O	O
restricted	NN	O	O
845	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
mutation	NN	O	O
.	NN	O	O

Genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
in	NN	O	O
attenuated	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
.	NN	O	O

Germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
of	NN	O	O
the	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
APC	NN	O	O
are	NN	O	O
implicated	NN	O	O
in	NN	O	O
attenuated	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
(	NN	O	O
AAPC	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
a	NN	O	O
variant	NN	O	O
of	NN	O	O
familial	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
(	NN	O	O
FAP	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

AAPC	NN	O	B-Disease
is	NN	O	O
recognized	NN	O	O
by	NN	O	O
the	NN	O	O
occurrence	NN	O	O
of	NN	O	O
<	NN	O	O
100	NN	O	O
colonic	NN	O	B-Disease
adenomas	NN	O	I-Disease
and	NN	O	O
a	NN	O	O
later	NN	O	O
onset	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
(	NN	O	O
age	NN	O	O
>	NN	O	O
40	NN	O	O
years	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
aim	NN	O	O
of	NN	O	O
this	NN	O	O
study	NN	O	O
was	NN	O	O
to	NN	O	O
assess	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
in	NN	O	O
AAPC	NN	O	B-Disease
families	NN	O	O
.	NN	O	O

By	NN	O	O
protein	NN	O	O
-	NN	O	O
truncation	NN	O	O
test	NN	O	O
(	NN	O	O
PTT	NN	O	O
)	NN	O	O
assay	NN	O	O
,	NN	O	O
the	NN	O	O
entire	NN	O	O
coding	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
was	NN	O	O
screened	NN	O	O
in	NN	O	O
affected	NN	O	O
individuals	NN	O	O
from	NN	O	O
11	NN	O	O
AAPC	NN	O	B-Disease
kindreds	NN	O	O
,	NN	O	O
and	NN	O	O
their	NN	O	O
phenotypic	NN	O	O
differences	NN	O	O
were	NN	O	O
examined	NN	O	O
.	NN	O	O

Five	NN	O	O
novel	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
APC	NN	O	B-Disease
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
seven	NN	O	O
kindreds	NN	O	O
.	NN	O	O

Mutations	NN	O	O
were	NN	O	O
located	NN	O	O
in	NN	O	O
three	NN	O	O
different	NN	O	O
regions	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
(	NN	O	O
1	NN	O	O
)	NN	O	O
at	NN	O	O
the	NN	O	O
5	NN	O	O
end	NN	O	O
spanning	NN	O	O
exons	NN	O	O
4	NN	O	O
and	NN	O	O
5	NN	O	O
,	NN	O	O
(	NN	O	O
2	NN	O	O
)	NN	O	O
within	NN	O	O
exon	NN	O	O
9	NN	O	O
,	NN	O	O
and	NN	O	O
(	NN	O	O
3	NN	O	O
)	NN	O	O
at	NN	O	O
the	NN	O	O
3	NN	O	O
distal	NN	O	O
end	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
.	NN	O	O

Variability	NN	O	O
in	NN	O	O
the	NN	O	O
number	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
adenomas	NN	O	I-Disease
was	NN	O	O
most	NN	O	O
apparent	NN	O	O
in	NN	O	O
individuals	NN	O	O
with	NN	O	O
mutations	NN	O	O
in	NN	O	O
region	NN	O	O
1	NN	O	O
,	NN	O	O
and	NN	O	O
upper	NN	O	O
-	NN	O	O
gastrointestinal	NN	O	O
manifestations	NN	O	O
were	NN	O	O
more	NN	O	O
severe	NN	O	O
in	NN	O	O
them	NN	O	O
.	NN	O	O

In	NN	O	O
individuals	NN	O	O
with	NN	O	O
mutations	NN	O	O
in	NN	O	O
either	NN	O	O
region	NN	O	O
2	NN	O	O
or	NN	O	O
region	NN	O	O
3	NN	O	O
,	NN	O	O
the	NN	O	O
average	NN	O	O
number	NN	O	O
of	NN	O	O
adenomas	NN	O	B-Disease
tended	NN	O	O
to	NN	O	O
be	NN	O	O
lower	NN	O	O
than	NN	O	O
those	NN	O	O
in	NN	O	O
individuals	NN	O	O
with	NN	O	O
mutations	NN	O	O
in	NN	O	O
region	NN	O	O
1	NN	O	O
,	NN	O	O
although	NN	O	O
age	NN	O	O
at	NN	O	O
diagnosis	NN	O	O
was	NN	O	O
similar	NN	O	O
.	NN	O	O

In	NN	O	O
all	NN	O	O
AAPC	NN	O	B-Disease
kindreds	NN	O	O
,	NN	O	O
a	NN	O	O
predominance	NN	O	O
of	NN	O	O
right	NN	O	O
-	NN	O	O
sided	NN	O	O
colorectal	NN	O	B-Disease
adenomas	NN	O	I-Disease
and	NN	O	O
rectal	NN	O	B-Disease
polyp	NN	O	I-Disease
sparing	NN	O	O
was	NN	O	O
observed	NN	O	O
.	NN	O	O

No	NN	O	O
desmoid	NN	O	B-Disease
tumors	NN	O	I-Disease
were	NN	O	O
found	NN	O	O
in	NN	O	O
these	NN	O	O
kindreds	NN	O	O
.	NN	O	O

Our	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
,	NN	O	O
in	NN	O	O
AAPC	NN	O	B-Disease
families	NN	O	O
,	NN	O	O
the	NN	O	O
location	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
mutation	NN	O	O
may	NN	O	O
partially	NN	O	O
predict	NN	O	O
specific	NN	O	O
phenotypic	NN	O	O
expression	NN	O	O
.	NN	O	O

This	NN	O	O
should	NN	O	O
help	NN	O	O
in	NN	O	O
the	NN	O	O
design	NN	O	O
of	NN	O	O
tailored	NN	O	O
clinical	NN	O	O
-	NN	O	O
management	NN	O	O
protocols	NN	O	O
in	NN	O	O
this	NN	O	O
subset	NN	O	O
of	NN	O	O
FAP	NN	O	B-Disease
patients	NN	O	O
.	NN	O	O
.	NN	O	O

Wilms	NN	O	B-Disease
'	NN	O	I-Disease
tumor	NN	O	I-Disease
1	NN	O	O
and	NN	O	O
Dax	NN	O	O
-	NN	O	O
1	NN	O	O
modulate	NN	O	O
the	NN	O	O
orphan	NN	O	O
nuclear	NN	O	O
receptor	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
in	NN	O	O
sex	NN	O	O
-	NN	O	O
specific	NN	O	O
gene	NN	O	O
expression	NN	O	O
.	NN	O	O

Products	NN	O	O
of	NN	O	O
steroidogenic	NN	O	O
factor	NN	O	O
1	NN	O	O
(	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
)	NN	O	O
and	NN	O	O
Wilms	NN	O	B-Disease
tumor	NN	O	I-Disease
1	NN	O	O
(	NN	O	O
WT1	NN	O	O
)	NN	O	O
genes	NN	O	O
are	NN	O	O
essential	NN	O	O
for	NN	O	O
mammalian	NN	O	O
gonadogenesis	NN	O	O
prior	NN	O	O
to	NN	O	O
sexual	NN	O	O
differentiation	NN	O	O
.	NN	O	O

In	NN	O	O
males	NN	O	O
,	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
participates	NN	O	O
in	NN	O	O
sexual	NN	O	O
development	NN	O	O
by	NN	O	O
regulating	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
polypeptide	NN	O	O
hormone	NN	O	O
Mullerian	NN	O	O
inhibiting	NN	O	O
substance	NN	O	O
(	NN	O	O
MIS	NN	O	O
)	NN	O	O
.	NN	O	O

Here	NN	O	O
,	NN	O	O
we	NN	O	O
show	NN	O	O
that	NN	O	O
WT1	NN	O	O
-	NN	O	O
KTS	NN	O	O
isoforms	NN	O	O
associate	NN	O	O
and	NN	O	O
synergize	NN	O	O
with	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
to	NN	O	O
promote	NN	O	O
MIS	NN	O	O
expression	NN	O	O
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
WT1	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
associated	NN	O	O
with	NN	O	O
male	NN	O	B-Disease
pseudohermaphroditism	NN	O	I-Disease
in	NN	O	O
Denys	NN	O	B-Disease
-	NN	O	I-Disease
Drash	NN	O	I-Disease
syndrome	NN	O	I-Disease
,	NN	O	O
fail	NN	O	O
to	NN	O	O
synergize	NN	O	O
with	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
.	NN	O	O

Additionally	NN	O	O
,	NN	O	O
the	NN	O	O
X	NN	O	O
-	NN	O	O
linked	NN	O	O
,	NN	O	O
candidate	NN	O	O
dosage	NN	O	O
-	NN	O	O
sensitive	NN	O	O
sex	NN	O	O
-	NN	O	O
reversal	NN	O	O
gene	NN	O	O
,	NN	O	O
Dax	NN	O	O
-	NN	O	O
1	NN	O	O
,	NN	O	O
antagonizes	NN	O	O
synergy	NN	O	O
between	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
and	NN	O	O
WT1	NN	O	O
,	NN	O	O
most	NN	O	O
likely	NN	O	O
through	NN	O	O
a	NN	O	O
direct	NN	O	O
interaction	NN	O	O
with	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
.	NN	O	O

We	NN	O	O
propose	NN	O	O
that	NN	O	O
WT1	NN	O	O
and	NN	O	O
Dax	NN	O	O
-	NN	O	O
1	NN	O	O
functionally	NN	O	O
oppose	NN	O	O
each	NN	O	O
other	NN	O	O
in	NN	O	O
testis	NN	O	O
development	NN	O	O
by	NN	O	O
modulating	NN	O	O
SF	NN	O	O
-	NN	O	O
1	NN	O	O
-	NN	O	O
mediated	NN	O	O
transactivation	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
mouse	NN	O	O
model	NN	O	O
for	NN	O	O
Prader	NN	O	B-Disease
-	NN	O	I-Disease
Willi	NN	O	I-Disease
syndrome	NN	O	I-Disease
imprinting	NN	O	O
-	NN	O	O
centre	NN	O	O
mutations	NN	O	O
.	NN	O	O

Imprinting	NN	O	O
in	NN	O	O
the	NN	O	O
15q11	NN	O	O
-	NN	O	O
q13	NN	O	O
region	NN	O	O
involves	NN	O	O
an	NN	O	O
imprinting	NN	O	O
centre	NN	O	O
(	NN	O	O
IC	NN	O	O
)	NN	O	O
,	NN	O	O
mapping	NN	O	O
in	NN	O	O
part	NN	O	O
to	NN	O	O
the	NN	O	O
promoter	NN	O	O
and	NN	O	O
first	NN	O	O
exon	NN	O	O
of	NN	O	O
SNRPN	NN	O	O
.	NN	O	O

Deletion	NN	O	O
of	NN	O	O
this	NN	O	O
IC	NN	O	O
abolishes	NN	O	O
local	NN	O	O
paternally	NN	O	O
derived	NN	O	O
gene	NN	O	O
expression	NN	O	O
and	NN	O	O
results	NN	O	O
in	NN	O	O
Prader	NN	O	B-Disease
-	NN	O	I-Disease
Willi	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
PWS	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
created	NN	O	O
two	NN	O	O
deletion	NN	O	O
mutations	NN	O	O
in	NN	O	O
mice	NN	O	O
to	NN	O	O
understand	NN	O	O
PWS	NN	O	B-Disease
and	NN	O	O
the	NN	O	O
mechanism	NN	O	O
of	NN	O	O
this	NN	O	O
IC	NN	O	O
.	NN	O	O

Mice	NN	O	O
harbouring	NN	O	O
an	NN	O	O
intragenic	NN	O	O
deletion	NN	O	O
in	NN	O	O
Snrpn	NN	O	O
are	NN	O	O
phenotypically	NN	O	O
normal	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
mutations	NN	O	O
of	NN	O	O
SNRPN	NN	O	O
are	NN	O	O
not	NN	O	O
sufficient	NN	O	O
to	NN	O	O
induce	NN	O	O
PWS	NN	O	B-Disease
.	NN	O	O

Mice	NN	O	O
with	NN	O	O
a	NN	O	O
larger	NN	O	O
deletion	NN	O	O
involving	NN	O	O
both	NN	O	O
Snrpn	NN	O	O
and	NN	O	O
the	NN	O	O
putative	NN	O	O
PWS	NN	O	O
-	NN	O	O
IC	NN	O	O
lack	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
imprinted	NN	O	O
genes	NN	O	O
Zfp127	NN	O	O
(	NN	O	O
mouse	NN	O	O
homologue	NN	O	O
of	NN	O	O
ZNF127	NN	O	O
)	NN	O	O
,	NN	O	O
Ndn	NN	O	O
and	NN	O	O
Ipw	NN	O	O
,	NN	O	O
and	NN	O	O
manifest	NN	O	O
several	NN	O	O
phenotypes	NN	O	O
common	NN	O	O
to	NN	O	O
PWS	NN	O	B-Disease
infants	NN	O	O
.	NN	O	O

These	NN	O	O
data	NN	O	O
demonstrate	NN	O	O
that	NN	O	O
both	NN	O	O
the	NN	O	O
position	NN	O	O
of	NN	O	O
the	NN	O	O
IC	NN	O	O
and	NN	O	O
its	NN	O	O
role	NN	O	O
in	NN	O	O
the	NN	O	O
coordinate	NN	O	O
expression	NN	O	O
of	NN	O	O
genes	NN	O	O
is	NN	O	O
conserved	NN	O	O
between	NN	O	O
mouse	NN	O	O
and	NN	O	O
human	NN	O	O
,	NN	O	O
and	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
mouse	NN	O	O
is	NN	O	O
a	NN	O	O
suitable	NN	O	O
model	NN	O	O
system	NN	O	O
in	NN	O	O
which	NN	O	O
to	NN	O	O
investigate	NN	O	O
the	NN	O	O
molecular	NN	O	O
mechanisms	NN	O	O
of	NN	O	O
imprinting	NN	O	O
in	NN	O	O
this	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
genome	NN	O	O
.	NN	O	O
.	NN	O	O

Mutations	NN	O	O
of	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
detected	NN	O	O
in	NN	O	O
Japanese	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
patients	NN	O	O
:	NN	O	O
possible	NN	O	O
preponderance	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
founder	NN	O	O
mutations	NN	O	O
4612del165	NN	O	O
and	NN	O	O
7883del5	NN	O	O
.	NN	O	O

The	NN	O	O
ATM	NN	O	O
(	NN	O	O
A	NN	O	O
-	NN	O	O
T	NN	O	O
,	NN	O	O
mutated	NN	O	O
)	NN	O	O
gene	NN	O	O
on	NN	O	O
human	NN	O	O
chromosome	NN	O	O
11q22	NN	O	O
.	NN	O	O

3	NN	O	O
has	NN	O	O
recently	NN	O	O
been	NN	O	O
identified	NN	O	O
as	NN	O	O
the	NN	O	O
gene	NN	O	O
responsible	NN	O	O
for	NN	O	O
the	NN	O	O
human	NN	O	O
recessive	NN	O	B-Disease
disease	NN	O	I-Disease
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
.	NN	O	O

In	NN	O	O
order	NN	O	O
to	NN	O	O
define	NN	O	O
the	NN	O	O
types	NN	O	O
of	NN	O	O
disease	NN	O	O
-	NN	O	O
causing	NN	O	O
ATM	NN	O	O
mutations	NN	O	O
in	NN	O	O
Japanese	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
to	NN	O	O
look	NN	O	O
for	NN	O	O
possible	NN	O	O
mutational	NN	O	O
hotspots	NN	O	O
,	NN	O	O
reverse	NN	O	O
-	NN	O	O
transcribed	NN	O	O
RNA	NN	O	O
derived	NN	O	O
from	NN	O	O
ten	NN	O	O
patients	NN	O	O
belonging	NN	O	O
to	NN	O	O
eight	NN	O	O
unrelated	NN	O	O
Japanese	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
families	NN	O	O
was	NN	O	O
analyzed	NN	O	O
for	NN	O	O
mutations	NN	O	O
by	NN	O	O
the	NN	O	O
restriction	NN	O	O
endonuclease	NN	O	O
fingerprinting	NN	O	O
method	NN	O	O
.	NN	O	O

As	NN	O	O
has	NN	O	O
been	NN	O	O
reported	NN	O	O
by	NN	O	O
others	NN	O	O
,	NN	O	O
mutations	NN	O	O
that	NN	O	O
lead	NN	O	O
to	NN	O	O
exon	NN	O	O
skipping	NN	O	O
or	NN	O	O
premature	NN	O	O
protein	NN	O	O
truncation	NN	O	O
were	NN	O	O
also	NN	O	O
predominant	NN	O	O
in	NN	O	O
our	NN	O	O
mutants	NN	O	O
.	NN	O	O

Six	NN	O	O
different	NN	O	O
mutations	NN	O	O
were	NN	O	O
identified	NN	O	O
on	NN	O	O
12	NN	O	O
of	NN	O	O
the	NN	O	O
16	NN	O	O
alleles	NN	O	O
examined	NN	O	O
.	NN	O	O

Four	NN	O	O
were	NN	O	O
deletions	NN	O	O
involving	NN	O	O
a	NN	O	O
loss	NN	O	O
of	NN	O	O
a	NN	O	O
single	NN	O	O
exon	NN	O	O
exon	NN	O	O
7	NN	O	O
,	NN	O	O
exon	NN	O	O
16	NN	O	O
,	NN	O	O
exon	NN	O	O
33	NN	O	O
or	NN	O	O
exon	NN	O	O
35	NN	O	O
.	NN	O	O

The	NN	O	O
others	NN	O	O
were	NN	O	O
minute	NN	O	O
deletions	NN	O	O
,	NN	O	O
4649delA	NN	O	O
in	NN	O	O
exon	NN	O	O
33	NN	O	O
and	NN	O	O
7883del5	NN	O	O
in	NN	O	O
exon	NN	O	O
55	NN	O	O
.	NN	O	O

The	NN	O	O
mutations	NN	O	O
4612del165	NN	O	O
and	NN	O	O
7883del5	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
more	NN	O	O
than	NN	O	O
two	NN	O	O
unrelated	NN	O	O
families	NN	O	O
;	NN	O	O
44	NN	O	O
%	NN	O	O
(	NN	O	O
7	NN	O	O
of	NN	O	O
16	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
had	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
two	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
4612del165	NN	O	O
mutations	NN	O	O
in	NN	O	O
three	NN	O	O
different	NN	O	O
families	NN	O	O
were	NN	O	O
all	NN	O	O
ascribed	NN	O	O
to	NN	O	O
the	NN	O	O
same	NN	O	O
T	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
A	NN	O	O
substitution	NN	O	O
at	NN	O	O
the	NN	O	O
splice	NN	O	O
donor	NN	O	O
site	NN	O	O
in	NN	O	O
intron	NN	O	O
33	NN	O	O
.	NN	O	O

Microsatellite	NN	O	O
genotyping	NN	O	O
around	NN	O	O
the	NN	O	O
ATM	NN	O	O
locus	NN	O	O
also	NN	O	O
indicated	NN	O	O
that	NN	O	O
a	NN	O	O
common	NN	O	O
haplotype	NN	O	O
was	NN	O	O
shared	NN	O	O
by	NN	O	O
the	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
in	NN	O	O
both	NN	O	O
mutations	NN	O	O
.	NN	O	O

This	NN	O	O
suggests	NN	O	O
that	NN	O	O
these	NN	O	O
two	NN	O	O
founder	NN	O	O
mutations	NN	O	O
may	NN	O	O
be	NN	O	O
predominant	NN	O	O
among	NN	O	O
Japanese	NN	O	O
ATM	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
.	NN	O	O

W474C	NN	O	O
amino	NN	O	O
acid	NN	O	O
substitution	NN	O	O
affects	NN	O	O
early	NN	O	O
processing	NN	O	O
of	NN	O	O
the	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
of	NN	O	O
beta	NN	O	O
-	NN	O	O
hexosaminidase	NN	O	O
A	NN	O	O
and	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
subacute	NN	O	O
G	NN	O	B-Disease
(	NN	O	I-Disease
M2	NN	O	I-Disease
)	NN	O	I-Disease
gangliosidosis	NN	O	I-Disease
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
HEXA	NN	O	O
gene	NN	O	O
,	NN	O	O
encoding	NN	O	O
the	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
of	NN	O	O
beta	NN	O	O
-	NN	O	O
hexosaminidase	NN	O	O
A	NN	O	O
(	NN	O	O
Hex	NN	O	O
A	NN	O	O
)	NN	O	O
,	NN	O	O
that	NN	O	O
abolish	NN	O	O
Hex	NN	O	O
A	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
cause	NN	O	O
Tay	NN	O	B-Disease
-	NN	O	I-Disease
Sachs	NN	O	I-Disease
disease	NN	O	I-Disease
(	NN	O	O
TSD	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
the	NN	O	O
fatal	NN	O	O
infantile	NN	O	B-Disease
form	NN	O	I-Disease
of	NN	O	I-Disease
G	NN	O	I-Disease
(	NN	O	I-Disease
M2	NN	O	I-Disease
)	NN	O	I-Disease
gangliosidosis	NN	O	I-Disease
,	NN	O	I-Disease
Type	NN	O	I-Disease
1	NN	O	I-Disease
.	NN	O	O

Less	NN	O	O
severe	NN	O	O
,	NN	O	O
subacute	NN	O	O
(	NN	O	O
juvenile	NN	O	O
-	NN	O	O
onset	NN	O	O
)	NN	O	O
and	NN	O	O
chronic	NN	O	O
(	NN	O	O
adult	NN	O	O
-	NN	O	O
onset	NN	O	O
)	NN	O	O
variants	NN	O	O
are	NN	O	O
characterized	NN	O	O
by	NN	O	O
a	NN	O	O
broad	NN	O	O
spectrum	NN	O	O
of	NN	O	O
clinical	NN	O	O
manifestations	NN	O	O
and	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
residual	NN	O	O
levels	NN	O	O
of	NN	O	O
Hex	NN	O	O
A	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
.	NN	O	O

We	NN	O	O
identified	NN	O	O
a	NN	O	O
1422	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
C	NN	O	O
(	NN	O	O
amino	NN	O	O
acid	NN	O	O
W474C	NN	O	O
)	NN	O	O
substitution	NN	O	O
in	NN	O	O
the	NN	O	O
first	NN	O	O
position	NN	O	O
of	NN	O	O
exon	NN	O	O
13	NN	O	O
of	NN	O	O
HEXA	NN	O	O
of	NN	O	O
a	NN	O	O
non	NN	O	O
-	NN	O	O
Jewish	NN	O	O
proband	NN	O	O
who	NN	O	O
manifested	NN	O	O
a	NN	O	O
subacute	NN	O	O
variant	NN	O	O
of	NN	O	O
G	NN	O	B-Disease
(	NN	O	I-Disease
M2	NN	O	I-Disease
)	NN	O	I-Disease
gangliosidosis	NN	O	I-Disease
.	NN	O	O

On	NN	O	O
the	NN	O	O
second	NN	O	O
maternally	NN	O	O
inherited	NN	O	O
allele	NN	O	O
,	NN	O	O
we	NN	O	O
identified	NN	O	O
the	NN	O	O
common	NN	O	O
infantile	NN	O	O
disease	NN	O	O
-	NN	O	O
causing	NN	O	O
4	NN	O	O
-	NN	O	O
bp	NN	O	O
insertion	NN	O	O
,	NN	O	O
+	NN	O	O
TATC	NN	O	O
1278	NN	O	O
,	NN	O	O
in	NN	O	O
exon	NN	O	O
11	NN	O	O
.	NN	O	O

Pulse	NN	O	O
-	NN	O	O
chase	NN	O	O
analysis	NN	O	O
using	NN	O	O
proband	NN	O	O
fibroblasts	NN	O	O
revealed	NN	O	O
that	NN	O	O
the	NN	O	O
W474C	NN	O	O
-	NN	O	O
containing	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
precursor	NN	O	O
was	NN	O	O
normally	NN	O	O
synthesized	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
phosphorylated	NN	O	O
or	NN	O	O
secreted	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
mature	NN	O	O
lysosomal	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
was	NN	O	O
not	NN	O	O
detected	NN	O	O
.	NN	O	O

When	NN	O	O
the	NN	O	O
W474C	NN	O	O
-	NN	O	O
containing	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
was	NN	O	O
transiently	NN	O	O
co	NN	O	O
-	NN	O	O
expressed	NN	O	O
with	NN	O	O
the	NN	O	O
beta	NN	O	O
-	NN	O	O
subunit	NN	O	O
to	NN	O	O
produce	NN	O	O
Hex	NN	O	O
A	NN	O	O
(	NN	O	O
alphabeta	NN	O	O
)	NN	O	O
in	NN	O	O
COS	NN	O	O
-	NN	O	O
7	NN	O	O
cells	NN	O	O
,	NN	O	O
the	NN	O	O
mature	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
was	NN	O	O
present	NN	O	O
,	NN	O	O
but	NN	O	O
its	NN	O	O
level	NN	O	O
was	NN	O	O
much	NN	O	O
lower	NN	O	O
than	NN	O	O
that	NN	O	O
from	NN	O	O
normal	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
transfections	NN	O	O
,	NN	O	O
although	NN	O	O
higher	NN	O	O
than	NN	O	O
in	NN	O	O
those	NN	O	O
cells	NN	O	O
transfected	NN	O	O
with	NN	O	O
an	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
associated	NN	O	O
with	NN	O	O
infantile	NN	O	O
TSD	NN	O	B-Disease
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
the	NN	O	O
precursor	NN	O	O
level	NN	O	O
of	NN	O	O
the	NN	O	O
W474C	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
accumulate	NN	O	O
in	NN	O	O
comparison	NN	O	O
to	NN	O	O
the	NN	O	O
normal	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
precursor	NN	O	O
levels	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
the	NN	O	O
1422	NN	O	O
G	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
C	NN	O	O
mutation	NN	O	O
is	NN	O	O
the	NN	O	O
cause	NN	O	O
of	NN	O	O
Hex	NN	O	B-Disease
A	NN	O	I-Disease
enzyme	NN	O	I-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
proband	NN	O	O
.	NN	O	O

The	NN	O	O
resulting	NN	O	O
W474C	NN	O	O
substitution	NN	O	O
clearly	NN	O	O
interferes	NN	O	O
with	NN	O	O
alpha	NN	O	O
-	NN	O	O
subunit	NN	O	O
processing	NN	O	O
,	NN	O	O
but	NN	O	O
because	NN	O	O
the	NN	O	O
base	NN	O	O
substitution	NN	O	O
falls	NN	O	O
at	NN	O	O
the	NN	O	O
first	NN	O	O
position	NN	O	O
of	NN	O	O
exon	NN	O	O
13	NN	O	O
,	NN	O	O
aberrant	NN	O	O
splicing	NN	O	O
may	NN	O	O
also	NN	O	O
contribute	NN	O	O
to	NN	O	O
Hex	NN	O	B-Disease
A	NN	O	I-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
this	NN	O	O
proband	NN	O	O
.	NN	O	O
.	NN	O	O

Two	NN	O	O
frequent	NN	O	O
missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
Pendred	NN	O	B-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Pendred	NN	O	B-Disease
syndrome	NN	O	I-Disease
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
disorder	NN	O	I-Disease
characterized	NN	O	O
by	NN	O	O
early	NN	O	O
childhood	NN	O	O
deafness	NN	O	B-Disease
and	NN	O	O
goiter	NN	O	B-Disease
.	NN	O	O

A	NN	O	O
century	NN	O	O
after	NN	O	O
its	NN	O	O
recognition	NN	O	O
as	NN	O	O
a	NN	O	O
syndrome	NN	O	O
by	NN	O	O
Vaughan	NN	O	O
Pendred	NN	O	O
,	NN	O	O
the	NN	O	O
disease	NN	O	O
gene	NN	O	O
(	NN	O	O
PDS	NN	O	O
)	NN	O	O
was	NN	O	O
mapped	NN	O	O
to	NN	O	O
chromosome	NN	O	O
7q22	NN	O	O
-	NN	O	O
q31	NN	O	O
.	NN	O	O

1	NN	O	O
and	NN	O	O
,	NN	O	O
recently	NN	O	O
,	NN	O	O
found	NN	O	O
to	NN	O	O
encode	NN	O	O
a	NN	O	O
putative	NN	O	O
sulfate	NN	O	O
transporter	NN	O	O
.	NN	O	O

We	NN	O	O
performed	NN	O	O
mutation	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
PDS	NN	O	B-Disease
gene	NN	O	O
in	NN	O	O
patients	NN	O	O
from	NN	O	O
14	NN	O	O
Pendred	NN	O	B-Disease
families	NN	O	O
originating	NN	O	O
from	NN	O	O
seven	NN	O	O
countries	NN	O	O
and	NN	O	O
identified	NN	O	O
all	NN	O	O
mutations	NN	O	O
.	NN	O	O

The	NN	O	O
mutations	NN	O	O
include	NN	O	O
three	NN	O	O
single	NN	O	O
base	NN	O	O
deletions	NN	O	O
,	NN	O	O
one	NN	O	O
splice	NN	O	O
site	NN	O	O
mutation	NN	O	O
and	NN	O	O
10	NN	O	O
missense	NN	O	O
mutations	NN	O	O
.	NN	O	O

One	NN	O	O
missense	NN	O	O
mutation	NN	O	O
(	NN	O	O
L236P	NN	O	O
)	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
a	NN	O	O
homozygous	NN	O	O
state	NN	O	O
in	NN	O	O
two	NN	O	O
consanguineous	NN	O	O
families	NN	O	O
and	NN	O	O
in	NN	O	O
a	NN	O	O
heterozygous	NN	O	O
state	NN	O	O
in	NN	O	O
five	NN	O	O
additional	NN	O	O
non	NN	O	O
-	NN	O	O
consanguineous	NN	O	O
families	NN	O	O
.	NN	O	O

Another	NN	O	O
missense	NN	O	O
mutation	NN	O	O
(	NN	O	O
T416P	NN	O	O
)	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
a	NN	O	O
homozygous	NN	O	O
state	NN	O	O
in	NN	O	O
one	NN	O	O
family	NN	O	O
and	NN	O	O
in	NN	O	O
a	NN	O	O
heterozygous	NN	O	O
state	NN	O	O
in	NN	O	O
four	NN	O	O
families	NN	O	O
.	NN	O	O

Pendred	NN	O	B-Disease
patients	NN	O	O
in	NN	O	O
three	NN	O	O
non	NN	O	O
-	NN	O	O
consanguineous	NN	O	O
families	NN	O	O
were	NN	O	O
shown	NN	O	O
to	NN	O	O
be	NN	O	O
compound	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
L236P	NN	O	O
and	NN	O	O
T416P	NN	O	O
.	NN	O	O

In	NN	O	O
total	NN	O	O
,	NN	O	O
one	NN	O	O
or	NN	O	O
both	NN	O	O
of	NN	O	O
these	NN	O	O
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
nine	NN	O	O
of	NN	O	O
the	NN	O	O
14	NN	O	O
families	NN	O	O
analyzed	NN	O	O
.	NN	O	O

The	NN	O	O
identification	NN	O	O
of	NN	O	O
two	NN	O	O
frequent	NN	O	O
PDS	NN	O	B-Disease
mutations	NN	O	O
will	NN	O	O
facilitate	NN	O	O
the	NN	O	O
molecular	NN	O	O
diagnosis	NN	O	O
of	NN	O	O
Pendred	NN	O	B-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Insertional	NN	O	O
mutation	NN	O	O
by	NN	O	O
transposable	NN	O	O
element	NN	O	O
,	NN	O	O
L1	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
DMD	NN	O	B-Disease
gene	NN	O	O
results	NN	O	O
in	NN	O	O
X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
dilated	NN	O	I-Disease
cardiomyopathy	NN	O	I-Disease
.	NN	O	O

X	NN	O	B-Disease
-	NN	O	I-Disease
linked	NN	O	I-Disease
dilated	NN	O	I-Disease
cardiomyopathy	NN	O	I-Disease
(	NN	O	O
XLDCM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
a	NN	O	O
clinical	NN	O	O
phenotype	NN	O	O
of	NN	O	O
dystrophinopathy	NN	O	B-Disease
which	NN	O	O
is	NN	O	O
characterized	NN	O	O
by	NN	O	O
preferential	NN	O	O
myocardial	NN	O	B-Disease
involvement	NN	O	I-Disease
without	NN	O	O
any	NN	O	O
overt	NN	O	O
clinical	NN	O	O
signs	NN	O	O
of	NN	O	O
skeletal	NN	O	B-Disease
myopathy	NN	O	I-Disease
.	NN	O	O

To	NN	O	O
date	NN	O	O
,	NN	O	O
several	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
Duchenne	NN	O	B-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
gene	NN	O	O
,	NN	O	O
DMD	NN	O	O
,	NN	O	O
have	NN	O	O
been	NN	O	O
identified	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
XLDCM	NN	O	B-Disease
,	NN	O	O
but	NN	O	O
a	NN	O	O
pathogenic	NN	O	O
correlation	NN	O	O
of	NN	O	O
these	NN	O	O
cardiospecific	NN	O	O
mutations	NN	O	O
in	NN	O	O
DMD	NN	O	O
with	NN	O	O
the	NN	O	O
XLDCM	NN	O	B-Disease
phenotype	NN	O	O
has	NN	O	O
remained	NN	O	O
to	NN	O	O
be	NN	O	O
elucidated	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
here	NN	O	O
the	NN	O	O
identification	NN	O	O
of	NN	O	O
a	NN	O	O
unique	NN	O	O
de	NN	O	O
novo	NN	O	O
L1	NN	O	O
insertion	NN	O	O
in	NN	O	O
the	NN	O	O
muscle	NN	O	O
exon	NN	O	O
1	NN	O	O
in	NN	O	O
DMD	NN	O	O
in	NN	O	O
three	NN	O	O
XLDCM	NN	O	B-Disease
patients	NN	O	O
from	NN	O	O
two	NN	O	O
unrelated	NN	O	O
Japanese	NN	O	O
families	NN	O	O
.	NN	O	O

The	NN	O	O
insertion	NN	O	O
was	NN	O	O
a	NN	O	O
5	NN	O	O
-	NN	O	O
truncated	NN	O	O
form	NN	O	O
of	NN	O	O
human	NN	O	O
L1	NN	O	O
inversely	NN	O	O
integrated	NN	O	O
in	NN	O	O
the	NN	O	O
5	NN	O	O
-	NN	O	O
untranslated	NN	O	O
region	NN	O	O
in	NN	O	O
the	NN	O	O
muscle	NN	O	O
exon	NN	O	O
1	NN	O	O
,	NN	O	O
which	NN	O	O
affected	NN	O	O
the	NN	O	O
transcription	NN	O	O
or	NN	O	O
the	NN	O	O
stability	NN	O	O
of	NN	O	O
the	NN	O	O
muscle	NN	O	O
form	NN	O	O
of	NN	O	O
dystrophin	NN	O	O
transcripts	NN	O	O
but	NN	O	O
not	NN	O	O
that	NN	O	O
of	NN	O	O
the	NN	O	O
brain	NN	O	O
or	NN	O	O
Purkinje	NN	O	O
cell	NN	O	O
form	NN	O	O
,	NN	O	O
probably	NN	O	O
due	NN	O	O
to	NN	O	O
its	NN	O	O
unique	NN	O	O
site	NN	O	O
of	NN	O	O
integration	NN	O	O
.	NN	O	O

We	NN	O	O
speculate	NN	O	O
that	NN	O	O
this	NN	O	O
insertion	NN	O	O
of	NN	O	O
an	NN	O	O
L1	NN	O	O
sequence	NN	O	O
in	NN	O	O
DMD	NN	O	O
is	NN	O	O
responsible	NN	O	O
for	NN	O	O
some	NN	O	O
of	NN	O	O
the	NN	O	O
population	NN	O	O
of	NN	O	O
Japanese	NN	O	O
patients	NN	O	O
with	NN	O	O
XLDCM	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Severe	NN	O	O
early	NN	O	O
-	NN	O	O
onset	NN	O	O
obesity	NN	O	B-Disease
,	NN	O	O
adrenal	NN	O	B-Disease
insufficiency	NN	O	I-Disease
and	NN	O	O
red	NN	O	O
hair	NN	O	O
pigmentation	NN	O	O
caused	NN	O	O
by	NN	O	O
POMC	NN	O	O
mutations	NN	O	O
in	NN	O	O
humans	NN	O	O
.	NN	O	O

Sequential	NN	O	O
cleavage	NN	O	O
of	NN	O	O
the	NN	O	O
precursor	NN	O	O
protein	NN	O	O
pre	NN	O	O
-	NN	O	O
pro	NN	O	O
-	NN	O	O
opiomelanocortin	NN	O	O
(	NN	O	O
POMC	NN	O	O
)	NN	O	O
generates	NN	O	O
the	NN	O	O
melanocortin	NN	O	O
peptides	NN	O	O
adrenocorticotrophin	NN	O	O
(	NN	O	O
ACTH	NN	O	O
)	NN	O	O
,	NN	O	O
melanocyte	NN	O	O
-	NN	O	O
stimulating	NN	O	O
hormones	NN	O	O
(	NN	O	O
MSH	NN	O	O
)	NN	O	O
alpha	NN	O	O
,	NN	O	O
beta	NN	O	O
and	NN	O	O
gamma	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
the	NN	O	O
opioid	NN	O	O
-	NN	O	O
receptor	NN	O	O
ligand	NN	O	O
beta	NN	O	O
-	NN	O	O
endorphin	NN	O	O
.	NN	O	O

While	NN	O	O
a	NN	O	O
few	NN	O	O
cases	NN	O	O
of	NN	O	O
isolated	NN	O	O
ACTH	NN	O	B-Disease
deficiency	NN	O	I-Disease
have	NN	O	O
been	NN	O	O
reported	NN	O	O
(	NN	O	O
OMIM	NN	O	O
201400	NN	O	O
)	NN	O	O
,	NN	O	O
an	NN	O	O
inherited	NN	O	O
POMC	NN	O	O
defect	NN	O	O
has	NN	O	O
not	NN	O	O
been	NN	O	O
described	NN	O	O
so	NN	O	O
far	NN	O	O
.	NN	O	O

Recent	NN	O	O
studies	NN	O	O
in	NN	O	O
animal	NN	O	O
models	NN	O	O
elucidated	NN	O	O
a	NN	O	O
central	NN	O	O
role	NN	O	O
of	NN	O	O
alpha	NN	O	O
-	NN	O	O
MSH	NN	O	O
in	NN	O	O
the	NN	O	O
regulation	NN	O	O
of	NN	O	O
food	NN	O	O
intake	NN	O	O
by	NN	O	O
activation	NN	O	O
of	NN	O	O
the	NN	O	O
brain	NN	O	O
melanocortin	NN	O	O
-	NN	O	O
4	NN	O	O
-	NN	O	O
receptor	NN	O	O
(	NN	O	O
MC4	NN	O	O
-	NN	O	O
R	NN	O	O
;	NN	O	O
refs	NN	O	O
3	NN	O	O
-	NN	O	O
5	NN	O	O
)	NN	O	O
and	NN	O	O
the	NN	O	O
linkage	NN	O	O
of	NN	O	O
human	NN	O	O
obesity	NN	O	B-Disease
to	NN	O	O
chromosome	NN	O	O
2	NN	O	O
in	NN	O	O
close	NN	O	O
proximity	NN	O	O
to	NN	O	O
the	NN	O	O
POMC	NN	O	O
locus	NN	O	O
,	NN	O	O
led	NN	O	O
to	NN	O	O
the	NN	O	O
proposal	NN	O	O
of	NN	O	O
an	NN	O	O
association	NN	O	O
of	NN	O	O
POMC	NN	O	O
with	NN	O	O
human	NN	O	O
obesity	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
dual	NN	O	O
role	NN	O	O
of	NN	O	O
alpha	NN	O	O
-	NN	O	O
MSH	NN	O	O
in	NN	O	O
regulating	NN	O	O
food	NN	O	O
intake	NN	O	O
and	NN	O	O
influencing	NN	O	O
hair	NN	O	O
pigmentation	NN	O	O
predicts	NN	O	O
that	NN	O	O
the	NN	O	O
phenotype	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
defect	NN	O	O
in	NN	O	O
POMC	NN	O	O
function	NN	O	O
would	NN	O	O
include	NN	O	O
obesity	NN	O	B-Disease
,	NN	O	O
alteration	NN	O	O
in	NN	O	O
pigmentation	NN	O	O
and	NN	O	O
ACTH	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
observation	NN	O	O
of	NN	O	O
these	NN	O	O
symptoms	NN	O	O
in	NN	O	O
two	NN	O	O
probands	NN	O	O
prompted	NN	O	O
us	NN	O	O
to	NN	O	O
search	NN	O	O
for	NN	O	O
mutations	NN	O	O
within	NN	O	O
their	NN	O	O
POMC	NN	O	O
genes	NN	O	O
.	NN	O	O

Patient	NN	O	O
1	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
a	NN	O	O
compound	NN	O	O
heterozygote	NN	O	O
for	NN	O	O
two	NN	O	O
mutations	NN	O	O
in	NN	O	O
exon	NN	O	O
3	NN	O	O
(	NN	O	O
G7013T	NN	O	O
,	NN	O	O
C7133delta	NN	O	O
)	NN	O	O
which	NN	O	O
interfere	NN	O	O
with	NN	O	O
appropriate	NN	O	O
synthesis	NN	O	O
of	NN	O	O
ACTH	NN	O	O
and	NN	O	O
alpha	NN	O	O
-	NN	O	O
MSH	NN	O	O
.	NN	O	O

Patient	NN	O	O
2	NN	O	O
was	NN	O	O
homozygous	NN	O	O
for	NN	O	O
a	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
2	NN	O	O
(	NN	O	O
C3804A	NN	O	O
)	NN	O	O
which	NN	O	O
abolishes	NN	O	O
POMC	NN	O	O
translation	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
represent	NN	O	O
the	NN	O	O
first	NN	O	O
examples	NN	O	O
of	NN	O	O
a	NN	O	O
genetic	NN	O	B-Disease
defect	NN	O	I-Disease
within	NN	O	O
the	NN	O	O
POMC	NN	O	O
gene	NN	O	O
and	NN	O	O
define	NN	O	O
a	NN	O	O
new	NN	O	O
monogenic	NN	O	B-Disease
endocrine	NN	O	I-Disease
disorder	NN	O	I-Disease
resulting	NN	O	O
in	NN	O	O
early	NN	O	O
-	NN	O	O
onset	NN	O	O
obesity	NN	O	B-Disease
,	NN	O	O
adrenal	NN	O	B-Disease
insufficiency	NN	O	I-Disease
and	NN	O	O
red	NN	O	O
hair	NN	O	O
pigmentation	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
European	NN	O	O
multicenter	NN	O	O
study	NN	O	O
of	NN	O	O
phenylalanine	NN	O	B-Disease
hydroxylase	NN	O	I-Disease
deficiency	NN	O	I-Disease
:	NN	O	O
classification	NN	O	O
of	NN	O	O
105	NN	O	O
mutations	NN	O	O
and	NN	O	O
a	NN	O	O
general	NN	O	O
system	NN	O	O
for	NN	O	O
genotype	NN	O	O
-	NN	O	O
based	NN	O	O
prediction	NN	O	O
of	NN	O	O
metabolic	NN	O	O
phenotype	NN	O	O
.	NN	O	O

Phenylketonuria	NN	O	B-Disease
(	NN	O	O
PKU	NN	O	B-Disease
)	NN	O	O
and	NN	O	O
mild	NN	O	B-Disease
hyperphenylalaninemia	NN	O	I-Disease
(	NN	O	O
MHP	NN	O	B-Disease
)	NN	O	O
are	NN	O	O
allelic	NN	O	B-Disease
disorders	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
gene	NN	O	O
encoding	NN	O	O
phenylalanine	NN	O	O
hydroxylase	NN	O	O
(	NN	O	O
PAH	NN	O	O
)	NN	O	O
.	NN	O	O

Previous	NN	O	O
studies	NN	O	O
have	NN	O	O
suggested	NN	O	O
that	NN	O	O
the	NN	O	O
highly	NN	O	O
variable	NN	O	O
metabolic	NN	O	O
phenotypes	NN	O	O
of	NN	O	O
PAH	NN	O	B-Disease
deficiency	NN	O	I-Disease
correlate	NN	O	O
with	NN	O	O
PAH	NN	O	O
genotypes	NN	O	O
.	NN	O	O

We	NN	O	O
identified	NN	O	O
both	NN	O	O
causative	NN	O	O
mutations	NN	O	O
in	NN	O	O
686	NN	O	O
patients	NN	O	O
from	NN	O	O
seven	NN	O	O
European	NN	O	O
centers	NN	O	O
.	NN	O	O

On	NN	O	O
the	NN	O	O
basis	NN	O	O
of	NN	O	O
the	NN	O	O
phenotypic	NN	O	O
characteristics	NN	O	O
of	NN	O	O
297	NN	O	O
functionally	NN	O	O
hemizygous	NN	O	O
patients	NN	O	O
,	NN	O	O
105	NN	O	O
of	NN	O	O
the	NN	O	O
mutations	NN	O	O
were	NN	O	O
assigned	NN	O	O
to	NN	O	O
one	NN	O	O
of	NN	O	O
four	NN	O	O
arbitrary	NN	O	O
phenotype	NN	O	O
categories	NN	O	O
.	NN	O	O

We	NN	O	O
proposed	NN	O	O
and	NN	O	O
tested	NN	O	O
a	NN	O	O
simple	NN	O	O
model	NN	O	O
for	NN	O	O
correlation	NN	O	O
between	NN	O	O
genotype	NN	O	O
and	NN	O	O
phenotypic	NN	O	O
outcome	NN	O	O
.	NN	O	O

The	NN	O	O
observed	NN	O	O
phenotype	NN	O	O
matched	NN	O	O
the	NN	O	O
predicted	NN	O	O
phenotype	NN	O	O
in	NN	O	O
79	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
cases	NN	O	O
,	NN	O	O
and	NN	O	O
in	NN	O	O
only	NN	O	O
5	NN	O	O
of	NN	O	O
184	NN	O	O
patients	NN	O	O
was	NN	O	O
the	NN	O	O
observed	NN	O	O
phenotype	NN	O	O
more	NN	O	O
than	NN	O	O
one	NN	O	O
category	NN	O	O
away	NN	O	O
from	NN	O	O
that	NN	O	O
expected	NN	O	O
.	NN	O	O

Among	NN	O	O
the	NN	O	O
seven	NN	O	O
contributing	NN	O	O
centers	NN	O	O
,	NN	O	O
the	NN	O	O
proportion	NN	O	O
of	NN	O	O
patients	NN	O	O
for	NN	O	O
whom	NN	O	O
the	NN	O	O
observed	NN	O	O
phenotype	NN	O	O
did	NN	O	O
not	NN	O	O
match	NN	O	O
the	NN	O	O
predicted	NN	O	O
phenotype	NN	O	O
was	NN	O	O
4	NN	O	O
%	NN	O	O
-	NN	O	O
23	NN	O	O
%	NN	O	O
(	NN	O	O
P	NN	O	O
<	NN	O	O
.	NN	O	O
0001	NN	O	O
)	NN	O	O
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
differences	NN	O	O
in	NN	O	O
methods	NN	O	O
used	NN	O	O
for	NN	O	O
mutation	NN	O	O
detection	NN	O	O
or	NN	O	O
phenotype	NN	O	O
classification	NN	O	O
may	NN	O	O
account	NN	O	O
for	NN	O	O
a	NN	O	O
considerable	NN	O	O
proportion	NN	O	O
of	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
inconsistencies	NN	O	O
.	NN	O	O

Our	NN	O	O
data	NN	O	O
indicate	NN	O	O
that	NN	O	O
the	NN	O	O
PAH	NN	O	O
-	NN	O	O
mutation	NN	O	O
genotype	NN	O	O
is	NN	O	O
the	NN	O	O
main	NN	O	O
determinant	NN	O	O
of	NN	O	O
metabolic	NN	O	O
phenotype	NN	O	O
in	NN	O	O
most	NN	O	O
patients	NN	O	O
with	NN	O	O
PAH	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
the	NN	O	O
present	NN	O	O
study	NN	O	O
,	NN	O	O
the	NN	O	O
classification	NN	O	O
of	NN	O	O
105	NN	O	O
PAH	NN	O	O
mutations	NN	O	O
may	NN	O	O
allow	NN	O	O
the	NN	O	O
prediction	NN	O	O
of	NN	O	O
the	NN	O	O
biochemical	NN	O	O
phenotype	NN	O	O
in	NN	O	O
>	NN	O	O
10	NN	O	O
,	NN	O	O
000	NN	O	O
genotypes	NN	O	O
,	NN	O	O
which	NN	O	O
may	NN	O	O
be	NN	O	O
useful	NN	O	O
for	NN	O	O
the	NN	O	O
management	NN	O	O
of	NN	O	O
hyperphenylalaninemia	NN	O	B-Disease
in	NN	O	O
newborns	NN	O	O
.	NN	O	O

Somatic	NN	O	O
instability	NN	O	O
of	NN	O	O
the	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
in	NN	O	O
mice	NN	O	O
transgenic	NN	O	O
for	NN	O	O
the	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
region	NN	O	O
is	NN	O	O
age	NN	O	O
dependent	NN	O	O
but	NN	O	O
not	NN	O	O
correlated	NN	O	O
to	NN	O	O
the	NN	O	O
relative	NN	O	O
intertissue	NN	O	O
transcription	NN	O	O
levels	NN	O	O
and	NN	O	O
proliferative	NN	O	O
capacities	NN	O	O
.	NN	O	O

A	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
nexpansion	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
-	NN	O	O
untranslated	NN	O	O
region	NN	O	O
(	NN	O	O
UTR	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
DM	NN	O	O
protein	NN	O	O
kinase	NN	O	O
gene	NN	O	O
(	NN	O	O
DMPK	NN	O	O
)	NN	O	O
is	NN	O	O
responsible	NN	O	O
for	NN	O	O
causing	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Major	NN	O	O
instability	NN	O	O
,	NN	O	O
with	NN	O	O
very	NN	O	O
large	NN	O	O
expansions	NN	O	O
between	NN	O	O
generations	NN	O	O
and	NN	O	O
high	NN	O	O
levels	NN	O	O
of	NN	O	O
somatic	NN	O	O
mosaicism	NN	O	O
,	NN	O	O
is	NN	O	O
observed	NN	O	O
in	NN	O	O
patients	NN	O	O
.	NN	O	O

There	NN	O	O
is	NN	O	O
a	NN	O	O
good	NN	O	O
correlation	NN	O	O
between	NN	O	O
repeat	NN	O	O
size	NN	O	O
(	NN	O	O
at	NN	O	O
least	NN	O	O
in	NN	O	O
leucocytes	NN	O	O
)	NN	O	O
,	NN	O	O
clinical	NN	O	O
severity	NN	O	O
and	NN	O	O
age	NN	O	O
of	NN	O	O
onset	NN	O	O
.	NN	O	O

The	NN	O	O
trinucleotide	NN	O	O
repeat	NN	O	O
instability	NN	O	O
mechanisms	NN	O	O
involved	NN	O	O
in	NN	O	O
DM	NN	O	B-Disease
and	NN	O	O
other	NN	O	O
human	NN	O	O
genetic	NN	O	B-Disease
diseases	NN	O	I-Disease
are	NN	O	O
unknown	NN	O	O
.	NN	O	O

We	NN	O	O
studied	NN	O	O
somatic	NN	O	O
instability	NN	O	O
by	NN	O	O
measuring	NN	O	O
the	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
length	NN	O	O
at	NN	O	O
several	NN	O	O
ages	NN	O	O
in	NN	O	O
various	NN	O	O
tissues	NN	O	O
of	NN	O	O
transgenic	NN	O	O
mice	NN	O	O
carrying	NN	O	O
a	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
55expansion	NN	O	O
surrounded	NN	O	O
by	NN	O	O
45	NN	O	O
kb	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
DM	NN	O	B-Disease
region	NN	O	O
,	NN	O	O
using	NN	O	O
small	NN	O	O
-	NN	O	O
pool	NN	O	O
PCR	NN	O	O
.	NN	O	O

These	NN	O	O
mice	NN	O	O
have	NN	O	O
been	NN	O	O
shown	NN	O	O
to	NN	O	O
reproduce	NN	O	O
the	NN	O	O
intergenerational	NN	O	O
and	NN	O	O
somatic	NN	O	O
instability	NN	O	O
of	NN	O	O
the	NN	O	O
55	NN	O	O
CTG	NN	O	O
repeat	NN	O	O
suggesting	NN	O	O
that	NN	O	O
surrounding	NN	O	O
sequences	NN	O	O
and	NN	O	O
the	NN	O	O
chromatin	NN	O	O
environment	NN	O	O
are	NN	O	O
involved	NN	O	O
in	NN	O	O
instability	NN	O	O
mechanisms	NN	O	O
.	NN	O	O

As	NN	O	O
observed	NN	O	O
in	NN	O	O
some	NN	O	O
of	NN	O	O
the	NN	O	O
tissues	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
patients	NN	O	O
,	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
tendency	NN	O	O
for	NN	O	O
repeat	NN	O	O
length	NN	O	O
and	NN	O	O
somatic	NN	O	O
mosaicism	NN	O	O
to	NN	O	O
increase	NN	O	O
with	NN	O	O
the	NN	O	O
age	NN	O	O
of	NN	O	O
the	NN	O	O
mouse	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
we	NN	O	O
observed	NN	O	O
no	NN	O	O
correlation	NN	O	O
between	NN	O	O
the	NN	O	O
somatic	NN	O	O
mutation	NN	O	O
rate	NN	O	O
and	NN	O	O
tissue	NN	O	O
proliferation	NN	O	O
capacity	NN	O	O
.	NN	O	O

The	NN	O	O
somatic	NN	O	O
mutation	NN	O	O
rates	NN	O	O
in	NN	O	O
different	NN	O	O
tissues	NN	O	O
were	NN	O	O
also	NN	O	O
not	NN	O	O
correlated	NN	O	O
to	NN	O	O
the	NN	O	O
relative	NN	O	O
inter	NN	O	O
-	NN	O	O
tissue	NN	O	O
difference	NN	O	O
in	NN	O	O
transcriptional	NN	O	O
levels	NN	O	O
of	NN	O	O
the	NN	O	O
three	NN	O	O
genes	NN	O	O
(	NN	O	O
DMAHP	NN	O	O
,	NN	O	O
DMPK	NN	O	O
and	NN	O	O
59	NN	O	O
)	NN	O	O
surrounding	NN	O	O
the	NN	O	O
repeat	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
novel	NN	O	O
missense	NN	O	O
mutation	NN	O	O
in	NN	O	O
patients	NN	O	O
from	NN	O	O
a	NN	O	O
retinoblastoma	NN	O	B-Disease
pedigree	NN	O	O
showing	NN	O	O
only	NN	O	O
mild	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
tumor	NN	O	B-Disease
phenotype	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
used	NN	O	O
single	NN	O	O
strand	NN	O	O
conformation	NN	O	O
polymorphism	NN	O	O
analysis	NN	O	O
to	NN	O	O
study	NN	O	O
the	NN	O	O
27	NN	O	O
exons	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
in	NN	O	O
individuals	NN	O	O
from	NN	O	O
a	NN	O	O
family	NN	O	O
showing	NN	O	O
mild	NN	O	O
expression	NN	O	O
of	NN	O	O
the	NN	O	O
retinoblastoma	NN	O	B-Disease
phenotype	NN	O	O
.	NN	O	O

In	NN	O	O
this	NN	O	O
family	NN	O	O
affected	NN	O	O
individuals	NN	O	O
developed	NN	O	O
unilateral	NN	O	B-Disease
tumors	NN	O	I-Disease
and	NN	O	O
,	NN	O	O
as	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
linkage	NN	O	O
analysis	NN	O	O
,	NN	O	O
unaffected	NN	O	O
mutation	NN	O	O
carriers	NN	O	O
were	NN	O	O
also	NN	O	O
identified	NN	O	O
within	NN	O	O
the	NN	O	O
pedigree	NN	O	O
.	NN	O	O

A	NN	O	O
single	NN	O	O
band	NN	O	O
shift	NN	O	O
using	NN	O	O
SSCP	NN	O	O
was	NN	O	O
identified	NN	O	O
in	NN	O	O
exon	NN	O	O
21	NN	O	O
which	NN	O	O
resulted	NN	O	O
in	NN	O	O
a	NN	O	O
missense	NN	O	O
mutation	NN	O	O
converting	NN	O	O
a	NN	O	O
cys	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
arg	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
position	NN	O	O
28	NN	O	O
in	NN	O	O
the	NN	O	O
exon	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
destroyed	NN	O	O
an	NN	O	O
NdeI	NN	O	O
restriction	NN	O	O
enzyme	NN	O	O
site	NN	O	O
.	NN	O	O

Analysis	NN	O	O
of	NN	O	O
all	NN	O	O
family	NN	O	O
members	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
the	NN	O	O
missense	NN	O	O
mutation	NN	O	O
co	NN	O	O
-	NN	O	O
segregated	NN	O	O
with	NN	O	O
patients	NN	O	O
with	NN	O	O
tumors	NN	O	B-Disease
or	NN	O	O
who	NN	O	O
,	NN	O	O
as	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
linkage	NN	O	O
analysis	NN	O	O
had	NN	O	O
been	NN	O	O
predicted	NN	O	O
to	NN	O	O
carry	NN	O	O
the	NN	O	O
predisposing	NN	O	O
mutation	NN	O	O
.	NN	O	O

These	NN	O	O
observations	NN	O	O
point	NN	O	O
to	NN	O	O
another	NN	O	O
region	NN	O	O
of	NN	O	O
the	NN	O	O
RB1	NN	O	O
gene	NN	O	O
where	NN	O	O
mutations	NN	O	O
only	NN	O	O
modify	NN	O	O
the	NN	O	O
function	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
and	NN	O	O
raise	NN	O	O
important	NN	O	O
questions	NN	O	O
for	NN	O	O
genetic	NN	O	O
counseling	NN	O	O
in	NN	O	O
families	NN	O	O
with	NN	O	O
these	NN	O	O
distinctive	NN	O	O
phenotypes	NN	O	O
.	NN	O	O
.	NN	O	O

Maternal	NN	O	B-Disease
disomy	NN	O	I-Disease
and	NN	O	O
Prader	NN	O	B-Disease
-	NN	O	I-Disease
Willi	NN	O	I-Disease
syndrome	NN	O	I-Disease
consistent	NN	O	O
with	NN	O	O
gamete	NN	O	O
complementation	NN	O	O
in	NN	O	O
a	NN	O	O
case	NN	O	O
of	NN	O	O
familial	NN	O	O
translocation	NN	O	O
(	NN	O	O
3	NN	O	O
;	NN	O	O
15	NN	O	O
)	NN	O	O
(	NN	O	O
p25	NN	O	O
;	NN	O	O
q11	NN	O	O
.	NN	O	O
2	NN	O	O
)	NN	O	O
.	NN	O	O

Maternal	NN	O	B-Disease
uniparental	NN	O	I-Disease
disomy	NN	O	I-Disease
(	NN	O	I-Disease
UPD	NN	O	I-Disease
)	NN	O	I-Disease
for	NN	O	I-Disease
chromosome	NN	O	I-Disease
15	NN	O	I-Disease
is	NN	O	O
responsible	NN	O	O
for	NN	O	O
an	NN	O	O
estimated	NN	O	O
30	NN	O	O
%	NN	O	O
of	NN	O	O
cases	NN	O	O
of	NN	O	O
Prader	NN	O	B-Disease
-	NN	O	I-Disease
Willi	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
PWS	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
on	NN	O	O
an	NN	O	O
unusual	NN	O	O
case	NN	O	O
of	NN	O	O
maternal	NN	O	B-Disease
disomy	NN	O	I-Disease
15	NN	O	I-Disease
in	NN	O	O
PWS	NN	O	B-Disease
that	NN	O	O
is	NN	O	O
most	NN	O	O
consistent	NN	O	O
with	NN	O	O
adjacent	NN	O	O
-	NN	O	O
1	NN	O	O
segregation	NN	O	O
of	NN	O	O
a	NN	O	O
paternal	NN	O	O
t	NN	O	O
(	NN	O	O
3	NN	O	O
;	NN	O	O
15	NN	O	O
)	NN	O	O
(	NN	O	O
p25	NN	O	O
;	NN	O	O
q11	NN	O	O
.	NN	O	O
2	NN	O	O
)	NN	O	O
with	NN	O	O
simultaneous	NN	O	O
maternal	NN	O	O
meiotic	NN	O	O
nondisjunction	NN	O	O
for	NN	O	O
chromosome	NN	O	O
15	NN	O	O
.	NN	O	O

The	NN	O	O
patient	NN	O	O
(	NN	O	O
J	NN	O	O
.	NN	O	O
B	NN	O	O
.	NN	O	O
)	NN	O	O
,	NN	O	O
a	NN	O	O
17	NN	O	O
-	NN	O	O
year	NN	O	O
-	NN	O	O
old	NN	O	O
white	NN	O	O
male	NN	O	O
with	NN	O	O
PWS	NN	O	B-Disease
,	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
have	NN	O	O
47	NN	O	O
chromosomes	NN	O	O
with	NN	O	O
a	NN	O	O
supernumerary	NN	O	O
,	NN	O	O
paternal	NN	O	O
der	NN	O	O
(	NN	O	O
15	NN	O	O
)	NN	O	O
consisting	NN	O	O
of	NN	O	O
the	NN	O	O
short	NN	O	O
arm	NN	O	O
and	NN	O	O
the	NN	O	O
proximal	NN	O	O
long	NN	O	O
arm	NN	O	O
of	NN	O	O
chromosome	NN	O	O
15	NN	O	O
,	NN	O	O
and	NN	O	O
distal	NN	O	O
chromosome	NN	O	O
arm	NN	O	O
3p	NN	O	O
.	NN	O	O

The	NN	O	O
t	NN	O	O
(	NN	O	O
3	NN	O	O
;	NN	O	O
15	NN	O	O
)	NN	O	O
was	NN	O	O
present	NN	O	O
in	NN	O	O
the	NN	O	O
balanced	NN	O	O
state	NN	O	O
in	NN	O	O
the	NN	O	O
patients	NN	O	O
father	NN	O	O
and	NN	O	O
a	NN	O	O
sister	NN	O	O
.	NN	O	O

Fluorescent	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
analysis	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
the	NN	O	O
PWS	NN	O	B-Disease
critical	NN	O	O
region	NN	O	O
resided	NN	O	O
on	NN	O	O
the	NN	O	O
derivative	NN	O	O
chromosome	NN	O	O
3	NN	O	O
and	NN	O	O
that	NN	O	O
there	NN	O	O
was	NN	O	O
no	NN	O	O
deletion	NN	O	O
of	NN	O	O
the	NN	O	O
PWS	NN	O	B-Disease
region	NN	O	O
on	NN	O	O
the	NN	O	O
normal	NN	O	O
pair	NN	O	O
of	NN	O	O
15s	NN	O	O
present	NN	O	O
in	NN	O	O
J	NN	O	O
.	NN	O	O

B	NN	O	O
.	NN	O	O

Methylation	NN	O	O
analysis	NN	O	O
at	NN	O	O
exon	NN	O	O
alpha	NN	O	O
of	NN	O	O
the	NN	O	O
small	NN	O	O
nuclear	NN	O	O
ribonucleoprotein	NN	O	O
-	NN	O	O
associated	NN	O	O
polypeptide	NN	O	O
N	NN	O	O
(	NN	O	O
SNRPN	NN	O	O
)	NN	O	O
gene	NN	O	O
showed	NN	O	O
a	NN	O	O
pattern	NN	O	O
characteristic	NN	O	O
of	NN	O	O
only	NN	O	O
the	NN	O	O
maternal	NN	O	O
chromosome	NN	O	O
15	NN	O	O
in	NN	O	O
J	NN	O	O
.	NN	O	O

B	NN	O	O
.	NN	O	O

Maternal	NN	O	B-Disease
disomy	NN	O	I-Disease
was	NN	O	O
confirmed	NN	O	O
by	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
analysis	NN	O	O
of	NN	O	O
microsatellite	NN	O	O
repeats	NN	O	O
at	NN	O	O
the	NN	O	O
gamma	NN	O	O
-	NN	O	O
aminobutyric	NN	O	O
acid	NN	O	O
receptor	NN	O	O
beta3	NN	O	O
subunit	NN	O	O
(	NN	O	O
GABRB3	NN	O	O
)	NN	O	O
locus	NN	O	O
.	NN	O	O

A	NN	O	O
niece	NN	O	O
(	NN	O	O
B	NN	O	O
.	NN	O	O
B	NN	O	O
.	NN	O	O
)	NN	O	O
with	NN	O	O
45	NN	O	O
chromosomes	NN	O	O
and	NN	O	O
the	NN	O	O
derivative	NN	O	O
3	NN	O	O
but	NN	O	O
without	NN	O	O
the	NN	O	O
der	NN	O	O
(	NN	O	O
15	NN	O	O
)	NN	O	O
demonstrated	NN	O	O
a	NN	O	O
phenotype	NN	O	O
consistent	NN	O	O
with	NN	O	O
that	NN	O	O
reported	NN	O	O
for	NN	O	O
haploinsufficiency	NN	O	O
of	NN	O	O
distal	NN	O	O
3	NN	O	O
p	NN	O	O
.	NN	O	O

Uniparental	NN	O	B-Disease
disomy	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
unbalanced	NN	O	O
segregation	NN	O	O
of	NN	O	O
non	NN	O	O
-	NN	O	O
Robertsonian	NN	O	O
translocations	NN	O	O
has	NN	O	O
been	NN	O	O
reported	NN	O	O
previously	NN	O	O
but	NN	O	O
has	NN	O	O
not	NN	O	O
,	NN	O	O
to	NN	O	O
our	NN	O	O
knowledge	NN	O	O
,	NN	O	O
been	NN	O	O
observed	NN	O	O
in	NN	O	O
a	NN	O	O
case	NN	O	O
of	NN	O	O
PWS	NN	O	B-Disease
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
our	NN	O	O
findings	NN	O	O
are	NN	O	O
best	NN	O	O
interpreted	NN	O	O
as	NN	O	O
true	NN	O	O
gamete	NN	O	O
complementation	NN	O	O
resulting	NN	O	O
in	NN	O	O
maternal	NN	O	B-Disease
UPD	NN	O	I-Disease
15	NN	O	I-Disease
and	NN	O	O
PWS	NN	O	B-Disease

Schwartz	NN	O	B-Disease
-	NN	O	I-Disease
Jampel	NN	O	I-Disease
syndrome	NN	O	I-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
and	NN	O	O
Stuve	NN	O	B-Disease
-	NN	O	I-Disease
Wiedemann	NN	O	I-Disease
syndrome	NN	O	I-Disease
:	NN	O	O
a	NN	O	O
case	NN	O	O
for	NN	O	O
"	NN	O	O
lumping	NN	O	O
"	NN	O	O
.	NN	O	O

Recent	NN	O	O
studies	NN	O	O
demonstrated	NN	O	O
the	NN	O	O
existence	NN	O	O
of	NN	O	O
a	NN	O	O
genetically	NN	O	O
distinct	NN	O	O
,	NN	O	O
usually	NN	O	O
lethal	NN	O	O
form	NN	O	O
of	NN	O	O
the	NN	O	O
Schwartz	NN	O	B-Disease
-	NN	O	I-Disease
Jampel	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
SJS	NN	O	B-Disease
)	NN	O	O
of	NN	O	O
myotonia	NN	O	B-Disease
and	NN	O	O
skeletal	NN	O	B-Disease
dysplasia	NN	O	I-Disease
,	NN	O	O
which	NN	O	O
we	NN	O	O
called	NN	O	O
SJS	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
.	NN	O	O

This	NN	O	O
disorder	NN	O	O
is	NN	O	O
reminiscent	NN	O	O
of	NN	O	O
another	NN	O	O
rare	NN	O	O
condition	NN	O	O
,	NN	O	O
the	NN	O	O
Stuve	NN	O	B-Disease
-	NN	O	I-Disease
Wiedemann	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
SWS	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
which	NN	O	O
comprises	NN	O	O
campomelia	NN	O	B-Disease
at	NN	O	O
birth	NN	O	O
with	NN	O	O
skeletal	NN	O	B-Disease
dysplasia	NN	O	I-Disease
,	NN	O	O
contractures	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
early	NN	O	B-Disease
death	NN	O	I-Disease
.	NN	O	O

To	NN	O	O
test	NN	O	O
for	NN	O	O
possible	NN	O	O
nosologic	NN	O	O
identity	NN	O	O
between	NN	O	O
these	NN	O	O
disorders	NN	O	O
,	NN	O	O
we	NN	O	O
reviewed	NN	O	O
the	NN	O	O
literature	NN	O	O
and	NN	O	O
obtained	NN	O	O
a	NN	O	O
follow	NN	O	O
-	NN	O	O
up	NN	O	O
of	NN	O	O
the	NN	O	O
only	NN	O	O
two	NN	O	O
surviving	NN	O	O
patients	NN	O	O
,	NN	O	O
one	NN	O	O
with	NN	O	O
SJS	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
at	NN	O	O
age	NN	O	O
10	NN	O	O
years	NN	O	O
and	NN	O	O
another	NN	O	O
with	NN	O	O
SWS	NN	O	B-Disease
at	NN	O	O
age	NN	O	O
7	NN	O	O
years	NN	O	O
.	NN	O	O

Patients	NN	O	O
reported	NN	O	O
as	NN	O	O
having	NN	O	O
either	NN	O	O
neonatal	NN	O	O
SJS	NN	O	B-Disease
or	NN	O	O
SWS	NN	O	B-Disease
presented	NN	O	O
a	NN	O	O
combination	NN	O	O
of	NN	O	O
a	NN	O	O
severe	NN	O	O
,	NN	O	O
prenatal	NN	O	O
-	NN	O	O
onset	NN	O	O
neuromuscular	NN	O	B-Disease
disorder	NN	O	I-Disease
(	NN	O	O
with	NN	O	O
congenital	NN	O	B-Disease
joint	NN	O	I-Disease
contractures	NN	O	I-Disease
,	NN	O	O
respiratory	NN	O	O
and	NN	O	O
feeding	NN	O	O
difficulties	NN	O	O
,	NN	O	O
tendency	NN	O	O
to	NN	O	O
hyperthermia	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
frequent	NN	O	O
death	NN	O	O
in	NN	O	O
infancy	NN	O	O
)	NN	O	O
with	NN	O	O
a	NN	O	O
distinct	NN	O	O
campomelic	NN	O	B-Disease
-	NN	O	I-Disease
metaphyseal	NN	O	I-Disease
skeletal	NN	O	I-Disease
dysplasia	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
similarity	NN	O	O
of	NN	O	O
the	NN	O	O
clinical	NN	O	O
and	NN	O	O
radiographic	NN	O	O
findings	NN	O	O
is	NN	O	O
so	NN	O	O
extensive	NN	O	O
that	NN	O	O
these	NN	O	O
disorders	NN	O	O
appear	NN	O	O
to	NN	O	O
be	NN	O	O
a	NN	O	O
single	NN	O	O
entity	NN	O	O
.	NN	O	O

The	NN	O	O
follow	NN	O	O
-	NN	O	O
up	NN	O	O
observation	NN	O	O
of	NN	O	O
an	NN	O	O
identical	NN	O	O
and	NN	O	O
unique	NN	O	O
pattern	NN	O	O
of	NN	O	O
progressive	NN	O	O
bone	NN	O	B-Disease
dysplasia	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
two	NN	O	O
patients	NN	O	O
(	NN	O	O
one	NN	O	O
with	NN	O	O
SJS	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
,	NN	O	O
one	NN	O	O
with	NN	O	O
SWS	NN	O	B-Disease
)	NN	O	O
surviving	NN	O	O
beyond	NN	O	O
infancy	NN	O	O
adds	NN	O	O
to	NN	O	O
the	NN	O	O
evidence	NN	O	O
in	NN	O	O
favor	NN	O	O
of	NN	O	O
identity	NN	O	O
.	NN	O	O

The	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
SWS	NN	O	B-Disease
and	NN	O	O
SJS	NN	O	B-Disease
type	NN	O	I-Disease
2	NN	O	I-Disease
are	NN	O	O
the	NN	O	O
same	NN	O	O
disorder	NN	O	O
should	NN	O	O
be	NN	O	O
testable	NN	O	O
by	NN	O	O
molecular	NN	O	O
methods	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
mouse	NN	O	O
model	NN	O	O
of	NN	O	O
severe	NN	O	O
von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
disease	NN	O	I-Disease
:	NN	O	O
defects	NN	O	O
in	NN	O	O
hemostasis	NN	O	O
and	NN	O	O
thrombosis	NN	O	B-Disease
.	NN	O	O

von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
factor	NN	O	I-Disease
(	NN	O	I-Disease
vWf	NN	O	I-Disease
)	NN	O	I-Disease
deficiency	NN	O	I-Disease
causes	NN	O	O
severe	NN	O	O
von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
disease	NN	O	I-Disease
in	NN	O	O
humans	NN	O	O
.	NN	O	O

We	NN	O	O
generated	NN	O	O
a	NN	O	O
mouse	NN	O	O
model	NN	O	O
for	NN	O	O
this	NN	O	O
disease	NN	O	O
by	NN	O	O
using	NN	O	O
gene	NN	O	O
targeting	NN	O	O
.	NN	O	O

vWf	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
mice	NN	O	O
appeared	NN	O	O
normal	NN	O	O
at	NN	O	O
birth	NN	O	O
;	NN	O	O
they	NN	O	O
were	NN	O	O
viable	NN	O	O
and	NN	O	O
fertile	NN	O	O
.	NN	O	O

Neither	NN	O	O
vWf	NN	O	O
nor	NN	O	O
vWf	NN	O	O
propolypeptide	NN	O	O
(	NN	O	O
von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
antigen	NN	O	O
II	NN	O	O
)	NN	O	O
were	NN	O	O
detectable	NN	O	O
in	NN	O	O
plasma	NN	O	O
,	NN	O	O
platelets	NN	O	O
,	NN	O	O
or	NN	O	O
endothelial	NN	O	O
cells	NN	O	O
of	NN	O	O
the	NN	O	O
homozygous	NN	O	O
mutant	NN	O	O
mice	NN	O	O
.	NN	O	O

The	NN	O	O
mutant	NN	O	O
mice	NN	O	O
exhibited	NN	O	O
defects	NN	O	O
in	NN	O	O
hemostasis	NN	O	O
with	NN	O	O
a	NN	O	O
highly	NN	O	O
prolonged	NN	O	O
bleeding	NN	O	O
time	NN	O	O
and	NN	O	O
spontaneous	NN	O	O
bleeding	NN	O	O
events	NN	O	O
in	NN	O	O
approximately	NN	O	O
10	NN	O	O
%	NN	O	O
of	NN	O	O
neonates	NN	O	O
.	NN	O	O

As	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
disease	NN	O	O
,	NN	O	O
the	NN	O	O
factor	NN	O	O
VIII	NN	O	O
level	NN	O	O
in	NN	O	O
these	NN	O	O
mice	NN	O	O
was	NN	O	O
reduced	NN	O	O
strongly	NN	O	O
as	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
the	NN	O	O
lack	NN	O	O
of	NN	O	O
protection	NN	O	O
provided	NN	O	O
by	NN	O	O
vWf	NN	O	O
.	NN	O	O

Defective	NN	O	O
thrombosis	NN	O	B-Disease
in	NN	O	O
mutant	NN	O	O
mice	NN	O	O
was	NN	O	O
also	NN	O	O
evident	NN	O	O
in	NN	O	O
an	NN	O	O
in	NN	O	O
vivo	NN	O	O
model	NN	O	O
of	NN	O	O
vascular	NN	O	B-Disease
injury	NN	O	I-Disease
.	NN	O	O

In	NN	O	O
this	NN	O	O
model	NN	O	O
,	NN	O	O
the	NN	O	O
exteriorized	NN	O	O
mesentery	NN	O	O
was	NN	O	O
superfused	NN	O	O
with	NN	O	O
ferric	NN	O	O
chloride	NN	O	O
and	NN	O	O
the	NN	O	O
accumulation	NN	O	O
of	NN	O	O
fluorescently	NN	O	O
labeled	NN	O	O
platelets	NN	O	O
was	NN	O	O
observed	NN	O	O
by	NN	O	O
intravital	NN	O	O
microscopy	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
these	NN	O	O
mice	NN	O	O
very	NN	O	O
closely	NN	O	O
mimic	NN	O	O
severe	NN	O	O
human	NN	O	O
von	NN	O	B-Disease
Willebrand	NN	O	I-Disease
disease	NN	O	I-Disease
and	NN	O	O
will	NN	O	O
be	NN	O	O
very	NN	O	O
useful	NN	O	O
for	NN	O	O
investigating	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
vWf	NN	O	O
in	NN	O	O
normal	NN	O	O
physiology	NN	O	O
and	NN	O	O
in	NN	O	O
disease	NN	O	O
models	NN	O	O
.	NN	O	O
.	NN	O	O

Oral	NN	O	O
contraceptives	NN	O	O
and	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Hereditary	NN	O	B-Disease
Ovarian	NN	O	I-Disease
Cancer	NN	O	I-Disease
Clinical	NN	O	O
Study	NN	O	O
Group	NN	O	O
.	NN	O	O

BACKGROUND	NN	O	O
Women	NN	O	O
with	NN	O	O
mutations	NN	O	O
in	NN	O	O
either	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
the	NN	O	O
BRCA2	NN	O	O
gene	NN	O	O
have	NN	O	O
a	NN	O	O
high	NN	O	O
lifetime	NN	O	O
risk	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

Oral	NN	O	O
contraceptives	NN	O	O
protect	NN	O	O
against	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
general	NN	O	O
,	NN	O	O
but	NN	O	O
it	NN	O	O
is	NN	O	O
not	NN	O	O
known	NN	O	O
whether	NN	O	O
they	NN	O	O
also	NN	O	O
protect	NN	O	O
against	NN	O	O
hereditary	NN	O	B-Disease
forms	NN	O	I-Disease
of	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

METHODS	NN	O	O
We	NN	O	O
enrolled	NN	O	O
207	NN	O	O
women	NN	O	O
with	NN	O	O
hereditary	NN	O	B-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
and	NN	O	O
161	NN	O	O
of	NN	O	O
their	NN	O	O
sisters	NN	O	O
as	NN	O	O
controls	NN	O	O
in	NN	O	O
a	NN	O	O
case	NN	O	O
-	NN	O	O
control	NN	O	O
study	NN	O	O
.	NN	O	O

All	NN	O	O
the	NN	O	O
patients	NN	O	O
carried	NN	O	O
a	NN	O	O
pathogenic	NN	O	O
mutation	NN	O	O
in	NN	O	O
either	NN	O	O
BRCA1	NN	O	O
(	NN	O	O
179	NN	O	O
women	NN	O	O
)	NN	O	O
or	NN	O	O
BRCA2	NN	O	O
(	NN	O	O
28	NN	O	O
women	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
control	NN	O	O
women	NN	O	O
were	NN	O	O
enrolled	NN	O	O
regardless	NN	O	O
of	NN	O	O
whether	NN	O	O
or	NN	O	O
not	NN	O	O
they	NN	O	O
had	NN	O	O
either	NN	O	O
mutation	NN	O	O
.	NN	O	O

Lifetime	NN	O	O
histories	NN	O	O
of	NN	O	O
oral	NN	O	O
-	NN	O	O
contraceptive	NN	O	O
use	NN	O	O
were	NN	O	O
obtained	NN	O	O
by	NN	O	O
interview	NN	O	O
or	NN	O	O
by	NN	O	O
written	NN	O	O
questionnaire	NN	O	O
and	NN	O	O
were	NN	O	O
compared	NN	O	O
between	NN	O	O
patients	NN	O	O
and	NN	O	O
control	NN	O	O
women	NN	O	O
,	NN	O	O
after	NN	O	O
adjustment	NN	O	O
for	NN	O	O
year	NN	O	O
of	NN	O	O
birth	NN	O	O
and	NN	O	O
parity	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
The	NN	O	O
adjusted	NN	O	O
odds	NN	O	O
ratio	NN	O	O
for	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
any	NN	O	O
past	NN	O	O
use	NN	O	O
of	NN	O	O
oral	NN	O	O
contraceptives	NN	O	O
was	NN	O	O
0	NN	O	O
.	NN	O	O

5	NN	O	O
(	NN	O	O
95	NN	O	O
percent	NN	O	O
confidence	NN	O	O
interval	NN	O	O
,	NN	O	O
0	NN	O	O
.	NN	O	O
3	NN	O	O
to	NN	O	O
0	NN	O	O
.	NN	O	O
8	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
risk	NN	O	O
decreased	NN	O	O
with	NN	O	O
increasing	NN	O	O
duration	NN	O	O
of	NN	O	O
use	NN	O	O
(	NN	O	O
P	NN	O	O
for	NN	O	O
trend	NN	O	O
,	NN	O	O
<	NN	O	O
0	NN	O	O
.	NN	O	O
001	NN	O	O
)	NN	O	O
;	NN	O	O
use	NN	O	O
for	NN	O	O
six	NN	O	O
or	NN	O	O
more	NN	O	O
years	NN	O	O
was	NN	O	O
associated	NN	O	O
with	NN	O	O
a	NN	O	O
60	NN	O	O
percent	NN	O	O
reduction	NN	O	O
in	NN	O	O
risk	NN	O	O
.	NN	O	O

Oral	NN	O	O
-	NN	O	O
contraceptive	NN	O	O
use	NN	O	O
protected	NN	O	O
against	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
both	NN	O	O
for	NN	O	O
carriers	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
mutation	NN	O	O
(	NN	O	O
odds	NN	O	O
ratio	NN	O	O
,	NN	O	O
0	NN	O	O
.	NN	O	O
5	NN	O	O
;	NN	O	O
95	NN	O	O
percent	NN	O	O
confidence	NN	O	O
interval	NN	O	O
,	NN	O	O
0	NN	O	O
.	NN	O	O
3	NN	O	O
to	NN	O	O
0	NN	O	O
.	NN	O	O
9	NN	O	O
)	NN	O	O
and	NN	O	O
for	NN	O	O
carriers	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA2	NN	O	O
mutation	NN	O	O
(	NN	O	O
odds	NN	O	O
ratio	NN	O	O
,	NN	O	O
0	NN	O	O
.	NN	O	O
4	NN	O	O
;	NN	O	O
95	NN	O	O
percent	NN	O	O
confidence	NN	O	O
interval	NN	O	O
,	NN	O	O
0	NN	O	O
.	NN	O	O
2	NN	O	O
to	NN	O	O
1	NN	O	O
.	NN	O	O
1	NN	O	O
)	NN	O	O
.	NN	O	O

CONCLUSIONS	NN	O	O
Oral	NN	O	O
-	NN	O	O
contraceptive	NN	O	O
use	NN	O	O
may	NN	O	O
reduce	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
women	NN	O	O
with	NN	O	O
pathogenic	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
or	NN	O	O
BRCA2	NN	O	O
gene	NN	O	O

A	NN	O	O
Japanese	NN	O	O
family	NN	O	O
with	NN	O	O
adrenoleukodystrophy	NN	O	B-Disease
with	NN	O	O
a	NN	O	O
codon	NN	O	O
291	NN	O	O
deletion	NN	O	O
:	NN	O	O
a	NN	O	O
clinical	NN	O	O
,	NN	O	O
biochemical	NN	O	O
,	NN	O	O
pathological	NN	O	O
,	NN	O	O
and	NN	O	O
genetic	NN	O	O
report	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
a	NN	O	O
Japanese	NN	O	O
family	NN	O	O
with	NN	O	O
adrenoleukodystrophy	NN	O	B-Disease
(	NN	O	O
ALD	NN	O	B-Disease
)	NN	O	O
with	NN	O	O
a	NN	O	O
three	NN	O	O
base	NN	O	O
pair	NN	O	O
deletion	NN	O	O
(	NN	O	O
delGAG	NN	O	O
291	NN	O	O
)	NN	O	O
in	NN	O	O
the	NN	O	O
ALD	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

A	NN	O	O
variety	NN	O	O
of	NN	O	O
phenotypes	NN	O	O
were	NN	O	O
observed	NN	O	O
within	NN	O	O
this	NN	O	O
family	NN	O	O
.	NN	O	O

While	NN	O	O
the	NN	O	O
proband	NN	O	O
(	NN	O	O
patient	NN	O	O
1	NN	O	O
)	NN	O	O
was	NN	O	O
classified	NN	O	O
as	NN	O	O
having	NN	O	O
a	NN	O	O
rare	NN	O	O
intermediate	NN	O	O
type	NN	O	O
of	NN	O	O
adult	NN	O	O
cerebral	NN	O	O
and	NN	O	O
cerebello	NN	O	O
-	NN	O	O
brain	NN	O	O
stem	NN	O	O
forms	NN	O	O
,	NN	O	O
his	NN	O	O
younger	NN	O	O
brother	NN	O	O
(	NN	O	O
patient	NN	O	O
2	NN	O	O
)	NN	O	O
and	NN	O	O
nephew	NN	O	O
(	NN	O	O
patient	NN	O	O
3	NN	O	O
)	NN	O	O
had	NN	O	O
a	NN	O	O
childhood	NN	O	O
ALD	NN	O	B-Disease
type	NN	O	O
.	NN	O	O

Another	NN	O	O
nephew	NN	O	O
(	NN	O	O
patient	NN	O	O
4	NN	O	O
)	NN	O	O
of	NN	O	O
patient	NN	O	O
1	NN	O	O
was	NN	O	O
classified	NN	O	O
as	NN	O	O
having	NN	O	O
an	NN	O	O
adolescent	NN	O	O
form	NN	O	O
.	NN	O	O

The	NN	O	O
tau	NN	O	O
level	NN	O	O
in	NN	O	O
the	NN	O	O
cerebrospinal	NN	O	O
fluid	NN	O	O
(	NN	O	O
CSF	NN	O	O
)	NN	O	O
in	NN	O	O
patient	NN	O	O
1	NN	O	O
was	NN	O	O
as	NN	O	O
high	NN	O	O
as	NN	O	O
that	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
Alzheimers	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
AD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

His	NN	O	O
brain	NN	O	O
magnetic	NN	O	O
resonance	NN	O	O
image	NN	O	O
(	NN	O	O
MRI	NN	O	O
)	NN	O	O
showed	NN	O	O
abnormalities	NN	O	B-Disease
in	NN	O	I-Disease
the	NN	O	I-Disease
bilateral	NN	O	I-Disease
cerebellar	NN	O	I-Disease
hemispheres	NN	O	I-Disease
and	NN	O	O
brain	NN	O	O
stem	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
in	NN	O	O
the	NN	O	O
cerebral	NN	O	O
white	NN	O	O
matter	NN	O	O
,	NN	O	O
where	NN	O	O
marked	NN	O	O
reductions	NN	O	O
of	NN	O	O
the	NN	O	O
cerebral	NN	O	O
blood	NN	O	O
flow	NN	O	O
and	NN	O	O
oxygen	NN	O	O
metabolism	NN	O	O
were	NN	O	O
clearly	NN	O	O
demonstrated	NN	O	O
by	NN	O	O
positron	NN	O	O
emission	NN	O	O
tomography	NN	O	O
(	NN	O	O
PET	NN	O	O
)	NN	O	O
.	NN	O	O

In	NN	O	O
patients	NN	O	O
2	NN	O	O
and	NN	O	O
3	NN	O	O
,	NN	O	O
the	NN	O	O
autopsy	NN	O	O
findings	NN	O	O
showed	NN	O	O
massive	NN	O	O
demyelination	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
cerebral	NN	O	I-Disease
white	NN	O	I-Disease
matter	NN	O	I-Disease
with	NN	O	O
sparing	NN	O	O
of	NN	O	O
the	NN	O	O
U	NN	O	O
-	NN	O	O
fibers	NN	O	O
,	NN	O	O
compatible	NN	O	O
with	NN	O	O
the	NN	O	O
findings	NN	O	O
of	NN	O	O
childhood	NN	O	O
ALD	NN	O	B-Disease
.	NN	O	O

Oleic	NN	O	O
and	NN	O	O
erucic	NN	O	O
acids	NN	O	O
(	NN	O	O
Lorenzos	NN	O	O
Oil	NN	O	O
)	NN	O	O
were	NN	O	O
administered	NN	O	O
to	NN	O	O
patients	NN	O	O
1	NN	O	O
and	NN	O	O
4	NN	O	O
,	NN	O	O
but	NN	O	O
sufficient	NN	O	O
effectiveness	NN	O	O
was	NN	O	O
not	NN	O	O
obtained	NN	O	O
.	NN	O	O

The	NN	O	O
findings	NN	O	O
in	NN	O	O
this	NN	O	O
family	NN	O	O
suggest	NN	O	O
that	NN	O	O
delGAG291	NN	O	O
is	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
cause	NN	O	O
of	NN	O	O
Japanese	NN	O	O
ALD	NN	O	B-Disease
with	NN	O	O
phenotypic	NN	O	O
variations	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
although	NN	O	O
the	NN	O	O
scale	NN	O	O
of	NN	O	O
the	NN	O	O
study	NN	O	O
is	NN	O	O
limited	NN	O	O
,	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
possibility	NN	O	O
that	NN	O	O
PET	NN	O	O
can	NN	O	O
detect	NN	O	O
an	NN	O	O
insidious	NN	O	B-Disease
lesion	NN	O	I-Disease
which	NN	O	O
is	NN	O	O
undetectable	NN	O	O
by	NN	O	O
computed	NN	O	O
tomogram	NN	O	O
(	NN	O	O
CT	NN	O	O
)	NN	O	O
or	NN	O	O
MRI	NN	O	O
analysis	NN	O	O
,	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
higher	NN	O	O
level	NN	O	O
of	NN	O	O
tau	NN	O	O
reflects	NN	O	O
the	NN	O	O
process	NN	O	O
of	NN	O	O
neuronal	NN	O	B-Disease
degeneration	NN	O	I-Disease
in	NN	O	O
ALD	NN	O	B-Disease
.	NN	O	O

Lorenzos	NN	O	O
Oil	NN	O	O
should	NN	O	O
be	NN	O	O
given	NN	O	O
in	NN	O	O
the	NN	O	O
early	NN	O	O
stage	NN	O	O
.	NN	O	O
.	NN	O	O

Nonsense	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
4	NN	O	O
of	NN	O	O
human	NN	O	O
complement	NN	O	O
C9	NN	O	O
gene	NN	O	O
is	NN	O	O
the	NN	O	O
major	NN	O	O
cause	NN	O	O
of	NN	O	O
Japanese	NN	O	O
complement	NN	O	B-Disease
C9	NN	O	I-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

Deficiency	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
ninth	NN	O	I-Disease
component	NN	O	I-Disease
of	NN	O	I-Disease
human	NN	O	I-Disease
complement	NN	O	I-Disease
(	NN	O	O
C9	NN	O	O
)	NN	O	O
is	NN	O	O
the	NN	O	O
most	NN	O	O
common	NN	O	O
complement	NN	O	B-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
Japan	NN	O	O
but	NN	O	O
is	NN	O	O
rare	NN	O	O
in	NN	O	O
other	NN	O	O
countries	NN	O	O
.	NN	O	O

We	NN	O	O
studied	NN	O	O
the	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
four	NN	O	O
Japanese	NN	O	O
C9	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
patients	NN	O	O
who	NN	O	O
had	NN	O	O
suffered	NN	O	O
from	NN	O	O
meningococcal	NN	O	B-Disease
meningitis	NN	O	I-Disease
.	NN	O	O

Direct	NN	O	O
sequencing	NN	O	O
of	NN	O	O
amplified	NN	O	O
C9	NN	O	O
cDNA	NN	O	O
and	NN	O	O
DNA	NN	O	O
revealed	NN	O	O
a	NN	O	O
nonsense	NN	O	O
substitution	NN	O	O
(	NN	O	O
CGA	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
TGA	NN	O	O
)	NN	O	O
at	NN	O	O
codon	NN	O	O
95	NN	O	O
in	NN	O	O
exon	NN	O	O
4	NN	O	O
in	NN	O	O
the	NN	O	O
four	NN	O	O
C9	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
individuals	NN	O	O
.	NN	O	O

An	NN	O	O
allele	NN	O	O
-	NN	O	O
specific	NN	O	O
polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
system	NN	O	O
designed	NN	O	O
to	NN	O	O
detect	NN	O	O
exclusively	NN	O	O
only	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
and	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
indicated	NN	O	O
that	NN	O	O
all	NN	O	O
the	NN	O	O
four	NN	O	O
patients	NN	O	O
were	NN	O	O
homozygous	NN	O	O
for	NN	O	O
the	NN	O	O
mutation	NN	O	O
in	NN	O	O
exon	NN	O	O
4	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
parents	NN	O	O
of	NN	O	O
patient	NN	O	O
2	NN	O	O
were	NN	O	O
heterozygous	NN	O	O
.	NN	O	O

The	NN	O	O
common	NN	O	O
mutation	NN	O	O
at	NN	O	O
codon	NN	O	O
95	NN	O	O
in	NN	O	O
exon	NN	O	O
4	NN	O	O
might	NN	O	O
be	NN	O	O
responsible	NN	O	O
for	NN	O	O
most	NN	O	O
Japanese	NN	O	O
C9	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
required	NN	O	O
for	NN	O	O
transcription	NN	O	O
-	NN	O	O
coupled	NN	O	O
repair	NN	O	O
of	NN	O	O
oxidative	NN	O	O
DNA	NN	O	O
damage	NN	O	O
.	NN	O	O

The	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
susceptibility	NN	O	O
gene	NN	O	O
BRCA1	NN	O	O
encodes	NN	O	O
a	NN	O	O
zinc	NN	O	O
finger	NN	O	O
protein	NN	O	O
of	NN	O	O
unknown	NN	O	O
function	NN	O	O
.	NN	O	O

Association	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
protein	NN	O	O
with	NN	O	O
the	NN	O	O
DNA	NN	O	O
repair	NN	O	O
protein	NN	O	O
Rad51	NN	O	O
and	NN	O	O
changes	NN	O	O
in	NN	O	O
the	NN	O	O
phosphorylation	NN	O	O
and	NN	O	O
cellular	NN	O	O
localization	NN	O	O
of	NN	O	O
the	NN	O	O
protein	NN	O	O
after	NN	O	O
exposure	NN	O	O
to	NN	O	O
DNA	NN	O	O
-	NN	O	O
damaging	NN	O	O
agents	NN	O	O
are	NN	O	O
consistent	NN	O	O
with	NN	O	O
a	NN	O	O
role	NN	O	O
for	NN	O	O
BRCA1	NN	O	O
in	NN	O	O
DNA	NN	O	O
repair	NN	O	O
.	NN	O	O

Here	NN	O	O
,	NN	O	O
it	NN	O	O
is	NN	O	O
shown	NN	O	O
that	NN	O	O
mouse	NN	O	O
embryonic	NN	O	O
stem	NN	O	O
cells	NN	O	O
deficient	NN	O	B-Disease
in	NN	O	I-Disease
BRCA1	NN	O	I-Disease
are	NN	O	O
defective	NN	O	O
in	NN	O	O
the	NN	O	O
ability	NN	O	O
to	NN	O	O
carry	NN	O	O
out	NN	O	O
transcription	NN	O	O
-	NN	O	O
coupled	NN	O	O
repair	NN	O	O
of	NN	O	O
oxidative	NN	O	O
DNA	NN	O	O
damage	NN	O	O
,	NN	O	O
and	NN	O	O
are	NN	O	O
hypersensitive	NN	O	O
to	NN	O	O
ionizing	NN	O	O
radiation	NN	O	O
and	NN	O	O
hydrogen	NN	O	O
peroxide	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
suggest	NN	O	O
that	NN	O	O
BRCA1	NN	O	O
participates	NN	O	O
,	NN	O	O
directly	NN	O	O
or	NN	O	O
indirectly	NN	O	O
,	NN	O	O
in	NN	O	O
transcription	NN	O	O
-	NN	O	O
coupled	NN	O	O
repair	NN	O	O
of	NN	O	O
oxidative	NN	O	O
DNA	NN	O	O
damage	NN	O	O
.	NN	O	O
.	NN	O	O

Truncation	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
transactivation	NN	O	O
region	NN	O	O
of	NN	O	O
PAX6	NN	O	O
result	NN	O	O
in	NN	O	O
dominant	NN	O	O
-	NN	O	O
negative	NN	O	O
mutants	NN	O	O
.	NN	O	O

PAX6	NN	O	O
is	NN	O	O
a	NN	O	O
transcription	NN	O	O
factor	NN	O	O
with	NN	O	O
two	NN	O	O
DNA	NN	O	O
-	NN	O	O
binding	NN	O	O
domains	NN	O	O
(	NN	O	O
paired	NN	O	O
box	NN	O	O
and	NN	O	O
homeobox	NN	O	O
)	NN	O	O
and	NN	O	O
a	NN	O	O
proline	NN	O	O
-	NN	O	O
serine	NN	O	O
-	NN	O	O
threonine	NN	O	O
(	NN	O	O
PST	NN	O	O
)	NN	O	O
-	NN	O	O
rich	NN	O	O
transactivation	NN	O	O
domain	NN	O	O
.	NN	O	O

PAX6	NN	O	O
regulates	NN	O	O
eye	NN	O	O
development	NN	O	O
in	NN	O	O
animals	NN	O	O
ranging	NN	O	O
from	NN	O	O
jellyfish	NN	O	O
to	NN	O	O
Drosophila	NN	O	O
to	NN	O	O
humans	NN	O	O
.	NN	O	O

Heterozygous	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
human	NN	O	O
PAX6	NN	O	O
gene	NN	O	O
result	NN	O	O
in	NN	O	O
various	NN	O	O
phenotypes	NN	O	O
,	NN	O	O
including	NN	O	O
aniridia	NN	O	B-Disease
,	NN	O	O
Peters	NN	O	B-Disease
anomaly	NN	O	I-Disease
,	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
keratitis	NN	O	I-Disease
,	NN	O	O
and	NN	O	O
familial	NN	O	B-Disease
foveal	NN	O	I-Disease
dysplasia	NN	O	I-Disease
.	NN	O	O

It	NN	O	O
is	NN	O	O
believed	NN	O	O
that	NN	O	O
the	NN	O	O
mutated	NN	O	O
allele	NN	O	O
of	NN	O	O
PAX6	NN	O	O
produces	NN	O	O
an	NN	O	O
inactive	NN	O	O
protein	NN	O	O
and	NN	O	O
aniridia	NN	O	B-Disease
is	NN	O	O
caused	NN	O	O
due	NN	O	O
to	NN	O	O
genetic	NN	O	O
haploinsufficiency	NN	O	O
.	NN	O	O

However	NN	O	O
,	NN	O	O
several	NN	O	O
truncation	NN	O	O
mutations	NN	O	O
have	NN	O	O
been	NN	O	O
found	NN	O	O
to	NN	O	O
occur	NN	O	O
in	NN	O	O
the	NN	O	O
C	NN	O	O
-	NN	O	O
terminal	NN	O	O
half	NN	O	O
of	NN	O	O
PAX6	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
Aniridia	NN	O	B-Disease
resulting	NN	O	O
in	NN	O	O
mutant	NN	O	O
proteins	NN	O	O
that	NN	O	O
retain	NN	O	O
the	NN	O	O
DNA	NN	O	O
-	NN	O	O
binding	NN	O	O
domains	NN	O	O
but	NN	O	O
have	NN	O	O
lost	NN	O	O
most	NN	O	O
of	NN	O	O
the	NN	O	O
transactivation	NN	O	O
domain	NN	O	O
.	NN	O	O

It	NN	O	O
is	NN	O	O
not	NN	O	O
clear	NN	O	O
whether	NN	O	O
such	NN	O	O
mutants	NN	O	O
really	NN	O	O
behave	NN	O	O
as	NN	O	O
loss	NN	O	O
-	NN	O	O
of	NN	O	O
-	NN	O	O
function	NN	O	O
mutants	NN	O	O
as	NN	O	O
predicted	NN	O	O
by	NN	O	O
haploinsufficiency	NN	O	O
.	NN	O	O

Contrary	NN	O	O
to	NN	O	O
this	NN	O	O
theory	NN	O	O
,	NN	O	O
our	NN	O	O
data	NN	O	O
showed	NN	O	O
that	NN	O	O
these	NN	O	O
mutants	NN	O	O
are	NN	O	O
dominant	NN	O	O
-	NN	O	O
negative	NN	O	O
in	NN	O	O
transient	NN	O	O
transfection	NN	O	O
assays	NN	O	O
when	NN	O	O
they	NN	O	O
are	NN	O	O
coexpressed	NN	O	O
with	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
PAX6	NN	O	O
.	NN	O	O

We	NN	O	O
found	NN	O	O
that	NN	O	O
the	NN	O	O
dominant	NN	O	O
-	NN	O	O
negative	NN	O	O
effects	NN	O	O
result	NN	O	O
from	NN	O	O
the	NN	O	O
enhanced	NN	O	O
DNA	NN	O	O
binding	NN	O	O
ability	NN	O	O
of	NN	O	O
these	NN	O	O
mutants	NN	O	O
.	NN	O	O

Kinetic	NN	O	O
studies	NN	O	O
of	NN	O	O
binding	NN	O	O
and	NN	O	O
dissociation	NN	O	O
revealed	NN	O	O
that	NN	O	O
various	NN	O	O
truncation	NN	O	O
mutants	NN	O	O
have	NN	O	O
3	NN	O	O
-	NN	O	O
5	NN	O	O
-	NN	O	O
fold	NN	O	O
higher	NN	O	O
affinity	NN	O	O
to	NN	O	O
various	NN	O	O
DNA	NN	O	O
-	NN	O	O
binding	NN	O	O
sites	NN	O	O
when	NN	O	O
compared	NN	O	O
with	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
PAX6	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
provide	NN	O	O
a	NN	O	O
new	NN	O	O
insight	NN	O	O
into	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
mutant	NN	O	O
PAX6	NN	O	O
in	NN	O	O
causing	NN	O	O
aniridia	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Reversal	NN	O	O
of	NN	O	O
severe	NN	O	O
hypertrophic	NN	O	B-Disease
cardiomyopathy	NN	O	I-Disease
and	NN	O	O
excellent	NN	O	O
neuropsychologic	NN	O	O
outcome	NN	O	O
in	NN	O	O
very	NN	O	B-Disease
-	NN	O	I-Disease
long	NN	O	I-Disease
-	NN	O	I-Disease
chain	NN	O	I-Disease
acyl	NN	O	I-Disease
-	NN	O	I-Disease
coenzyme	NN	O	I-Disease
A	NN	O	I-Disease
dehydrogenase	NN	O	I-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

Very	NN	O	B-Disease
-	NN	O	I-Disease
long	NN	O	I-Disease
-	NN	O	I-Disease
chain	NN	O	I-Disease
acyl	NN	O	I-Disease
-	NN	O	I-Disease
coenzyme	NN	O	I-Disease
A	NN	O	I-Disease
dehydrogenase	NN	O	I-Disease
(	NN	O	I-Disease
VLCAD	NN	O	I-Disease
)	NN	O	I-Disease
deficiency	NN	O	I-Disease
is	NN	O	O
a	NN	O	O
disorder	NN	O	O
of	NN	O	O
fatty	NN	O	O
acid	NN	O	O
beta	NN	O	O
oxidation	NN	O	O
that	NN	O	O
reportedly	NN	O	O
has	NN	O	O
high	NN	O	O
rates	NN	O	O
of	NN	O	O
morbidity	NN	O	O
and	NN	O	O
mortality	NN	O	O
.	NN	O	O

We	NN	O	O
describe	NN	O	O
the	NN	O	O
outcome	NN	O	O
of	NN	O	O
a	NN	O	O
5	NN	O	O
-	NN	O	O
year	NN	O	O
-	NN	O	O
old	NN	O	O
girl	NN	O	O
with	NN	O	O
VLCAD	NN	O	B-Disease
deficiency	NN	O	I-Disease
who	NN	O	O
was	NN	O	O
first	NN	O	O
seen	NN	O	O
at	NN	O	O
5	NN	O	O
months	NN	O	O
of	NN	O	O
age	NN	O	O
with	NN	O	O
severe	NN	O	O
hypertrophic	NN	O	B-Disease
cardiomyopathy	NN	O	I-Disease
,	NN	O	O
hepatomegaly	NN	O	B-Disease
,	NN	O	O
encephalopathy	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
hypotonia	NN	O	B-Disease
.	NN	O	O

Biochemical	NN	O	O
studies	NN	O	O
indicated	NN	O	O
VLCAD	NN	O	B-Disease
deficiency	NN	O	I-Disease
caused	NN	O	O
by	NN	O	O
a	NN	O	O
stable	NN	O	O
yet	NN	O	O
inactive	NN	O	O
enzyme	NN	O	O
.	NN	O	O

Molecular	NN	O	O
genetic	NN	O	O
analysis	NN	O	O
of	NN	O	O
her	NN	O	O
VLCAD	NN	O	O
gene	NN	O	O
revealed	NN	O	O
a	NN	O	O
T1372C	NN	O	O
(	NN	O	O
F458L	NN	O	O
)	NN	O	O
missense	NN	O	O
mutation	NN	O	O
and	NN	O	O
a	NN	O	O
1668	NN	O	O
ACAG	NN	O	O
1669	NN	O	O
splice	NN	O	O
site	NN	O	O
mutation	NN	O	O
.	NN	O	O

After	NN	O	O
initial	NN	O	O
treatment	NN	O	O
with	NN	O	O
intravenous	NN	O	O
glucose	NN	O	O
and	NN	O	O
carnitine	NN	O	O
,	NN	O	O
the	NN	O	O
patient	NN	O	O
has	NN	O	O
thrived	NN	O	O
on	NN	O	O
a	NN	O	O
low	NN	O	O
-	NN	O	O
fat	NN	O	O
diet	NN	O	O
supplemented	NN	O	O
with	NN	O	O
medium	NN	O	O
-	NN	O	O
chain	NN	O	O
triglyceride	NN	O	O
oil	NN	O	O
and	NN	O	O
carnitine	NN	O	O
and	NN	O	O
avoidance	NN	O	O
of	NN	O	O
fasting	NN	O	O
.	NN	O	O

Her	NN	O	O
ventricular	NN	O	O
hypertrophy	NN	O	O
resolved	NN	O	O
significantly	NN	O	O
over	NN	O	O
1	NN	O	O
year	NN	O	O
,	NN	O	O
and	NN	O	O
cognitively	NN	O	O
,	NN	O	O
she	NN	O	O
is	NN	O	O
in	NN	O	O
the	NN	O	O
superior	NN	O	O
range	NN	O	O
for	NN	O	O
age	NN	O	O
.	NN	O	O

Clinical	NN	O	O
recognition	NN	O	O
of	NN	O	O
VLCAD	NN	O	B-Disease
deficiency	NN	O	I-Disease
is	NN	O	O
important	NN	O	O
because	NN	O	O
it	NN	O	O
is	NN	O	O
one	NN	O	O
of	NN	O	O
the	NN	O	O
few	NN	O	O
directly	NN	O	O
treatable	NN	O	O
causes	NN	O	O
of	NN	O	O
cardiomyopathy	NN	O	B-Disease
in	NN	O	O
children	NN	O	O
.	NN	O	O
.	NN	O	O

Cloning	NN	O	O
of	NN	O	O
a	NN	O	O
novel	NN	O	O
member	NN	O	O
of	NN	O	O
the	NN	O	O
low	NN	O	O
-	NN	O	O
density	NN	O	O
lipoprotein	NN	O	O
receptor	NN	O	O
family	NN	O	O
.	NN	O	O

A	NN	O	O
gene	NN	O	O
encoding	NN	O	O
a	NN	O	O
novel	NN	O	O
transmembrane	NN	O	O
protein	NN	O	O
was	NN	O	O
identified	NN	O	O
by	NN	O	O
DNA	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
within	NN	O	O
the	NN	O	O
insulin	NN	O	B-Disease
-	NN	O	I-Disease
dependent	NN	O	I-Disease
diabetes	NN	O	I-Disease
mellitus	NN	O	I-Disease
(	NN	O	O
IDDM	NN	O	B-Disease
)	NN	O	O
locus	NN	O	O
IDDM4	NN	O	O
on	NN	O	O
chromosome	NN	O	O
11q13	NN	O	O
.	NN	O	O

Based	NN	O	O
on	NN	O	O
its	NN	O	O
chromosomal	NN	O	O
position	NN	O	O
,	NN	O	O
this	NN	O	O
gene	NN	O	O
is	NN	O	O
a	NN	O	O
candidate	NN	O	O
for	NN	O	O
conferring	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
diabetes	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
gene	NN	O	O
,	NN	O	O
termed	NN	O	O
low	NN	O	O
-	NN	O	O
density	NN	O	O
lipoprotein	NN	O	O
receptor	NN	O	O
related	NN	O	O
protein	NN	O	O
5	NN	O	O
(	NN	O	O
LRP5	NN	O	O
)	NN	O	O
,	NN	O	O
encodes	NN	O	O
a	NN	O	O
protein	NN	O	O
of	NN	O	O
1615	NN	O	O
amino	NN	O	O
acids	NN	O	O
that	NN	O	O
contains	NN	O	O
conserved	NN	O	O
modules	NN	O	O
which	NN	O	O
are	NN	O	O
characteristic	NN	O	O
of	NN	O	O
the	NN	O	O
low	NN	O	O
-	NN	O	O
density	NN	O	O
lipoprotein	NN	O	O
(	NN	O	O
LDL	NN	O	O
)	NN	O	O
receptor	NN	O	O
family	NN	O	O
.	NN	O	O

These	NN	O	O
modules	NN	O	O
include	NN	O	O
a	NN	O	O
putative	NN	O	O
signal	NN	O	O
peptide	NN	O	O
for	NN	O	O
protein	NN	O	O
export	NN	O	O
,	NN	O	O
four	NN	O	O
epidermal	NN	O	O
growth	NN	O	O
factor	NN	O	O
(	NN	O	O
EGF	NN	O	O
)	NN	O	O
repeats	NN	O	O
with	NN	O	O
associated	NN	O	O
spacer	NN	O	O
domains	NN	O	O
,	NN	O	O
three	NN	O	O
LDL	NN	O	O
-	NN	O	O
receptor	NN	O	O
(	NN	O	O
LDLR	NN	O	O
)	NN	O	O
repeats	NN	O	O
,	NN	O	O
a	NN	O	O
single	NN	O	O
transmembrane	NN	O	O
spanning	NN	O	O
domain	NN	O	O
,	NN	O	O
and	NN	O	O
a	NN	O	O
cytoplasmic	NN	O	O
domain	NN	O	O
.	NN	O	O

The	NN	O	O
encoded	NN	O	O
protein	NN	O	O
has	NN	O	O
a	NN	O	O
unique	NN	O	O
organization	NN	O	O
of	NN	O	O
EGF	NN	O	O
and	NN	O	O
LDLR	NN	O	O
repeats	NN	O	O
;	NN	O	O
therefore	NN	O	O
,	NN	O	O
LRP5	NN	O	O
likely	NN	O	O
represents	NN	O	O
a	NN	O	O
new	NN	O	O
category	NN	O	O
of	NN	O	O
the	NN	O	O
LDLR	NN	O	O
family	NN	O	O
.	NN	O	O

Both	NN	O	O
human	NN	O	O
and	NN	O	O
mouse	NN	O	O
LRP5	NN	O	O
cDNAs	NN	O	O
have	NN	O	O
been	NN	O	O
isolated	NN	O	O
and	NN	O	O
the	NN	O	O
encoded	NN	O	O
mature	NN	O	O
proteins	NN	O	O
are	NN	O	O
95	NN	O	O
%	NN	O	O
identical	NN	O	O
,	NN	O	O
indicating	NN	O	O
a	NN	O	O
high	NN	O	O
degree	NN	O	O
of	NN	O	O
evolutionary	NN	O	O
conservation	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
APC	NN	O	B-Disease
variants	NN	O	O
I1307K	NN	O	O
and	NN	O	O
E1317Q	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
colorectal	NN	O	B-Disease
tumors	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
not	NN	O	O
always	NN	O	O
with	NN	O	O
a	NN	O	O
family	NN	O	O
history	NN	O	O
.	NN	O	O

Classical	NN	O	O
familial	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
(	NN	O	O
FAP	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
a	NN	O	O
high	NN	O	O
-	NN	O	O
penetrance	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disease	NN	O	I-Disease
that	NN	O	O
predisposes	NN	O	O
to	NN	O	O
hundreds	NN	O	O
or	NN	O	O
thousands	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
adenomas	NN	O	I-Disease
and	NN	O	I-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
that	NN	O	O
results	NN	O	O
from	NN	O	O
truncating	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

A	NN	O	O
variant	NN	O	O
of	NN	O	O
FAP	NN	O	B-Disease
is	NN	O	O
attenuated	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
,	NN	O	O
which	NN	O	O
results	NN	O	O
from	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
5	NN	O	O
and	NN	O	O
3	NN	O	O
regions	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

Attenuated	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
patients	NN	O	O
have	NN	O	O
"	NN	O	O
multiple	NN	O	O
"	NN	O	O
colorectal	NN	O	B-Disease
adenomas	NN	O	I-Disease
(	NN	O	O
typically	NN	O	O
fewer	NN	O	O
than	NN	O	O
100	NN	O	O
)	NN	O	O
without	NN	O	O
the	NN	O	O
florid	NN	O	O
phenotype	NN	O	O
of	NN	O	O
classical	NN	O	O
FAP	NN	O	B-Disease
.	NN	O	O

Another	NN	O	O
group	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
multiple	NN	O	O
adenomas	NN	O	B-Disease
has	NN	O	O
no	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
,	NN	O	O
and	NN	O	O
their	NN	O	O
phenotype	NN	O	O
probably	NN	O	O
results	NN	O	O
from	NN	O	O
variation	NN	O	O
at	NN	O	O
a	NN	O	O
locus	NN	O	O
,	NN	O	O
or	NN	O	O
loci	NN	O	O
,	NN	O	O
elsewhere	NN	O	O
in	NN	O	O
the	NN	O	O
genome	NN	O	O
.	NN	O	O

Recently	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
a	NN	O	O
missense	NN	O	O
variant	NN	O	O
of	NN	O	O
APC	NN	O	B-Disease
(	NN	O	O
I1307K	NN	O	O
)	NN	O	O
was	NN	O	O
described	NN	O	O
that	NN	O	O
confers	NN	O	O
an	NN	O	O
increased	NN	O	O
risk	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
tumors	NN	O	I-Disease
,	NN	O	O
including	NN	O	O
multiple	NN	O	O
adenomas	NN	O	B-Disease
,	NN	O	O
in	NN	O	O
Ashkenazim	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
studied	NN	O	O
a	NN	O	O
set	NN	O	O
of	NN	O	O
164	NN	O	O
patients	NN	O	O
with	NN	O	O
multiple	NN	O	O
colorectal	NN	O	B-Disease
adenomas	NN	O	I-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
carcinoma	NN	O	I-Disease
and	NN	O	O
analyzed	NN	O	O
codons	NN	O	O
1263	NN	O	O
-	NN	O	O
1377	NN	O	O
(	NN	O	O
exon	NN	O	O
15G	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
for	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
variants	NN	O	O
.	NN	O	O

Three	NN	O	O
patients	NN	O	O
with	NN	O	O
the	NN	O	O
I1307K	NN	O	O
allele	NN	O	O
were	NN	O	O
detected	NN	O	O
,	NN	O	O
each	NN	O	O
of	NN	O	O
Ashkenazi	NN	O	O
descent	NN	O	O
.	NN	O	O

Four	NN	O	O
patients	NN	O	O
had	NN	O	O
a	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
E1317Q	NN	O	O
missense	NN	O	O
variant	NN	O	O
of	NN	O	O
APC	NN	O	O
that	NN	O	O
was	NN	O	O
not	NN	O	O
present	NN	O	O
in	NN	O	O
controls	NN	O	O
;	NN	O	O
one	NN	O	O
of	NN	O	O
these	NN	O	O
individuals	NN	O	O
had	NN	O	O
an	NN	O	O
unusually	NN	O	O
large	NN	O	O
number	NN	O	O
of	NN	O	O
metaplastic	NN	O	B-Disease
polyps	NN	O	I-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
colorectum	NN	O	I-Disease
.	NN	O	O

There	NN	O	O
is	NN	O	O
increasing	NN	O	O
evidence	NN	O	O
that	NN	O	O
there	NN	O	O
exist	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
variants	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
that	NN	O	O
predispose	NN	O	O
to	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
multiple	NN	O	O
colorectal	NN	O	B-Disease
adenomas	NN	O	I-Disease
and	NN	O	I-Disease
carcinoma	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
without	NN	O	O
the	NN	O	O
florid	NN	O	O
phenotype	NN	O	O
of	NN	O	O
classical	NN	O	O
FAP	NN	O	B-Disease
,	NN	O	O
and	NN	O	O
possibly	NN	O	O
with	NN	O	O
importance	NN	O	O
for	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
risk	NN	O	O
in	NN	O	O
the	NN	O	O
general	NN	O	O
population	NN	O	O
.	NN	O	O
.	NN	O	O

Genomic	NN	O	O
structure	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
congenital	NN	O	B-Disease
chloride	NN	O	I-Disease
diarrhea	NN	O	I-Disease
(	NN	O	O
CLD	NN	O	B-Disease
)	NN	O	O
gene	NN	O	O
.	NN	O	O

Congenital	NN	O	B-Disease
chloride	NN	O	I-Disease
diarrhea	NN	O	I-Disease
(	NN	O	O
CLD	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
mutations	NN	O	O
in	NN	O	O
a	NN	O	O
gene	NN	O	O
which	NN	O	O
encodes	NN	O	O
an	NN	O	O
intestinal	NN	O	O
anion	NN	O	O
transporter	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
here	NN	O	O
the	NN	O	O
complete	NN	O	O
genomic	NN	O	O
organization	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
CLD	NN	O	B-Disease
gene	NN	O	O
which	NN	O	O
spans	NN	O	O
approximately	NN	O	O
39kb	NN	O	O
,	NN	O	O
and	NN	O	O
comprises	NN	O	O
21	NN	O	O
exons	NN	O	O
.	NN	O	O

All	NN	O	O
exon	NN	O	O
/	NN	O	O
intron	NN	O	O
boundaries	NN	O	O
conform	NN	O	O
to	NN	O	O
the	NN	O	O
GT	NN	O	O
/	NN	O	O
AG	NN	O	O
rule	NN	O	O
.	NN	O	O

An	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
putative	NN	O	O
promoter	NN	O	O
region	NN	O	O
sequence	NN	O	O
shows	NN	O	O
a	NN	O	O
putative	NN	O	O
TATA	NN	O	O
box	NN	O	O
and	NN	O	O
predicts	NN	O	O
multiple	NN	O	O
transcription	NN	O	O
factor	NN	O	O
binding	NN	O	O
sites	NN	O	O
.	NN	O	O

The	NN	O	O
genomic	NN	O	O
structure	NN	O	O
was	NN	O	O
determined	NN	O	O
using	NN	O	O
DNA	NN	O	O
from	NN	O	O
several	NN	O	O
sources	NN	O	O
including	NN	O	O
multiple	NN	O	O
large	NN	O	O
-	NN	O	O
insert	NN	O	O
libaries	NN	O	O
and	NN	O	O
genomic	NN	O	O
DNA	NN	O	O
from	NN	O	O
Finnish	NN	O	O
CLD	NN	O	B-Disease
patients	NN	O	O
and	NN	O	O
controls	NN	O	O
.	NN	O	O

Exon	NN	O	O
-	NN	O	O
specific	NN	O	O
primers	NN	O	O
developed	NN	O	O
in	NN	O	O
this	NN	O	O
study	NN	O	O
will	NN	O	O
facilitate	NN	O	O
mutation	NN	O	O
screening	NN	O	O
studies	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O

Genomic	NN	O	O
sequencing	NN	O	O
of	NN	O	O
a	NN	O	O
BAC	NN	O	O
clone	NN	O	O
H	NN	O	O
_	NN	O	O
RG364P16	NN	O	O
revealed	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
another	NN	O	O
,	NN	O	O
highly	NN	O	O
homologous	NN	O	O
gene	NN	O	O
3	NN	O	O
of	NN	O	O
the	NN	O	O
CLD	NN	O	B-Disease
gene	NN	O	O
,	NN	O	O
with	NN	O	O
a	NN	O	O
similar	NN	O	O
genomic	NN	O	O
structure	NN	O	O
,	NN	O	O
recently	NN	O	O
identified	NN	O	O
as	NN	O	O
the	NN	O	O
Pendred	NN	O	B-Disease
syndrome	NN	O	I-Disease
gene	NN	O	O
(	NN	O	O
PDS	NN	O	B-Disease
)	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
APCI1307K	NN	O	O
allele	NN	O	O
and	NN	O	O
cancer	NN	O	B-Disease
risk	NN	O	O
in	NN	O	O
a	NN	O	O
community	NN	O	O
-	NN	O	O
based	NN	O	O
study	NN	O	O
of	NN	O	O
Ashkenazi	NN	O	O
Jews	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
APC	NN	O	O
are	NN	O	O
classically	NN	O	O
associated	NN	O	O
with	NN	O	O
familial	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
(	NN	O	O
FAP	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
a	NN	O	O
highly	NN	O	O
penetrant	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disorder	NN	O	I-Disease
characterized	NN	O	O
by	NN	O	O
multiple	NN	O	O
intestinal	NN	O	O
polyps	NN	O	B-Disease
and	NN	O	O
,	NN	O	O
without	NN	O	O
surgical	NN	O	O
intervention	NN	O	O
,	NN	O	O
the	NN	O	O
development	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
(	NN	O	O
CRC	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

APC	NN	O	B-Disease
is	NN	O	O
a	NN	O	O
tumour	NN	O	O
-	NN	O	O
suppressor	NN	O	O
gene	NN	O	O
,	NN	O	O
and	NN	O	O
somatic	NN	O	O
loss	NN	O	O
occurs	NN	O	O
in	NN	O	O
tumours	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
germline	NN	O	O
T	NN	O	O
-	NN	O	O
to	NN	O	O
-	NN	O	O
A	NN	O	O
transversion	NN	O	O
responsible	NN	O	O
for	NN	O	O
the	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
allele	NN	O	O
converts	NN	O	O
the	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
sequence	NN	O	O
to	NN	O	O
a	NN	O	O
homopolymer	NN	O	O
tract	NN	O	O
(	NN	O	O
A8	NN	O	O
)	NN	O	O
that	NN	O	O
is	NN	O	O
genetically	NN	O	O
unstable	NN	O	O
and	NN	O	O
prone	NN	O	O
to	NN	O	O
somatic	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
I1307K	NN	O	O
allele	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
6	NN	O	O
.	NN	O	O

1	NN	O	O
%	NN	O	O
of	NN	O	O
unselected	NN	O	O
Ashkenazi	NN	O	O
Jews	NN	O	O
and	NN	O	O
higher	NN	O	O
proportions	NN	O	O
of	NN	O	O
Ashkenazim	NN	O	O
with	NN	O	O
family	NN	O	O
or	NN	O	O
personal	NN	O	O
histories	NN	O	O
of	NN	O	O
CRC	NN	O	B-Disease
(	NN	O	O
ref	NN	O	O
.	NN	O	O
2	NN	O	O
)	NN	O	O
.	NN	O	O

To	NN	O	O
evaluate	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
I1307K	NN	O	O
in	NN	O	O
cancer	NN	O	B-Disease
,	NN	O	O
we	NN	O	O
genotyped	NN	O	O
5	NN	O	O
,	NN	O	O
081	NN	O	O
Ashkenazi	NN	O	O
volunteers	NN	O	O
in	NN	O	O
a	NN	O	O
community	NN	O	O
survey	NN	O	O
.	NN	O	O

Risk	NN	O	O
of	NN	O	O
developing	NN	O	O
colorectal	NN	O	B-Disease
,	NN	O	I-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
other	NN	O	I-Disease
cancers	NN	O	I-Disease
were	NN	O	O
compared	NN	O	O
between	NN	O	O
genotyped	NN	O	O
I1307K	NN	O	O
carriers	NN	O	O
and	NN	O	O
non	NN	O	O
-	NN	O	O
carriers	NN	O	O
and	NN	O	O
their	NN	O	O
first	NN	O	O
-	NN	O	O
degree	NN	O	O
relatives	NN	O	O
.	NN	O	O

Sperm	NN	O	O
DNA	NN	O	O
analysis	NN	O	O
in	NN	O	O
a	NN	O	O
Friedreich	NN	O	B-Disease
ataxia	NN	O	I-Disease
premutation	NN	O	O
carrier	NN	O	O
suggests	NN	O	O
both	NN	O	O
meiotic	NN	O	O
and	NN	O	O
mitotic	NN	O	O
expansion	NN	O	O
in	NN	O	O
the	NN	O	O
FRDA	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

Friedreich	NN	O	B-Disease
ataxia	NN	O	I-Disease
is	NN	O	O
usually	NN	O	O
caused	NN	O	O
by	NN	O	O
an	NN	O	O
expansion	NN	O	O
of	NN	O	O
a	NN	O	O
GAA	NN	O	O
trinucleotide	NN	O	O
repeat	NN	O	O
in	NN	O	O
intron	NN	O	O
1	NN	O	O
of	NN	O	O
the	NN	O	O
FRDA	NN	O	B-Disease
gene	NN	O	O
.	NN	O	O

Occasionally	NN	O	O
,	NN	O	O
a	NN	O	O
fully	NN	O	O
expanded	NN	O	O
allele	NN	O	O
has	NN	O	O
been	NN	O	O
found	NN	O	O
to	NN	O	O
arise	NN	O	O
from	NN	O	O
a	NN	O	O
premutation	NN	O	O
of	NN	O	O
100	NN	O	O
or	NN	O	O
less	NN	O	O
triplet	NN	O	O
repeats	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
examined	NN	O	O
the	NN	O	O
sperm	NN	O	O
DNA	NN	O	O
of	NN	O	O
a	NN	O	O
premutation	NN	O	O
carrier	NN	O	O
.	NN	O	O

This	NN	O	O
mans	NN	O	O
leucocyte	NN	O	O
DNA	NN	O	O
showed	NN	O	O
one	NN	O	O
normal	NN	O	O
allele	NN	O	O
and	NN	O	O
one	NN	O	O
allele	NN	O	O
of	NN	O	O
approximately	NN	O	O
100	NN	O	O
repeats	NN	O	O
.	NN	O	O

His	NN	O	O
sperm	NN	O	O
showed	NN	O	O
an	NN	O	O
expanded	NN	O	O
allele	NN	O	O
in	NN	O	O
a	NN	O	O
tight	NN	O	O
range	NN	O	O
centering	NN	O	O
on	NN	O	O
a	NN	O	O
size	NN	O	O
of	NN	O	O
approximately	NN	O	O
320	NN	O	O
trinucleotide	NN	O	O
repeats	NN	O	O
.	NN	O	O

His	NN	O	O
affected	NN	O	O
son	NN	O	O
has	NN	O	O
repeat	NN	O	O
sizes	NN	O	O
of	NN	O	O
1040	NN	O	O
and	NN	O	O
540	NN	O	O
.	NN	O	O

These	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
expansion	NN	O	O
occurs	NN	O	O
in	NN	O	O
two	NN	O	O
stages	NN	O	O
,	NN	O	O
the	NN	O	O
first	NN	O	O
during	NN	O	O
meiosis	NN	O	O
followed	NN	O	O
by	NN	O	O
a	NN	O	O
second	NN	O	O
mitotic	NN	O	O
expansion	NN	O	O
.	NN	O	O

We	NN	O	O
also	NN	O	O
show	NN	O	O
that	NN	O	O
in	NN	O	O
all	NN	O	O
informative	NN	O	O
carrier	NN	O	O
father	NN	O	O
to	NN	O	O
affected	NN	O	O
child	NN	O	O
transmissions	NN	O	O
,	NN	O	O
with	NN	O	O
the	NN	O	O
notable	NN	O	O
exception	NN	O	O
of	NN	O	O
the	NN	O	O
premutation	NN	O	O
carrier	NN	O	O
,	NN	O	O
the	NN	O	O
expansion	NN	O	O
size	NN	O	O
decreases	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
R496H	NN	O	O
mutation	NN	O	O
of	NN	O	O
arylsulfatase	NN	O	O
A	NN	O	O
does	NN	O	O
not	NN	O	O
cause	NN	O	O
metachromatic	NN	O	B-Disease
leukodystrophy	NN	O	I-Disease
.	NN	O	O

Deficiency	NN	O	B-Disease
of	NN	O	I-Disease
arylsulfatase	NN	O	I-Disease
A	NN	O	I-Disease
(	NN	O	O
ARSA	NN	O	O
)	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
causes	NN	O	O
metachromatic	NN	O	B-Disease
leukodystrophy	NN	O	I-Disease
(	NN	O	O
MLD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

A	NN	O	O
number	NN	O	O
of	NN	O	O
ARSA	NN	O	O
gene	NN	O	O
mutations	NN	O	O
responsible	NN	O	O
for	NN	O	O
MLD	NN	O	B-Disease
have	NN	O	O
been	NN	O	O
identified	NN	O	O
.	NN	O	O

Recently	NN	O	O
,	NN	O	O
the	NN	O	O
R496H	NN	O	O
mutation	NN	O	O
of	NN	O	O
ARSA	NN	O	O
was	NN	O	O
proposed	NN	O	O
to	NN	O	O
be	NN	O	O
a	NN	O	O
cause	NN	O	O
of	NN	O	O
MLD	NN	O	B-Disease
(	NN	O	O
Draghia	NN	O	O
et	NN	O	O
al	NN	O	O
.	NN	O	O
,	NN	O	O
1997	NN	O	O
)	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
investigated	NN	O	O
the	NN	O	O
R496H	NN	O	O
mutation	NN	O	O
and	NN	O	O
found	NN	O	O
this	NN	O	O
mutation	NN	O	O
at	NN	O	O
a	NN	O	O
relatively	NN	O	O
high	NN	O	O
frequency	NN	O	O
in	NN	O	O
an	NN	O	O
African	NN	O	O
American	NN	O	O
population	NN	O	O
(	NN	O	O
f	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
09	NN	O	O
,	NN	O	O
n	NN	O	O
=	NN	O	O
61	NN	O	O
subjects	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
ARSA	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
in	NN	O	O
subjects	NN	O	O
with	NN	O	O
and	NN	O	O
without	NN	O	O
the	NN	O	O
R496H	NN	O	O
mutation	NN	O	O
was	NN	O	O
determined	NN	O	O
and	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
normal	NN	O	O
.	NN	O	O

It	NN	O	O
is	NN	O	O
therefore	NN	O	O
concluded	NN	O	O
that	NN	O	O
the	NN	O	O
R496H	NN	O	O
mutation	NN	O	O
of	NN	O	O
ARSA	NN	O	O
does	NN	O	O
not	NN	O	O
negatively	NN	O	O
influence	NN	O	O
the	NN	O	O
activity	NN	O	O
of	NN	O	O
ARSA	NN	O	O
and	NN	O	O
is	NN	O	O
not	NN	O	O
a	NN	O	O
cause	NN	O	O
of	NN	O	O
MLD	NN	O	B-Disease

Down	NN	O	O
-	NN	O	O
regulation	NN	O	O
of	NN	O	O
transmembrane	NN	O	O
carbonic	NN	O	O
anhydrases	NN	O	O
in	NN	O	O
renal	NN	O	B-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
by	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
transgenes	NN	O	O
.	NN	O	O

To	NN	O	O
discover	NN	O	O
genes	NN	O	O
involved	NN	O	O
in	NN	O	O
von	NN	O	B-Disease
Hippel	NN	O	I-Disease
-	NN	O	I-Disease
Lindau	NN	O	I-Disease
(	NN	O	O
VHL	NN	O	B-Disease
)	NN	O	O
-	NN	O	O
mediated	NN	O	O
carcinogenesis	NN	O	O
,	NN	O	O
we	NN	O	O
used	NN	O	O
renal	NN	O	B-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
stably	NN	O	O
transfected	NN	O	O
with	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
VHL	NN	O	O
-	NN	O	O
expressing	NN	O	O
transgenes	NN	O	O
.	NN	O	O

Large	NN	O	O
-	NN	O	O
scale	NN	O	O
RNA	NN	O	O
differential	NN	O	O
display	NN	O	O
technology	NN	O	O
applied	NN	O	O
to	NN	O	O
these	NN	O	O
cell	NN	O	O
lines	NN	O	O
identified	NN	O	O
several	NN	O	O
differentially	NN	O	O
expressed	NN	O	O
genes	NN	O	O
,	NN	O	O
including	NN	O	O
an	NN	O	O
alpha	NN	O	O
carbonic	NN	O	O
anhydrase	NN	O	O
gene	NN	O	O
,	NN	O	O
termed	NN	O	O
CA12	NN	O	O
.	NN	O	O

The	NN	O	O
deduced	NN	O	O
protein	NN	O	O
sequence	NN	O	O
was	NN	O	O
classified	NN	O	O
as	NN	O	O
a	NN	O	O
one	NN	O	O
-	NN	O	O
pass	NN	O	O
transmembrane	NN	O	O
CA	NN	O	O
possessing	NN	O	O
an	NN	O	O
apparently	NN	O	O
intact	NN	O	O
catalytic	NN	O	O
domain	NN	O	O
in	NN	O	O
the	NN	O	O
extracellular	NN	O	O
CA	NN	O	O
module	NN	O	O
.	NN	O	O

Reintroduced	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
VHL	NN	O	B-Disease
strongly	NN	O	O
inhibited	NN	O	O
the	NN	O	O
overexpression	NN	O	O
of	NN	O	O
the	NN	O	O
CA12	NN	O	O
gene	NN	O	O
in	NN	O	O
the	NN	O	O
parental	NN	O	O
renal	NN	O	B-Disease
cell	NN	O	I-Disease
carcinoma	NN	O	I-Disease
cell	NN	O	O
lines	NN	O	O
.	NN	O	O

Similar	NN	O	O
results	NN	O	O
were	NN	O	O
obtained	NN	O	O
with	NN	O	O
CA9	NN	O	O
,	NN	O	O
encoding	NN	O	O
another	NN	O	O
transmembrane	NN	O	O
CA	NN	O	O
with	NN	O	O
an	NN	O	O
intact	NN	O	O
catalytic	NN	O	O
domain	NN	O	O
.	NN	O	O

Although	NN	O	O
both	NN	O	O
domains	NN	O	O
of	NN	O	O
the	NN	O	O
VHL	NN	O	B-Disease
protein	NN	O	O
contribute	NN	O	O
to	NN	O	O
regulation	NN	O	O
of	NN	O	O
CA12	NN	O	O
expression	NN	O	O
,	NN	O	O
the	NN	O	O
elongin	NN	O	O
binding	NN	O	O
domain	NN	O	O
alone	NN	O	O
could	NN	O	O
effectively	NN	O	O
regulate	NN	O	O
CA9	NN	O	O
expression	NN	O	O
.	NN	O	O

We	NN	O	O
mapped	NN	O	O
CA12	NN	O	O
and	NN	O	O
CA9	NN	O	O
loci	NN	O	O
to	NN	O	O
chromosome	NN	O	O
bands	NN	O	O
15q22	NN	O	O
and	NN	O	O
17q21	NN	O	O
.	NN	O	O

2	NN	O	O
respectively	NN	O	O
,	NN	O	O
regions	NN	O	O
prone	NN	O	O
to	NN	O	O
amplification	NN	O	O
in	NN	O	O
some	NN	O	O
human	NN	O	O
cancers	NN	O	B-Disease
.	NN	O	O

Additional	NN	O	O
experiments	NN	O	O
are	NN	O	O
needed	NN	O	O
to	NN	O	O
define	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
CA	NN	O	O
IX	NN	O	O
and	NN	O	O
CA	NN	O	O
XII	NN	O	O
enzymes	NN	O	O
in	NN	O	O
the	NN	O	O
regulation	NN	O	O
of	NN	O	O
pH	NN	O	O
in	NN	O	O
the	NN	O	O
extracellular	NN	O	O
microenvironment	NN	O	O
and	NN	O	O
its	NN	O	O
potential	NN	O	O
impact	NN	O	O
on	NN	O	O
cancer	NN	O	B-Disease
cell	NN	O	O
growth	NN	O	O
.	NN	O	O

A	NN	O	O
gene	NN	O	O
encoding	NN	O	O
a	NN	O	O
transmembrane	NN	O	O
protein	NN	O	O
is	NN	O	O
mutated	NN	O	O
in	NN	O	O
patients	NN	O	O
with	NN	O	O
diabetes	NN	O	B-Disease
mellitus	NN	O	I-Disease
and	NN	O	O
optic	NN	O	B-Disease
atrophy	NN	O	I-Disease
(	NN	O	O
Wolfram	NN	O	B-Disease
syndrome	NN	O	I-Disease
)	NN	O	O
.	NN	O	O

Wolfram	NN	O	B-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
WFS	NN	O	B-Disease
;	NN	O	O
OMIM	NN	O	O
222300	NN	O	O
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
neurodegenerative	NN	O	I-Disease
disorder	NN	O	I-Disease
defined	NN	O	O
by	NN	O	O
young	NN	O	O
-	NN	O	O
onset	NN	O	O
non	NN	O	O
-	NN	O	O
immune	NN	O	O
insulin	NN	O	B-Disease
-	NN	O	I-Disease
dependent	NN	O	I-Disease
diabetes	NN	O	I-Disease
mellitus	NN	O	I-Disease
and	NN	O	O
progressive	NN	O	O
optic	NN	O	B-Disease
atrophy	NN	O	I-Disease
.	NN	O	O

Linkage	NN	O	O
to	NN	O	O
markers	NN	O	O
on	NN	O	O
chromosome	NN	O	O
4p	NN	O	O
was	NN	O	O
confirmed	NN	O	O
in	NN	O	O
five	NN	O	O
families	NN	O	O
.	NN	O	O

On	NN	O	O
the	NN	O	O
basis	NN	O	O
of	NN	O	O
meiotic	NN	O	O
recombinants	NN	O	O
and	NN	O	O
disease	NN	O	O
-	NN	O	O
associated	NN	O	O
haplotypes	NN	O	O
,	NN	O	O
the	NN	O	O
WFS	NN	O	B-Disease
gene	NN	O	O
was	NN	O	O
localized	NN	O	O
to	NN	O	O
a	NN	O	O
BAC	NN	O	O
/	NN	O	O
P1	NN	O	O
contig	NN	O	O
of	NN	O	O
less	NN	O	O
than	NN	O	O
250	NN	O	O
kb	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
a	NN	O	O
novel	NN	O	O
gene	NN	O	O
(	NN	O	O
WFS1	NN	O	O
)	NN	O	O
encoding	NN	O	O
a	NN	O	O
putative	NN	O	O
transmembrane	NN	O	O
protein	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
all	NN	O	O
affected	NN	O	O
individuals	NN	O	O
in	NN	O	O
six	NN	O	O
WFS	NN	O	B-Disease
families	NN	O	O
,	NN	O	O
and	NN	O	O
these	NN	O	O
mutations	NN	O	O
were	NN	O	O
associated	NN	O	O
with	NN	O	O
the	NN	O	O
disease	NN	O	O
phenotype	NN	O	O
.	NN	O	O

WFS1	NN	O	O
appears	NN	O	O
to	NN	O	O
function	NN	O	O
in	NN	O	O
survival	NN	O	O
of	NN	O	O
islet	NN	O	O
beta	NN	O	O
-	NN	O	O
cells	NN	O	O
and	NN	O	O
neurons	NN	O	O
.	NN	O	O
.	NN	O	O

Stable	NN	O	O
interaction	NN	O	O
between	NN	O	O
the	NN	O	O
products	NN	O	O
of	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
tumor	NN	O	B-Disease
suppressor	NN	O	O
genes	NN	O	O
in	NN	O	O
mitotic	NN	O	O
and	NN	O	O
meiotic	NN	O	O
cells	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
account	NN	O	O
for	NN	O	O
most	NN	O	O
cases	NN	O	O
of	NN	O	O
familial	NN	O	O
,	NN	O	O
early	NN	O	O
onset	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
and	NN	O	O
encode	NN	O	O
products	NN	O	O
that	NN	O	O
each	NN	O	O
interact	NN	O	O
with	NN	O	O
hRAD51	NN	O	O
.	NN	O	O

Results	NN	O	O
presented	NN	O	O
here	NN	O	O
show	NN	O	O
that	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
coexist	NN	O	O
in	NN	O	O
a	NN	O	O
biochemical	NN	O	O
complex	NN	O	O
and	NN	O	O
colocalize	NN	O	O
in	NN	O	O
subnuclear	NN	O	O
foci	NN	O	O
in	NN	O	O
somatic	NN	O	O
cells	NN	O	O
and	NN	O	O
on	NN	O	O
the	NN	O	O
axial	NN	O	O
elements	NN	O	O
of	NN	O	O
developing	NN	O	O
synaptonemal	NN	O	O
complexes	NN	O	O
.	NN	O	O

Like	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
RAD51	NN	O	O
,	NN	O	O
BRCA2	NN	O	O
relocates	NN	O	O
to	NN	O	O
PCNA	NN	O	O
+	NN	O	O
replication	NN	O	O
sites	NN	O	O
following	NN	O	O
exposure	NN	O	O
of	NN	O	O
S	NN	O	O
phase	NN	O	O
cells	NN	O	O
to	NN	O	O
hydroxyurea	NN	O	O
or	NN	O	O
UV	NN	O	O
irradiation	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
participate	NN	O	O
,	NN	O	O
together	NN	O	O
,	NN	O	O
in	NN	O	O
a	NN	O	O
pathway	NN	O	O
(	NN	O	O
s	NN	O	O
)	NN	O	O
associated	NN	O	O
with	NN	O	O
the	NN	O	O
activation	NN	O	O
of	NN	O	O
double	NN	O	O
-	NN	O	O
strand	NN	O	O
break	NN	O	O
repair	NN	O	O
and	NN	O	O
/	NN	O	O
or	NN	O	O
homologous	NN	O	O
recombination	NN	O	O
.	NN	O	O

Dysfunction	NN	O	O
of	NN	O	O
this	NN	O	O
pathway	NN	O	O
may	NN	O	O
be	NN	O	O
a	NN	O	O
general	NN	O	O
phenomenon	NN	O	O
in	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
cases	NN	O	O
of	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
/	NN	O	I-Disease
or	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

A	NN	O	O
novel	NN	O	O
Arg362Ser	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
sterol	NN	O	O
27	NN	O	O
-	NN	O	O
hydroxylase	NN	O	O
gene	NN	O	O
(	NN	O	O
CYP27	NN	O	O
)	NN	O	O
:	NN	O	O
its	NN	O	O
effects	NN	O	O
on	NN	O	O
pre	NN	O	O
-	NN	O	O
mRNA	NN	O	O
splicing	NN	O	O
and	NN	O	O
enzyme	NN	O	O
activity	NN	O	O
.	NN	O	O

A	NN	O	O
novel	NN	O	O
C	NN	O	O
to	NN	O	O
A	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
sterol	NN	O	O
27	NN	O	O
-	NN	O	O
hydroxylase	NN	O	O
gene	NN	O	O
(	NN	O	O
CYP27	NN	O	O
)	NN	O	O
was	NN	O	O
identified	NN	O	O
by	NN	O	O
sequencing	NN	O	O
amplified	NN	O	O
CYP27	NN	O	O
gene	NN	O	O
products	NN	O	O
from	NN	O	O
a	NN	O	O
patient	NN	O	O
with	NN	O	O
cerebrotendinous	NN	O	B-Disease
xanthomatosis	NN	O	I-Disease
(	NN	O	O
CTX	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

The	NN	O	O
mutation	NN	O	O
changed	NN	O	O
the	NN	O	O
adrenodoxin	NN	O	O
cofactor	NN	O	O
binding	NN	O	O
residue	NN	O	O
362Arg	NN	O	O
to	NN	O	O
362Ser	NN	O	O
(	NN	O	O
CGT	NN	O	O
362Arg	NN	O	O
to	NN	O	O
AGT	NN	O	O
362Ser	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
was	NN	O	O
responsible	NN	O	O
for	NN	O	O
deficiency	NN	O	O
in	NN	O	O
the	NN	O	O
sterol	NN	O	O
27	NN	O	O
-	NN	O	O
hydroxylase	NN	O	O
activity	NN	O	O
,	NN	O	O
as	NN	O	O
confirmed	NN	O	O
by	NN	O	O
expression	NN	O	O
of	NN	O	O
mutant	NN	O	O
cDNA	NN	O	O
into	NN	O	O
COS	NN	O	O
-	NN	O	O
1	NN	O	O
cells	NN	O	O
.	NN	O	O

Quantitative	NN	O	O
analysis	NN	O	O
showed	NN	O	O
that	NN	O	O
the	NN	O	O
expression	NN	O	O
of	NN	O	O
CYP27	NN	O	O
gene	NN	O	O
mRNA	NN	O	O
in	NN	O	O
the	NN	O	O
patient	NN	O	O
represented	NN	O	O
52	NN	O	O
.	NN	O	O

5	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
level	NN	O	O
.	NN	O	O

As	NN	O	O
the	NN	O	O
mutation	NN	O	O
occurred	NN	O	O
at	NN	O	O
the	NN	O	O
penultimate	NN	O	O
nucleotide	NN	O	O
of	NN	O	O
exon	NN	O	O
6	NN	O	O
(	NN	O	O
-	NN	O	O
2	NN	O	O
position	NN	O	O
of	NN	O	O
exon	NN	O	O
6	NN	O	O
-	NN	O	O
intron	NN	O	O
6	NN	O	O
splice	NN	O	O
site	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
,	NN	O	O
we	NN	O	O
hypothesized	NN	O	O
that	NN	O	O
the	NN	O	O
mutation	NN	O	O
may	NN	O	O
partially	NN	O	O
affect	NN	O	O
the	NN	O	O
normal	NN	O	O
splicing	NN	O	O
efficiency	NN	O	O
in	NN	O	O
exon	NN	O	O
6	NN	O	O
and	NN	O	O
cause	NN	O	O
alternative	NN	O	O
splicing	NN	O	O
elsewhere	NN	O	O
,	NN	O	O
which	NN	O	O
resulted	NN	O	O
in	NN	O	O
decreased	NN	O	O
transcript	NN	O	O
in	NN	O	O
the	NN	O	O
patient	NN	O	O
.	NN	O	O

Transfection	NN	O	O
of	NN	O	O
constructed	NN	O	O
minigenes	NN	O	O
,	NN	O	O
with	NN	O	O
or	NN	O	O
without	NN	O	O
the	NN	O	O
mutation	NN	O	O
,	NN	O	O
into	NN	O	O
COS	NN	O	O
-	NN	O	O
1	NN	O	O
cells	NN	O	O
confirmed	NN	O	O
that	NN	O	O
the	NN	O	O
mutant	NN	O	O
minigene	NN	O	O
was	NN	O	O
responsible	NN	O	O
for	NN	O	O
a	NN	O	O
mRNA	NN	O	O
species	NN	O	O
alternatively	NN	O	O
spliced	NN	O	O
at	NN	O	O
an	NN	O	O
activated	NN	O	O
cryptic	NN	O	O
5	NN	O	O
splice	NN	O	O
site	NN	O	O
88	NN	O	O
bp	NN	O	O
upstream	NN	O	O
from	NN	O	O
the	NN	O	O
3	NN	O	O
end	NN	O	O
of	NN	O	O
exon	NN	O	O
6	NN	O	O
.	NN	O	O

Our	NN	O	O
data	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
C	NN	O	O
to	NN	O	O
A	NN	O	O
mutation	NN	O	O
at	NN	O	O
the	NN	O	O
penultimate	NN	O	O
nucleotide	NN	O	O
of	NN	O	O
exon	NN	O	O
6	NN	O	O
of	NN	O	O
the	NN	O	O
CYP27	NN	O	O
gene	NN	O	O
not	NN	O	O
only	NN	O	O
causes	NN	O	O
the	NN	O	O
deficiency	NN	O	B-Disease
in	NN	O	I-Disease
the	NN	O	I-Disease
sterol	NN	O	I-Disease
27	NN	O	I-Disease
-	NN	O	I-Disease
hydroxylase	NN	O	I-Disease
activity	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
also	NN	O	O
partially	NN	O	O
leads	NN	O	O
to	NN	O	O
alternative	NN	O	O
pre	NN	O	O
-	NN	O	O
mRNA	NN	O	O
splicing	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
.	NN	O	O

To	NN	O	O
our	NN	O	O
knowledge	NN	O	O
,	NN	O	O
this	NN	O	O
is	NN	O	O
the	NN	O	O
first	NN	O	O
report	NN	O	O
regarding	NN	O	O
effects	NN	O	O
on	NN	O	O
pre	NN	O	O
-	NN	O	O
mRNA	NN	O	O
splicing	NN	O	O
of	NN	O	O
a	NN	O	O
mutation	NN	O	O
at	NN	O	O
the	NN	O	O
-	NN	O	O
2	NN	O	O
position	NN	O	O
of	NN	O	O
a	NN	O	O
5	NN	O	O
splice	NN	O	O
site	NN	O	O
.	NN	O	O

ATM	NN	O	O
germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
classical	NN	O	O
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
patients	NN	O	O
in	NN	O	O
the	NN	O	O
Dutch	NN	O	O
population	NN	O	O
.	NN	O	O

Germline	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
ATM	NN	O	O
gene	NN	O	O
are	NN	O	O
responsible	NN	O	O
for	NN	O	O
the	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
disorder	NN	O	I-Disease
ataxia	NN	O	B-Disease
-	NN	O	I-Disease
telangiectasia	NN	O	I-Disease
(	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
)	NN	O	O
.	NN	O	O

In	NN	O	O
our	NN	O	O
study	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
determined	NN	O	O
the	NN	O	O
ATM	NN	O	O
mutation	NN	O	O
spectrum	NN	O	O
in	NN	O	O
19	NN	O	O
classical	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
patients	NN	O	O
,	NN	O	O
including	NN	O	O
some	NN	O	O
immigrant	NN	O	O
populations	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
12	NN	O	O
of	NN	O	O
Dutch	NN	O	O
ethnic	NN	O	O
origin	NN	O	O
.	NN	O	O

Both	NN	O	O
the	NN	O	O
protein	NN	O	O
truncation	NN	O	O
test	NN	O	O
(	NN	O	O
PTT	NN	O	O
)	NN	O	O
and	NN	O	O
the	NN	O	O
restriction	NN	O	O
endonuclease	NN	O	O
fingerprinting	NN	O	O
(	NN	O	O
REF	NN	O	O
)	NN	O	O
method	NN	O	O
were	NN	O	O
used	NN	O	O
and	NN	O	O
compared	NN	O	O
for	NN	O	O
their	NN	O	O
detection	NN	O	O
efficiency	NN	O	O
,	NN	O	O
identifying	NN	O	O
76	NN	O	O
%	NN	O	O
and	NN	O	O
60	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
mutations	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

Most	NN	O	O
patients	NN	O	O
were	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
compound	NN	O	O
heterozygote	NN	O	O
.	NN	O	O

Seventeen	NN	O	O
mutations	NN	O	O
were	NN	O	O
distinct	NN	O	O
,	NN	O	O
of	NN	O	O
which	NN	O	O
10	NN	O	O
were	NN	O	O
not	NN	O	O
reported	NN	O	O
previously	NN	O	O
.	NN	O	O

Mutations	NN	O	O
are	NN	O	O
small	NN	O	O
deletions	NN	O	O
or	NN	O	O
point	NN	O	O
mutations	NN	O	O
frequently	NN	O	O
affecting	NN	O	O
splice	NN	O	O
sites	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
a	NN	O	O
16	NN	O	O
.	NN	O	O

7	NN	O	O
-	NN	O	O
kb	NN	O	O
genomic	NN	O	O
deletion	NN	O	O
of	NN	O	O
the	NN	O	O
3	NN	O	O
end	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
,	NN	O	O
most	NN	O	O
likely	NN	O	O
a	NN	O	O
result	NN	O	O
of	NN	O	O
recombination	NN	O	O
between	NN	O	O
two	NN	O	O
LINE	NN	O	O
elements	NN	O	O
,	NN	O	O
was	NN	O	O
identified	NN	O	O
.	NN	O	O

The	NN	O	O
most	NN	O	O
frequently	NN	O	O
found	NN	O	O
mutation	NN	O	O
,	NN	O	O
identified	NN	O	O
in	NN	O	O
three	NN	O	O
unrelated	NN	O	O
Turkish	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
individuals	NN	O	O
,	NN	O	O
was	NN	O	O
previously	NN	O	O
described	NN	O	O
to	NN	O	O
be	NN	O	O
a	NN	O	O
Turkish	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
founder	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
founder	NN	O	O
mutation	NN	O	O
among	NN	O	O
relatively	NN	O	O
small	NN	O	O
ethnic	NN	O	O
population	NN	O	O
groups	NN	O	O
in	NN	O	O
Western	NN	O	O
Europe	NN	O	O
could	NN	O	O
indicate	NN	O	O
a	NN	O	O
high	NN	O	O
carrier	NN	O	O
frequency	NN	O	O
in	NN	O	O
such	NN	O	O
communities	NN	O	O
.	NN	O	O

In	NN	O	O
patients	NN	O	O
of	NN	O	O
Dutch	NN	O	O
ethnic	NN	O	O
origin	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
no	NN	O	O
significant	NN	O	O
founder	NN	O	O
effect	NN	O	O
could	NN	O	O
be	NN	O	O
identified	NN	O	O
.	NN	O	O

The	NN	O	O
observed	NN	O	O
genetic	NN	O	O
heterogeneity	NN	O	O
including	NN	O	O
the	NN	O	O
relative	NN	O	O
high	NN	O	O
percentage	NN	O	O
of	NN	O	O
splice	NN	O	O
-	NN	O	O
site	NN	O	O
mutations	NN	O	O
had	NN	O	O
no	NN	O	O
reflection	NN	O	O
on	NN	O	O
the	NN	O	O
phenotype	NN	O	O
.	NN	O	O

All	NN	O	O
patients	NN	O	O
manifested	NN	O	O
classical	NN	O	O
A	NN	O	B-Disease
-	NN	O	I-Disease
T	NN	O	I-Disease
and	NN	O	O
increased	NN	O	O
cellular	NN	O	O
radioresistant	NN	O	O
DNA	NN	O	O
synthesis	NN	O	O
.	NN	O	O

Determination	NN	O	O
of	NN	O	O
the	NN	O	O
genomic	NN	O	O
structure	NN	O	O
of	NN	O	O
the	NN	O	O
COL4A4	NN	O	O
gene	NN	O	O
and	NN	O	O
of	NN	O	O
novel	NN	O	O
mutations	NN	O	O
causing	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
Alport	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
Alport	NN	O	I-Disease
syndrome	NN	O	I-Disease
is	NN	O	O
a	NN	O	O
progressive	NN	O	O
hematuric	NN	O	B-Disease
glomerulonephritis	NN	O	I-Disease
characterized	NN	O	O
by	NN	O	O
glomerular	NN	O	B-Disease
basement	NN	O	I-Disease
membrane	NN	O	I-Disease
abnormalities	NN	O	I-Disease
and	NN	O	O
associated	NN	O	O
with	NN	O	O
mutations	NN	O	O
in	NN	O	O
either	NN	O	O
the	NN	O	O
COL4A3	NN	O	O
or	NN	O	O
the	NN	O	O
COL4A4	NN	O	O
gene	NN	O	O
,	NN	O	O
which	NN	O	O
encode	NN	O	O
the	NN	O	O
alpha3	NN	O	O
and	NN	O	O
alpha4	NN	O	O
type	NN	O	O
IV	NN	O	O
collagen	NN	O	O
chains	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

To	NN	O	O
date	NN	O	O
,	NN	O	O
mutation	NN	O	O
screening	NN	O	O
in	NN	O	O
the	NN	O	O
two	NN	O	O
genes	NN	O	O
has	NN	O	O
been	NN	O	O
hampered	NN	O	O
by	NN	O	O
the	NN	O	O
lack	NN	O	O
of	NN	O	O
genomic	NN	O	O
structure	NN	O	O
information	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
here	NN	O	O
the	NN	O	O
complete	NN	O	O
characterization	NN	O	O
of	NN	O	O
the	NN	O	O
48	NN	O	O
exons	NN	O	O
of	NN	O	O
the	NN	O	O
COL4A4	NN	O	O
gene	NN	O	O
,	NN	O	O
a	NN	O	O
comprehensive	NN	O	O
gene	NN	O	O
screen	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
subsequent	NN	O	O
detection	NN	O	O
of	NN	O	O
10	NN	O	O
novel	NN	O	O
mutations	NN	O	O
in	NN	O	O
eight	NN	O	O
patients	NN	O	O
diagnosed	NN	O	O
with	NN	O	O
autosomal	NN	O	B-Disease
recessive	NN	O	I-Disease
Alport	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
we	NN	O	O
identified	NN	O	O
a	NN	O	O
glycine	NN	O	O
to	NN	O	O
alanine	NN	O	O
substitution	NN	O	O
in	NN	O	O
the	NN	O	O
collagenous	NN	O	O
domain	NN	O	O
that	NN	O	O
is	NN	O	O
apparently	NN	O	O
silent	NN	O	O
in	NN	O	O
the	NN	O	O
heterozygous	NN	O	O
carriers	NN	O	O
,	NN	O	O
in	NN	O	O
11	NN	O	O
.	NN	O	O

5	NN	O	O
%	NN	O	O
of	NN	O	O
all	NN	O	O
control	NN	O	O
individuals	NN	O	O
,	NN	O	O
and	NN	O	O
in	NN	O	O
one	NN	O	O
control	NN	O	O
individual	NN	O	O
homozygous	NN	O	O
for	NN	O	O
this	NN	O	O
glycine	NN	O	O
substitution	NN	O	O
.	NN	O	O

There	NN	O	O
has	NN	O	O
been	NN	O	O
no	NN	O	O
previous	NN	O	O
finding	NN	O	O
of	NN	O	O
a	NN	O	O
glycine	NN	O	O
substitution	NN	O	O
that	NN	O	O
is	NN	O	O
not	NN	O	O
associated	NN	O	O
with	NN	O	O
any	NN	O	O
obvious	NN	O	O
phenotype	NN	O	O
in	NN	O	O
homozygous	NN	O	O
individuals	NN	O	O
.	NN	O	O

Founder	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
in	NN	O	O
French	NN	O	O
Canadian	NN	O	O
breast	NN	O	B-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
identified	NN	O	O
four	NN	O	O
mutations	NN	O	O
in	NN	O	O
each	NN	O	O
of	NN	O	O
the	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
-	NN	O	O
susceptibility	NN	O	O
genes	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
,	NN	O	O
in	NN	O	O
French	NN	O	O
Canadian	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
from	NN	O	O
Quebec	NN	O	O
.	NN	O	O

To	NN	O	O
identify	NN	O	O
founder	NN	O	O
effects	NN	O	O
,	NN	O	O
we	NN	O	O
examined	NN	O	O
independently	NN	O	O
ascertained	NN	O	O
French	NN	O	O
Canadian	NN	O	O
cancer	NN	O	B-Disease
families	NN	O	O
for	NN	O	O
the	NN	O	O
distribution	NN	O	O
of	NN	O	O
these	NN	O	O
eight	NN	O	O
mutations	NN	O	O
.	NN	O	O

Mutations	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
41	NN	O	O
of	NN	O	O
97	NN	O	O
families	NN	O	O
.	NN	O	O

Six	NN	O	O
of	NN	O	O
eight	NN	O	O
mutations	NN	O	O
were	NN	O	O
observed	NN	O	O
at	NN	O	O
least	NN	O	O
twice	NN	O	O
.	NN	O	O

The	NN	O	O
BRCA1	NN	O	O
C4446T	NN	O	O
mutation	NN	O	O
was	NN	O	O
the	NN	O	O
most	NN	O	O
common	NN	O	O
mutation	NN	O	O
found	NN	O	O
,	NN	O	O
followed	NN	O	O
by	NN	O	O
the	NN	O	O
BRCA2	NN	O	O
8765delAG	NN	O	O
mutation	NN	O	O
.	NN	O	O

Together	NN	O	O
,	NN	O	O
these	NN	O	O
mutations	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
28	NN	O	O
of	NN	O	O
41	NN	O	O
families	NN	O	O
identified	NN	O	O
to	NN	O	O
have	NN	O	O
a	NN	O	O
mutation	NN	O	O
.	NN	O	O

The	NN	O	O
odds	NN	O	O
of	NN	O	O
detection	NN	O	O
of	NN	O	O
any	NN	O	O
of	NN	O	O
the	NN	O	O
four	NN	O	O
BRCA1	NN	O	O
mutations	NN	O	O
was	NN	O	O
18	NN	O	O
.	NN	O	O

7x	NN	O	O
greater	NN	O	O
if	NN	O	O
one	NN	O	O
or	NN	O	O
more	NN	O	O
cases	NN	O	O
of	NN	O	O
ovarian	NN	O	B-Disease
cancer	NN	O	I-Disease
were	NN	O	O
also	NN	O	O
present	NN	O	O
in	NN	O	O
the	NN	O	O
family	NN	O	O
.	NN	O	O

The	NN	O	O
odds	NN	O	O
of	NN	O	O
detection	NN	O	O
of	NN	O	O
any	NN	O	O
of	NN	O	O
the	NN	O	O
four	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
was	NN	O	O
5	NN	O	O
.	NN	O	O

3x	NN	O	O
greater	NN	O	O
if	NN	O	O
there	NN	O	O
were	NN	O	O
at	NN	O	O
least	NN	O	O
five	NN	O	O
cases	NN	O	O
of	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
family	NN	O	O
.	NN	O	O

Interestingly	NN	O	O
,	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
case	NN	O	O
<	NN	O	O
36	NN	O	O
years	NN	O	O
of	NN	O	O
age	NN	O	O
was	NN	O	O
strongly	NN	O	O
predictive	NN	O	O
of	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
any	NN	O	O
of	NN	O	O
the	NN	O	O
eight	NN	O	O
mutations	NN	O	O
screened	NN	O	O
.	NN	O	O

Carriers	NN	O	O
of	NN	O	O
the	NN	O	O
same	NN	O	O
mutation	NN	O	O
,	NN	O	O
from	NN	O	O
different	NN	O	O
families	NN	O	O
,	NN	O	O
shared	NN	O	O
similar	NN	O	O
haplotypes	NN	O	O
,	NN	O	O
indicating	NN	O	O
that	NN	O	O
the	NN	O	O
mutant	NN	O	O
alleles	NN	O	O
were	NN	O	O
likely	NN	O	O
to	NN	O	O
be	NN	O	O
identical	NN	O	O
by	NN	O	O
descent	NN	O	O
for	NN	O	O
a	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
founder	NN	O	O
population	NN	O	O
.	NN	O	O

The	NN	O	O
identification	NN	O	O
of	NN	O	O
common	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
BRCA2	NN	O	O
mutations	NN	O	O
will	NN	O	O
facilitate	NN	O	O
carrier	NN	O	O
detection	NN	O	O
in	NN	O	O
French	NN	O	O
Canadian	NN	O	O
breast	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
breast	NN	O	B-Disease
/	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
families	NN	O	O
.	NN	O	O

Are	NN	O	O
Dp71	NN	O	O
and	NN	O	O
Dp140	NN	O	O
brain	NN	O	O
dystrophin	NN	O	O
isoforms	NN	O	O
related	NN	O	O
to	NN	O	O
cognitive	NN	O	B-Disease
impairment	NN	O	I-Disease
in	NN	O	O
Duchenne	NN	O	B-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
?	NN	O	O

Molecular	NN	O	O
study	NN	O	O
and	NN	O	O
neuropsychological	NN	O	O
analysis	NN	O	O
were	NN	O	O
performed	NN	O	O
concurrently	NN	O	O
on	NN	O	O
49	NN	O	O
patients	NN	O	O
with	NN	O	O
Duchenne	NN	O	B-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DMD	NN	O	B-Disease
)	NN	O	O
in	NN	O	O
order	NN	O	O
to	NN	O	O
find	NN	O	O
a	NN	O	O
molecular	NN	O	O
explanation	NN	O	O
for	NN	O	O
the	NN	O	O
cognitive	NN	O	B-Disease
impairment	NN	O	I-Disease
observed	NN	O	O
in	NN	O	O
most	NN	O	O
DMD	NN	O	B-Disease
patients	NN	O	O
.	NN	O	O

Complete	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
dystrophin	NN	O	O
gene	NN	O	O
was	NN	O	O
performed	NN	O	O
to	NN	O	O
define	NN	O	O
the	NN	O	O
localization	NN	O	O
of	NN	O	O
deletions	NN	O	O
and	NN	O	O
duplications	NN	O	O
in	NN	O	O
relation	NN	O	O
to	NN	O	O
the	NN	O	O
different	NN	O	O
DMD	NN	O	B-Disease
promoters	NN	O	O
.	NN	O	O

Qualitative	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
Dp71	NN	O	O
transcript	NN	O	O
and	NN	O	O
testing	NN	O	O
for	NN	O	O
the	NN	O	O
specific	NN	O	O
first	NN	O	O
exon	NN	O	O
of	NN	O	O
Dp140	NN	O	O
were	NN	O	O
also	NN	O	O
carried	NN	O	O
out	NN	O	O
.	NN	O	O

Neuropsychological	NN	O	O
analysis	NN	O	O
assessed	NN	O	O
verbal	NN	O	O
and	NN	O	O
visuospatial	NN	O	O
intelligence	NN	O	O
,	NN	O	O
verbal	NN	O	O
memory	NN	O	O
,	NN	O	O
and	NN	O	O
reading	NN	O	O
skills	NN	O	O
.	NN	O	O

Comparison	NN	O	O
of	NN	O	O
molecular	NN	O	O
and	NN	O	O
psychometric	NN	O	O
findings	NN	O	O
demonstrated	NN	O	O
that	NN	O	O
deletions	NN	O	O
and	NN	O	O
duplications	NN	O	O
that	NN	O	O
were	NN	O	O
localized	NN	O	O
in	NN	O	O
the	NN	O	O
distal	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
seemed	NN	O	O
to	NN	O	O
be	NN	O	O
preferentially	NN	O	O
associated	NN	O	O
with	NN	O	O
cognitive	NN	O	B-Disease
impairment	NN	O	I-Disease
.	NN	O	O

Two	NN	O	O
altered	NN	O	O
Dp71	NN	O	O
transcripts	NN	O	O
and	NN	O	O
two	NN	O	O
deleted	NN	O	O
Dp140	NN	O	O
DNA	NN	O	O
sequences	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
four	NN	O	O
patients	NN	O	O
with	NN	O	O
severe	NN	O	O
cerebral	NN	O	B-Disease
dysfunction	NN	O	I-Disease
.	NN	O	O

These	NN	O	O
findings	NN	O	O
suggest	NN	O	O
that	NN	O	O
some	NN	O	O
sequences	NN	O	O
located	NN	O	O
in	NN	O	O
the	NN	O	O
distal	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
and	NN	O	O
,	NN	O	O
in	NN	O	O
particular	NN	O	O
,	NN	O	O
some	NN	O	O
DMD	NN	O	B-Disease
isoforms	NN	O	O
expressed	NN	O	O
in	NN	O	O
the	NN	O	O
brain	NN	O	O
may	NN	O	O
be	NN	O	O
related	NN	O	O
to	NN	O	O
the	NN	O	O
cognitive	NN	O	B-Disease
impairment	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
DMD	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

I1307K	NN	O	O
APC	NN	O	O
and	NN	O	O
hMLH1	NN	O	O
mutations	NN	O	O
in	NN	O	O
a	NN	O	O
non	NN	O	O
-	NN	O	O
Jewish	NN	O	O
family	NN	O	O
with	NN	O	O
hereditary	NN	O	B-Disease
non	NN	O	I-Disease
-	NN	O	I-Disease
polyposis	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
describe	NN	O	O
a	NN	O	O
French	NN	O	O
Canadian	NN	O	O
hereditary	NN	O	B-Disease
non	NN	O	I-Disease
-	NN	O	I-Disease
polyposis	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
(	NN	O	O
HNPCC	NN	O	B-Disease
)	NN	O	O
kindred	NN	O	O
which	NN	O	O
carries	NN	O	O
a	NN	O	O
novel	NN	O	O
truncating	NN	O	O
mutation	NN	O	O
in	NN	O	O
hMLH1	NN	O	O
.	NN	O	O

Interestingly	NN	O	O
,	NN	O	O
the	NN	O	O
I1307K	NN	O	O
APC	NN	O	O
polymorphism	NN	O	O
,	NN	O	O
associated	NN	O	O
with	NN	O	O
an	NN	O	O
increased	NN	O	O
risk	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
is	NN	O	O
also	NN	O	O
present	NN	O	O
in	NN	O	O
this	NN	O	O
family	NN	O	O
.	NN	O	O

The	NN	O	O
I1307K	NN	O	O
polymorphism	NN	O	O
has	NN	O	O
previously	NN	O	O
only	NN	O	O
been	NN	O	O
identified	NN	O	O
in	NN	O	O
individuals	NN	O	O
of	NN	O	O
self	NN	O	O
-	NN	O	O
reported	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
origins	NN	O	O
.	NN	O	O

In	NN	O	O
addition	NN	O	O
,	NN	O	O
in	NN	O	O
this	NN	O	O
family	NN	O	O
,	NN	O	O
there	NN	O	O
appears	NN	O	O
to	NN	O	O
be	NN	O	O
no	NN	O	O
relationship	NN	O	O
between	NN	O	O
the	NN	O	O
I1307K	NN	O	O
polymorphism	NN	O	O
and	NN	O	O
the	NN	O	O
presence	NN	O	O
or	NN	O	O
absence	NN	O	O
of	NN	O	O
cancer	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Identification	NN	O	O
of	NN	O	O
a	NN	O	O
novel	NN	O	O
mutation	NN	O	O
of	NN	O	O
the	NN	O	O
CPO	NN	O	O
gene	NN	O	O
in	NN	O	O
a	NN	O	O
Japanese	NN	O	O
hereditary	NN	O	B-Disease
coproporphyria	NN	O	I-Disease
family	NN	O	O
.	NN	O	O

Hereditary	NN	O	B-Disease
coproporphyria	NN	O	I-Disease
(	NN	O	O
HCP	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disease	NN	O	I-Disease
characterized	NN	O	O
by	NN	O	O
a	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
coproporphyrinogen	NN	O	I-Disease
oxidase	NN	O	I-Disease
(	NN	O	O
CPO	NN	O	O
)	NN	O	O
caused	NN	O	O
by	NN	O	O
a	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
CPO	NN	O	O
gene	NN	O	O
.	NN	O	O

Only	NN	O	O
11	NN	O	O
mutations	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
have	NN	O	O
been	NN	O	O
reported	NN	O	O
in	NN	O	O
HCP	NN	O	B-Disease
patients	NN	O	O
.	NN	O	O

We	NN	O	O
report	NN	O	O
another	NN	O	O
mutation	NN	O	O
in	NN	O	O
a	NN	O	O
Japanese	NN	O	O
family	NN	O	O
.	NN	O	O

Polymerase	NN	O	O
chain	NN	O	O
reaction	NN	O	O
-	NN	O	O
single	NN	O	O
strand	NN	O	O
conformational	NN	O	O
polymorphism	NN	O	O
and	NN	O	O
direct	NN	O	O
sequence	NN	O	O
analyses	NN	O	O
demonstrated	NN	O	O
a	NN	O	O
C	NN	O	O
to	NN	O	O
T	NN	O	O
substitution	NN	O	O
in	NN	O	O
exon	NN	O	O
1	NN	O	O
of	NN	O	O
the	NN	O	O
CPO	NN	O	O
gene	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
position	NN	O	O
85	NN	O	O
,	NN	O	O
which	NN	O	O
lies	NN	O	O
in	NN	O	O
the	NN	O	O
putative	NN	O	O
presequence	NN	O	O
for	NN	O	O
targeting	NN	O	O
to	NN	O	O
mitochondria	NN	O	O
.	NN	O	O

This	NN	O	O
mutation	NN	O	O
changes	NN	O	O
the	NN	O	O
codon	NN	O	O
for	NN	O	O
glutamine	NN	O	O
to	NN	O	O
a	NN	O	O
termination	NN	O	O
codon	NN	O	O
at	NN	O	O
amino	NN	O	O
acid	NN	O	O
position	NN	O	O
29	NN	O	O
.	NN	O	O

MaeI	NN	O	O
restriction	NN	O	O
analysis	NN	O	O
showed	NN	O	O
two	NN	O	O
other	NN	O	O
carriers	NN	O	O
in	NN	O	O
the	NN	O	O
family	NN	O	O
.	NN	O	O

The	NN	O	O
C	NN	O	O
-	NN	O	O
T	NN	O	O
mutation	NN	O	O
is	NN	O	O
located	NN	O	O
within	NN	O	O
a	NN	O	O
recently	NN	O	O
proposed	NN	O	O
putative	NN	O	O
alternative	NN	O	O
translation	NN	O	O
initiation	NN	O	O
codon	NN	O	O
(	NN	O	O
TIC	NN	O	O
-	NN	O	O
1	NN	O	O
)	NN	O	O
,	NN	O	O
supporting	NN	O	O
that	NN	O	O
TIC	NN	O	O
-	NN	O	O
1	NN	O	O
is	NN	O	O
the	NN	O	O
real	NN	O	O
TIC	NN	O	O
rather	NN	O	O
than	NN	O	O
TIC	NN	O	O
-	NN	O	O
2	NN	O	O
.	NN	O	O
.	NN	O	O

Human	NN	O	B-Disease
complement	NN	O	I-Disease
factor	NN	O	I-Disease
H	NN	O	I-Disease
deficiency	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
hemolytic	NN	O	B-Disease
uremic	NN	O	I-Disease
syndrome	NN	O	I-Disease
.	NN	O	O

This	NN	O	O
study	NN	O	O
reports	NN	O	O
on	NN	O	O
six	NN	O	O
cases	NN	O	O
of	NN	O	O
deficiency	NN	O	B-Disease
in	NN	O	I-Disease
the	NN	O	I-Disease
human	NN	O	I-Disease
complement	NN	O	I-Disease
regulatory	NN	O	I-Disease
protein	NN	O	I-Disease
Factor	NN	O	I-Disease
H	NN	O	I-Disease
(	NN	O	O
FH	NN	O	O
)	NN	O	O
in	NN	O	O
the	NN	O	O
context	NN	O	O
of	NN	O	O
an	NN	O	O
acute	NN	O	B-Disease
renal	NN	O	I-Disease
disease	NN	O	I-Disease
.	NN	O	O

Five	NN	O	O
of	NN	O	O
the	NN	O	O
cases	NN	O	O
were	NN	O	O
observed	NN	O	O
in	NN	O	O
children	NN	O	O
presenting	NN	O	O
with	NN	O	O
idiopathic	NN	O	O
hemolytic	NN	O	B-Disease
uremic	NN	O	I-Disease
syndrome	NN	O	I-Disease
(	NN	O	O
HUS	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Two	NN	O	O
of	NN	O	O
the	NN	O	O
children	NN	O	O
exhibited	NN	O	O
a	NN	O	O
homozygous	NN	O	O
deficiency	NN	O	O
characterized	NN	O	O
by	NN	O	O
the	NN	O	O
absence	NN	O	O
of	NN	O	O
the	NN	O	O
150	NN	O	O
-	NN	O	O
kD	NN	O	O
form	NN	O	O
of	NN	O	O
Factor	NN	O	O
H	NN	O	O
and	NN	O	O
the	NN	O	O
presence	NN	O	O
,	NN	O	O
upon	NN	O	O
immunoblotting	NN	O	O
,	NN	O	O
of	NN	O	O
the	NN	O	O
42	NN	O	O
-	NN	O	O
kD	NN	O	O
Factor	NN	O	O
H	NN	O	O
-	NN	O	O
like	NN	O	O
protein	NN	O	O
1	NN	O	O
(	NN	O	O
FHL	NN	O	O
-	NN	O	O
1	NN	O	O
)	NN	O	O
and	NN	O	O
other	NN	O	O
FH	NN	O	O
-	NN	O	O
related	NN	O	O
protein	NN	O	O
(	NN	O	O
FHR	NN	O	O
)	NN	O	O
bands	NN	O	O
.	NN	O	O

Southern	NN	O	O
blot	NN	O	O
and	NN	O	O
PCR	NN	O	O
analysis	NN	O	O
of	NN	O	O
DNA	NN	O	O
of	NN	O	O
one	NN	O	O
patient	NN	O	O
with	NN	O	O
homozygous	NN	O	O
deficiency	NN	O	O
ruled	NN	O	O
out	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
a	NN	O	O
large	NN	O	O
deletion	NN	O	O
of	NN	O	O
the	NN	O	O
FH	NN	O	O
gene	NN	O	O
as	NN	O	O
the	NN	O	O
underlying	NN	O	O
defect	NN	O	O
for	NN	O	O
the	NN	O	O
deficiency	NN	O	O
.	NN	O	O

The	NN	O	O
other	NN	O	O
four	NN	O	O
children	NN	O	O
presented	NN	O	O
with	NN	O	O
heterozygous	NN	O	O
deficiency	NN	O	O
and	NN	O	O
exhibited	NN	O	O
a	NN	O	O
normal	NN	O	O
immunoblotting	NN	O	O
pattern	NN	O	O
of	NN	O	O
proteins	NN	O	O
of	NN	O	O
the	NN	O	O
FH	NN	O	O
family	NN	O	O
.	NN	O	O

Factor	NN	O	B-Disease
H	NN	O	I-Disease
deficiency	NN	O	I-Disease
is	NN	O	O
the	NN	O	O
only	NN	O	O
complement	NN	O	B-Disease
deficiency	NN	O	I-Disease
associated	NN	O	O
with	NN	O	O
HUS	NN	O	B-Disease
.	NN	O	O

These	NN	O	O
observations	NN	O	O
suggest	NN	O	O
a	NN	O	O
role	NN	O	O
for	NN	O	O
FH	NN	O	O
and	NN	O	O
/	NN	O	O
or	NN	O	O
FH	NN	O	O
receptors	NN	O	O
in	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
idiopathic	NN	O	O
HUS	NN	O	B-Disease
.	NN	O	O
.	NN	O	O

Further	NN	O	O
evidence	NN	O	O
for	NN	O	O
a	NN	O	O
major	NN	O	O
ancient	NN	O	O
mutation	NN	O	O
underlying	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
from	NN	O	O
linkage	NN	O	O
disequilibrium	NN	O	O
studies	NN	O	O
in	NN	O	O
the	NN	O	O
Japanese	NN	O	O
population	NN	O	O
.	NN	O	O

The	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
mutation	NN	O	O
is	NN	O	O
an	NN	O	O
unstable	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
,	NN	O	O
present	NN	O	O
at	NN	O	O
a	NN	O	O
copy	NN	O	O
number	NN	O	O
of	NN	O	O
5	NN	O	O
-	NN	O	O
37	NN	O	O
repeats	NN	O	O
on	NN	O	O
normal	NN	O	O
chromosomes	NN	O	O
but	NN	O	O
amplified	NN	O	O
to	NN	O	O
50	NN	O	O
-	NN	O	O
3000	NN	O	O
copies	NN	O	O
on	NN	O	O
DM	NN	O	B-Disease
chromosomes	NN	O	O
.	NN	O	O

Previous	NN	O	O
findings	NN	O	O
in	NN	O	O
Caucasian	NN	O	O
populations	NN	O	O
of	NN	O	O
a	NN	O	O
DM	NN	O	B-Disease
founder	NN	O	O
chromosome	NN	O	O
raise	NN	O	O
a	NN	O	O
question	NN	O	O
about	NN	O	O
the	NN	O	O
molecular	NN	O	O
events	NN	O	O
involved	NN	O	O
in	NN	O	O
the	NN	O	O
expansion	NN	O	O
mutation	NN	O	O
.	NN	O	O

To	NN	O	O
investigate	NN	O	O
whether	NN	O	O
a	NN	O	O
founder	NN	O	O
chromosome	NN	O	O
for	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
mutation	NN	O	O
exists	NN	O	O
in	NN	O	O
the	NN	O	O
Japanese	NN	O	O
population	NN	O	O
,	NN	O	O
we	NN	O	O
genotyped	NN	O	O
families	NN	O	O
using	NN	O	O
polymorphic	NN	O	O
markers	NN	O	O
near	NN	O	O
the	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
region	NN	O	O
and	NN	O	O
constructed	NN	O	O
haplotypes	NN	O	O
.	NN	O	O

Six	NN	O	O
different	NN	O	O
haplotypes	NN	O	O
were	NN	O	O
found	NN	O	O
and	NN	O	O
DM	NN	O	B-Disease
alleles	NN	O	O
were	NN	O	O
always	NN	O	O
haplotype	NN	O	O
A	NN	O	O
.	NN	O	O

To	NN	O	O
find	NN	O	O
an	NN	O	O
origin	NN	O	O
of	NN	O	O
the	NN	O	O
(	NN	O	O
CTG	NN	O	O
)	NN	O	O
n	NN	O	O
repeat	NN	O	O
mutation	NN	O	O
and	NN	O	O
to	NN	O	O
investigate	NN	O	O
the	NN	O	O
mechanism	NN	O	O
of	NN	O	O
the	NN	O	O
expansion	NN	O	O
mutation	NN	O	O
in	NN	O	O
the	NN	O	O
Japanese	NN	O	O
population	NN	O	O
we	NN	O	O
have	NN	O	O
studied	NN	O	O
90	NN	O	O
Japanese	NN	O	O
DM	NN	O	B-Disease
families	NN	O	O
comprising	NN	O	O
190	NN	O	O
affected	NN	O	O
and	NN	O	O
130	NN	O	O
unaffected	NN	O	O
members	NN	O	O
.	NN	O	O

The	NN	O	O
results	NN	O	O
suggest	NN	O	O
that	NN	O	O
a	NN	O	O
few	NN	O	O
common	NN	O	O
ancestral	NN	O	O
mutations	NN	O	O
in	NN	O	O
both	NN	O	O
Caucasian	NN	O	O
and	NN	O	O
Japanese	NN	O	O
populations	NN	O	O
have	NN	O	O
originated	NN	O	O
by	NN	O	O
expansion	NN	O	O
of	NN	O	O
an	NN	O	O
ancestral	NN	O	O
n	NN	O	O
=	NN	O	O
5	NN	O	O
repeat	NN	O	O
to	NN	O	O
n	NN	O	O
=	NN	O	O
19	NN	O	O
-	NN	O	O
37	NN	O	O
copies	NN	O	O
.	NN	O	O

These	NN	O	O
data	NN	O	O
support	NN	O	O
multistep	NN	O	O
models	NN	O	O
of	NN	O	O
triplet	NN	O	O
repeat	NN	O	O
expansion	NN	O	O
that	NN	O	O
have	NN	O	O
been	NN	O	O
proposed	NN	O	O
for	NN	O	O
both	NN	O	O
DM	NN	O	B-Disease
and	NN	O	O
Friedreichs	NN	O	B-Disease
ataxia	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

The	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
C6	NN	O	B-Disease
deficiency	NN	O	I-Disease
in	NN	O	O
the	NN	O	O
western	NN	O	O
Cape	NN	O	O
,	NN	O	O
South	NN	O	O
Africa	NN	O	O
.	NN	O	O

Deficiency	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
sixth	NN	O	I-Disease
component	NN	O	I-Disease
of	NN	O	I-Disease
human	NN	O	I-Disease
complement	NN	O	I-Disease
(	NN	O	O
C6	NN	O	O
)	NN	O	O
has	NN	O	O
been	NN	O	O
reported	NN	O	O
in	NN	O	O
a	NN	O	O
number	NN	O	O
of	NN	O	O
families	NN	O	O
from	NN	O	O
the	NN	O	O
western	NN	O	O
Cape	NN	O	O
,	NN	O	O
South	NN	O	O
Africa	NN	O	O
.	NN	O	O

Meningococcal	NN	O	B-Disease
disease	NN	O	I-Disease
is	NN	O	O
endemic	NN	O	O
in	NN	O	O
the	NN	O	O
Cape	NN	O	O
and	NN	O	O
almost	NN	O	O
all	NN	O	O
pedigrees	NN	O	O
of	NN	O	O
total	NN	O	O
C6	NN	O	B-Disease
deficiency	NN	O	I-Disease
(	NN	O	O
C6Q0	NN	O	O
)	NN	O	O
have	NN	O	O
been	NN	O	O
ascertained	NN	O	O
because	NN	O	O
of	NN	O	O
recurrent	NN	O	O
disease	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
sequenced	NN	O	O
the	NN	O	O
expressed	NN	O	O
exons	NN	O	O
of	NN	O	O
the	NN	O	O
C6	NN	O	O
gene	NN	O	O
from	NN	O	O
selected	NN	O	O
cases	NN	O	O
and	NN	O	O
have	NN	O	O
found	NN	O	O
three	NN	O	O
molecular	NN	O	O
defects	NN	O	O
leading	NN	O	O
to	NN	O	O
total	NN	O	O
deficiency	NN	O	O
879delG	NN	O	O
,	NN	O	O
which	NN	O	O
is	NN	O	O
the	NN	O	O
common	NN	O	O
defect	NN	O	O
in	NN	O	O
the	NN	O	O
Cape	NN	O	O
and	NN	O	O
hitherto	NN	O	O
unreported	NN	O	O
,	NN	O	O
and	NN	O	O
1195delC	NN	O	O
and	NN	O	O
1936delG	NN	O	O
,	NN	O	O
which	NN	O	O
have	NN	O	O
been	NN	O	O
previously	NN	O	O
reported	NN	O	O
in	NN	O	O
African	NN	O	O
-	NN	O	O
Americans	NN	O	O
.	NN	O	O

We	NN	O	O
also	NN	O	O
show	NN	O	O
that	NN	O	O
the	NN	O	O
879delG	NN	O	O
and	NN	O	O
1195delC	NN	O	O
defects	NN	O	O
are	NN	O	O
associated	NN	O	O
with	NN	O	O
characteristic	NN	O	O
C6	NN	O	O
/	NN	O	O
C7	NN	O	O
region	NN	O	O
DNA	NN	O	O
marker	NN	O	O
haplotypes	NN	O	O
,	NN	O	O
although	NN	O	O
small	NN	O	O
variations	NN	O	O
were	NN	O	O
observed	NN	O	O
.	NN	O	O

The	NN	O	O
1936delG	NN	O	O
defect	NN	O	O
was	NN	O	O
observed	NN	O	O
only	NN	O	O
once	NN	O	O
in	NN	O	O
the	NN	O	O
Cape	NN	O	O
,	NN	O	O
but	NN	O	O
its	NN	O	O
associated	NN	O	O
haplotype	NN	O	O
could	NN	O	O
be	NN	O	O
deduced	NN	O	O
.	NN	O	O

The	NN	O	O
data	NN	O	O
from	NN	O	O
the	NN	O	O
haplotypes	NN	O	O
indicate	NN	O	O
that	NN	O	O
these	NN	O	O
three	NN	O	O
molecular	NN	O	O
defects	NN	O	O
account	NN	O	O
for	NN	O	O
the	NN	O	O
defects	NN	O	O
in	NN	O	O
all	NN	O	O
the	NN	O	O
38	NN	O	O
unrelated	NN	O	O
C6Q0	NN	O	O
individuals	NN	O	O
we	NN	O	O
have	NN	O	O
studied	NN	O	O
from	NN	O	O
the	NN	O	O
Cape	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
also	NN	O	O
observed	NN	O	O
the	NN	O	O
879delG	NN	O	O
defect	NN	O	O
in	NN	O	O
two	NN	O	O
Dutch	NN	O	O
C6	NN	O	B-Disease
-	NN	O	I-Disease
deficient	NN	O	I-Disease
kindreds	NN	O	O
,	NN	O	O
but	NN	O	O
the	NN	O	O
879delG	NN	O	O
defect	NN	O	O
in	NN	O	O
the	NN	O	O
Cape	NN	O	O
probably	NN	O	O
did	NN	O	O
not	NN	O	O
come	NN	O	O
from	NN	O	O
The	NN	O	O
Netherlands	NN	O	O
.	NN	O	O
.	NN	O	O

Complement	NN	O	B-Disease
C7	NN	O	I-Disease
deficiency	NN	O	I-Disease
:	NN	O	O
seven	NN	O	O
further	NN	O	O
molecular	NN	O	O
defects	NN	O	O
and	NN	O	O
their	NN	O	O
associated	NN	O	O
marker	NN	O	O
haplotypes	NN	O	O
.	NN	O	O

Seven	NN	O	O
further	NN	O	O
molecular	NN	O	O
bases	NN	O	O
of	NN	O	O
C7	NN	O	B-Disease
deficiency	NN	O	I-Disease
are	NN	O	O
described	NN	O	O
.	NN	O	O

All	NN	O	O
these	NN	O	O
new	NN	O	O
molecular	NN	O	O
defects	NN	O	O
involve	NN	O	O
single	NN	O	O
-	NN	O	O
nucleotide	NN	O	O
events	NN	O	O
,	NN	O	O
deletions	NN	O	O
and	NN	O	O
substitutions	NN	O	O
,	NN	O	O
some	NN	O	O
of	NN	O	O
which	NN	O	O
alter	NN	O	O
splice	NN	O	O
sites	NN	O	O
,	NN	O	O
and	NN	O	O
others	NN	O	O
codons	NN	O	O
.	NN	O	O

They	NN	O	O
are	NN	O	O
distributed	NN	O	O
along	NN	O	O
the	NN	O	O
C7	NN	O	O
gene	NN	O	O
,	NN	O	O
but	NN	O	O
predominantly	NN	O	O
towards	NN	O	O
the	NN	O	O
3	NN	O	O
end	NN	O	O
.	NN	O	O

All	NN	O	O
were	NN	O	O
found	NN	O	O
in	NN	O	O
compound	NN	O	O
heterozygous	NN	O	O
individuals	NN	O	O
.	NN	O	O

The	NN	O	O
C6	NN	O	O
/	NN	O	O
C7	NN	O	O
marker	NN	O	O
haplotypes	NN	O	O
associated	NN	O	O
with	NN	O	O
most	NN	O	O
C7	NN	O	B-Disease
defects	NN	O	I-Disease
are	NN	O	O
tabulated	NN	O	O
.	NN	O	O
.	NN	O	O

A	NN	O	O
genome	NN	O	O
-	NN	O	O
wide	NN	O	O
search	NN	O	O
for	NN	O	O
chromosomal	NN	O	O
loci	NN	O	O
linked	NN	O	O
to	NN	O	O
mental	NN	O	O
health	NN	O	O
wellness	NN	O	O
in	NN	O	O
relatives	NN	O	O
at	NN	O	O
high	NN	O	O
risk	NN	O	O
for	NN	O	O
bipolar	NN	O	B-Disease
affective	NN	O	I-Disease
disorder	NN	O	I-Disease
among	NN	O	O
the	NN	O	O
Old	NN	O	O
Order	NN	O	O
Amish	NN	O	O
.	NN	O	O

Bipolar	NN	O	B-Disease
affective	NN	O	I-Disease
disorder	NN	O	I-Disease
(	NN	O	O
BPAD	NN	O	B-Disease
;	NN	O	O
manic	NN	O	B-Disease
-	NN	O	I-Disease
depressive	NN	O	I-Disease
illness	NN	O	I-Disease
)	NN	O	O
is	NN	O	O
characterized	NN	O	O
by	NN	O	O
episodes	NN	O	O
of	NN	O	O
mania	NN	O	B-Disease
and	NN	O	O
/	NN	O	O
or	NN	O	O
hypomania	NN	O	B-Disease
interspersed	NN	O	O
with	NN	O	O
periods	NN	O	O
of	NN	O	O
depression	NN	O	B-Disease
.	NN	O	O

Compelling	NN	O	O
evidence	NN	O	O
supports	NN	O	O
a	NN	O	O
significant	NN	O	O
genetic	NN	O	O
component	NN	O	O
in	NN	O	O
the	NN	O	O
susceptibility	NN	O	O
to	NN	O	O
develop	NN	O	O
BPAD	NN	O	B-Disease
.	NN	O	O

To	NN	O	O
date	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
linkage	NN	O	O
studies	NN	O	O
have	NN	O	O
attempted	NN	O	O
only	NN	O	O
to	NN	O	O
identify	NN	O	O
chromosomal	NN	O	O
loci	NN	O	O
that	NN	O	O
cause	NN	O	O
or	NN	O	O
increase	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
developing	NN	O	O
BPAD	NN	O	B-Disease
.	NN	O	O

To	NN	O	O
determine	NN	O	O
whether	NN	O	O
there	NN	O	O
could	NN	O	O
be	NN	O	O
protective	NN	O	O
alleles	NN	O	O
that	NN	O	O
prevent	NN	O	O
or	NN	O	O
reduce	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
developing	NN	O	O
BPAD	NN	O	B-Disease
,	NN	O	O
similar	NN	O	O
to	NN	O	O
what	NN	O	O
is	NN	O	O
observed	NN	O	O
in	NN	O	O
other	NN	O	O
genetic	NN	O	B-Disease
disorders	NN	O	I-Disease
,	NN	O	O
we	NN	O	O
used	NN	O	O
mental	NN	O	O
health	NN	O	O
wellness	NN	O	O
(	NN	O	O
absence	NN	O	O
of	NN	O	O
any	NN	O	O
psychiatric	NN	O	B-Disease
disorder	NN	O	I-Disease
)	NN	O	O
as	NN	O	O
the	NN	O	O
phenotype	NN	O	O
in	NN	O	O
our	NN	O	O
genome	NN	O	O
-	NN	O	O
wide	NN	O	O
linkage	NN	O	O
scan	NN	O	O
of	NN	O	O
several	NN	O	O
large	NN	O	O
multigeneration	NN	O	O
Old	NN	O	O
Order	NN	O	O
Amish	NN	O	O
pedigrees	NN	O	O
exhibiting	NN	O	O
an	NN	O	O
extremely	NN	O	O
high	NN	O	O
incidence	NN	O	O
of	NN	O	O
BPAD	NN	O	B-Disease
.	NN	O	O

We	NN	O	O
have	NN	O	O
found	NN	O	O
strong	NN	O	O
evidence	NN	O	O
for	NN	O	O
a	NN	O	O
locus	NN	O	O
on	NN	O	O
chromosome	NN	O	O
4p	NN	O	O
at	NN	O	O
D4S2949	NN	O	O
(	NN	O	O
maximum	NN	O	O
GENEHUNTER	NN	O	O
-	NN	O	O
PLUS	NN	O	O
nonparametric	NN	O	O
linkage	NN	O	O
score	NN	O	O
=	NN	O	O
4	NN	O	O
.	NN	O	O
05	NN	O	O
,	NN	O	O
P	NN	O	O
=	NN	O	O
5	NN	O	O
.	NN	O	O
22	NN	O	O
x	NN	O	O
10	NN	O	O
(	NN	O	O
-	NN	O	O
4	NN	O	O
)	NN	O	O
;	NN	O	O
SIBPAL	NN	O	O
Pempirical	NN	O	O
value	NN	O	O
<	NN	O	O
3	NN	O	O
x	NN	O	O
10	NN	O	O
(	NN	O	O
-	NN	O	O
5	NN	O	O
)	NN	O	O
)	NN	O	O
and	NN	O	O
suggestive	NN	O	O
evidence	NN	O	O
for	NN	O	O
a	NN	O	O
locus	NN	O	O
on	NN	O	O
chromosome	NN	O	O
4q	NN	O	O
at	NN	O	O
D4S397	NN	O	O
(	NN	O	O
maximum	NN	O	O
GENEHUNTER	NN	O	O
-	NN	O	O
PLUS	NN	O	O
nonparametric	NN	O	O
linkage	NN	O	O
score	NN	O	O
=	NN	O	O
3	NN	O	O
.	NN	O	O
29	NN	O	O
,	NN	O	O
P	NN	O	O
=	NN	O	O
2	NN	O	O
.	NN	O	O
57	NN	O	O
x	NN	O	O
10	NN	O	O
(	NN	O	O
-	NN	O	O
3	NN	O	O
)	NN	O	O
;	NN	O	O
SIBPAL	NN	O	O
Pempirical	NN	O	O
value	NN	O	O
<	NN	O	O
1	NN	O	O
x	NN	O	O
10	NN	O	O
(	NN	O	O
-	NN	O	O
3	NN	O	O
)	NN	O	O
)	NN	O	O
that	NN	O	O
are	NN	O	O
linked	NN	O	O
to	NN	O	O
mental	NN	O	O
health	NN	O	O
wellness	NN	O	O
.	NN	O	O

These	NN	O	O
findings	NN	O	O
are	NN	O	O
consistent	NN	O	O
with	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
certain	NN	O	O
alleles	NN	O	O
could	NN	O	O
prevent	NN	O	O
or	NN	O	O
modify	NN	O	O
the	NN	O	O
clinical	NN	O	O
manifestations	NN	O	O
of	NN	O	O
BPAD	NN	O	B-Disease
and	NN	O	O
perhaps	NN	O	O
other	NN	O	O
related	NN	O	O
affective	NN	O	B-Disease
disorders	NN	O	I-Disease
.	NN	O	O

Segregation	NN	O	O
distortion	NN	O	O
in	NN	O	O
myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
.	NN	O	O

Myotonic	NN	O	B-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
DM	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
autosomal	NN	O	B-Disease
dominant	NN	O	I-Disease
disease	NN	O	I-Disease
which	NN	O	O
,	NN	O	O
in	NN	O	O
the	NN	O	O
typical	NN	O	O
pedigree	NN	O	O
,	NN	O	O
shows	NN	O	O
a	NN	O	O
three	NN	O	O
generation	NN	O	O
anticipation	NN	O	O
cascade	NN	O	O
.	NN	O	O

This	NN	O	O
results	NN	O	O
in	NN	O	O
infertility	NN	O	B-Disease
and	NN	O	O
congenital	NN	O	B-Disease
myotonic	NN	O	I-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
CDM	NN	O	B-Disease
)	NN	O	O
with	NN	O	O
the	NN	O	O
disappearance	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
in	NN	O	O
that	NN	O	O
pedigree	NN	O	O
.	NN	O	O

The	NN	O	O
concept	NN	O	O
of	NN	O	O
segregation	NN	O	O
distortion	NN	O	O
,	NN	O	O
where	NN	O	O
there	NN	O	O
is	NN	O	O
preferential	NN	O	O
transmission	NN	O	O
of	NN	O	O
the	NN	O	O
larger	NN	O	O
allele	NN	O	O
at	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
locus	NN	O	O
,	NN	O	O
has	NN	O	O
been	NN	O	O
put	NN	O	O
forward	NN	O	O
to	NN	O	O
explain	NN	O	O
partially	NN	O	O
the	NN	O	O
maintenance	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
in	NN	O	O
the	NN	O	O
population	NN	O	O
.	NN	O	O

In	NN	O	O
a	NN	O	O
survey	NN	O	O
of	NN	O	O
DM	NN	O	B-Disease
in	NN	O	O
Northern	NN	O	O
Ireland	NN	O	O
,	NN	O	O
59	NN	O	O
pedigrees	NN	O	O
were	NN	O	O
ascertained	NN	O	O
.	NN	O	O

Sibships	NN	O	O
where	NN	O	O
the	NN	O	O
status	NN	O	O
of	NN	O	O
all	NN	O	O
the	NN	O	O
members	NN	O	O
had	NN	O	O
been	NN	O	O
identified	NN	O	O
were	NN	O	O
examined	NN	O	O
to	NN	O	O
determine	NN	O	O
the	NN	O	O
transmission	NN	O	O
of	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
expansion	NN	O	O
from	NN	O	O
affected	NN	O	O
parents	NN	O	O
to	NN	O	O
their	NN	O	O
offspring	NN	O	O
.	NN	O	O

Where	NN	O	O
the	NN	O	O
transmitting	NN	O	O
parent	NN	O	O
was	NN	O	O
male	NN	O	O
,	NN	O	O
58	NN	O	O
.	NN	O	O

3	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
offspring	NN	O	O
were	NN	O	O
affected	NN	O	O
,	NN	O	O
and	NN	O	O
in	NN	O	O
the	NN	O	O
case	NN	O	O
of	NN	O	O
a	NN	O	O
female	NN	O	O
transmitting	NN	O	O
parent	NN	O	O
,	NN	O	O
68	NN	O	O
.	NN	O	O

7	NN	O	O
%	NN	O	O
were	NN	O	O
affected	NN	O	O
.	NN	O	O

Studies	NN	O	O
on	NN	O	O
meiotic	NN	O	O
drive	NN	O	O
in	NN	O	O
DM	NN	O	B-Disease
have	NN	O	O
shown	NN	O	O
increased	NN	O	O
transmission	NN	O	O
of	NN	O	O
the	NN	O	O
larger	NN	O	O
allele	NN	O	O
at	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
locus	NN	O	O
in	NN	O	O
non	NN	O	O
-	NN	O	O
DM	NN	O	O
heterozygotes	NN	O	O
for	NN	O	O
CTGn	NN	O	O
.	NN	O	O

This	NN	O	O
study	NN	O	O
provides	NN	O	O
further	NN	O	O
evidence	NN	O	O
that	NN	O	O
the	NN	O	O
DM	NN	O	B-Disease
expansion	NN	O	O
tends	NN	O	O
to	NN	O	O
be	NN	O	O
transmitted	NN	O	O
preferentially	NN	O	O
.	NN	O	O

Diagnosis	NN	O	O
of	NN	O	O
hemochromatosis	NN	O	B-Disease
.	NN	O	O

If	NN	O	O
untreated	NN	O	O
,	NN	O	O
hemochromatosis	NN	O	B-Disease
can	NN	O	O
cause	NN	O	O
serious	NN	O	O
illness	NN	O	O
and	NN	O	O
early	NN	O	B-Disease
death	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
the	NN	O	O
disease	NN	O	O
is	NN	O	O
still	NN	O	O
substantially	NN	O	O
under	NN	O	O
-	NN	O	O
diagnosed	NN	O	O
.	NN	O	O

The	NN	O	O
cornerstone	NN	O	O
of	NN	O	O
screening	NN	O	O
and	NN	O	O
case	NN	O	O
detection	NN	O	O
is	NN	O	O
the	NN	O	O
measurement	NN	O	O
of	NN	O	O
serum	NN	O	O
transferrin	NN	O	O
saturation	NN	O	O
and	NN	O	O
the	NN	O	O
serum	NN	O	O
ferritin	NN	O	O
level	NN	O	O
.	NN	O	O

Once	NN	O	O
the	NN	O	O
diagnosis	NN	O	O
is	NN	O	O
suspected	NN	O	O
,	NN	O	O
physicians	NN	O	O
must	NN	O	O
use	NN	O	O
serum	NN	O	O
ferritin	NN	O	O
levels	NN	O	O
and	NN	O	O
hepatic	NN	O	O
iron	NN	O	O
stores	NN	O	O
on	NN	O	O
liver	NN	O	O
biopsy	NN	O	O
specimens	NN	O	O
to	NN	O	O
assess	NN	O	O
patients	NN	O	O
for	NN	O	O
the	NN	O	O
presence	NN	O	O
of	NN	O	O
iron	NN	O	B-Disease
overload	NN	O	I-Disease
.	NN	O	O

Liver	NN	O	O
biopsy	NN	O	O
is	NN	O	O
also	NN	O	O
used	NN	O	O
to	NN	O	O
establish	NN	O	O
the	NN	O	O
presence	NN	O	O
or	NN	O	O
absence	NN	O	O
of	NN	O	O
cirrhosis	NN	O	B-Disease
,	NN	O	O
which	NN	O	O
can	NN	O	O
affect	NN	O	O
prognosis	NN	O	O
and	NN	O	O
management	NN	O	O
.	NN	O	O

A	NN	O	O
DNA	NN	O	O
-	NN	O	O
based	NN	O	O
test	NN	O	O
for	NN	O	O
the	NN	O	O
HFE	NN	O	O
gene	NN	O	O
is	NN	O	O
commercially	NN	O	O
available	NN	O	O
,	NN	O	O
but	NN	O	O
its	NN	O	O
place	NN	O	O
in	NN	O	O
the	NN	O	O
diagnosis	NN	O	O
of	NN	O	O
hemochromatosis	NN	O	B-Disease
is	NN	O	O
still	NN	O	O
being	NN	O	O
evaluated	NN	O	O
.	NN	O	O

Currently	NN	O	O
,	NN	O	O
the	NN	O	O
most	NN	O	O
useful	NN	O	O
role	NN	O	O
for	NN	O	O
this	NN	O	O
test	NN	O	O
is	NN	O	O
in	NN	O	O
the	NN	O	O
detection	NN	O	O
of	NN	O	O
hemochromatosis	NN	O	B-Disease
in	NN	O	O
the	NN	O	O
family	NN	O	O
members	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
a	NN	O	O
proven	NN	O	O
case	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O

It	NN	O	O
is	NN	O	O
crucial	NN	O	O
to	NN	O	O
diagnose	NN	O	O
hemochromatosis	NN	O	B-Disease
before	NN	O	O
hepatic	NN	O	B-Disease
cirrhosis	NN	O	I-Disease
develops	NN	O	O
because	NN	O	O
phlebotomy	NN	O	O
therapy	NN	O	O
can	NN	O	O
avert	NN	O	O
serious	NN	O	O
chronic	NN	O	O
disease	NN	O	O
and	NN	O	O
can	NN	O	O
even	NN	O	O
lead	NN	O	O
to	NN	O	O
normal	NN	O	O
life	NN	O	O
expectancy	NN	O	O
.	NN	O	O
.	NN	O	O

Prevalence	NN	O	O
of	NN	O	O
the	NN	O	O
I1307K	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
variant	NN	O	O
in	NN	O	O
Israeli	NN	O	O
Jews	NN	O	O
of	NN	O	O
differing	NN	O	O
ethnic	NN	O	O
origin	NN	O	O
and	NN	O	O
risk	NN	O	O
for	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

BACKGROUND	NN	O	O
&	NN	O	O
AIMS	NN	O	O
Israeli	NN	O	O
Jews	NN	O	O
of	NN	O	O
European	NN	O	O
birth	NN	O	O
,	NN	O	O
i	NN	O	O
.	NN	O	O
e	NN	O	O
.	NN	O	O
,	NN	O	O
Ashkenazim	NN	O	O
,	NN	O	O
have	NN	O	O
the	NN	O	O
highest	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
incidence	NN	O	O
of	NN	O	O
any	NN	O	O
Israeli	NN	O	O
ethnic	NN	O	O
group	NN	O	O
.	NN	O	O

The	NN	O	O
I1307K	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
variant	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
6	NN	O	O
.	NN	O	O

1	NN	O	O
%	NN	O	O
of	NN	O	O
American	NN	O	O
Jews	NN	O	O
,	NN	O	O
28	NN	O	O
%	NN	O	O
of	NN	O	O
their	NN	O	O
familial	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
cases	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
in	NN	O	O
non	NN	O	O
-	NN	O	O
Jews	NN	O	O
.	NN	O	O

We	NN	O	O
assessed	NN	O	O
the	NN	O	O
I1307K	NN	O	O
prevalence	NN	O	O
in	NN	O	O
Israeli	NN	O	O
Jews	NN	O	O
of	NN	O	O
differing	NN	O	O
ethnic	NN	O	O
origin	NN	O	O
and	NN	O	O
risk	NN	O	O
for	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

METHODS	NN	O	O
DNA	NN	O	O
samples	NN	O	O
from	NN	O	O
500	NN	O	O
unrelated	NN	O	O
Jews	NN	O	O
of	NN	O	O
European	NN	O	O
or	NN	O	O
non	NN	O	O
-	NN	O	O
European	NN	O	O
origin	NN	O	O
,	NN	O	O
with	NN	O	O
or	NN	O	O
without	NN	O	O
a	NN	O	O
personal	NN	O	O
and	NN	O	O
/	NN	O	O
or	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
neoplasia	NN	O	B-Disease
,	NN	O	O
were	NN	O	O
examined	NN	O	O
for	NN	O	O
the	NN	O	O
I1307K	NN	O	O
variant	NN	O	O
by	NN	O	O
the	NN	O	O
allele	NN	O	O
-	NN	O	O
specific	NN	O	O
oligonucleotide	NN	O	O
(	NN	O	O
ASO	NN	O	O
)	NN	O	O
method	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
In	NN	O	O
persons	NN	O	O
at	NN	O	O
average	NN	O	O
risk	NN	O	O
for	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	O
I1307K	NN	O	O
was	NN	O	O
found	NN	O	O
in	NN	O	O
5	NN	O	O
.	NN	O	O

0	NN	O	O
%	NN	O	O
of	NN	O	O
120	NN	O	O
European	NN	O	O
and	NN	O	O
1	NN	O	O
.	NN	O	O

6	NN	O	O
%	NN	O	O
of	NN	O	O
188	NN	O	O
non	NN	O	O
-	NN	O	O
European	NN	O	O
Jews	NN	O	O
(	NN	O	O
P	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
08	NN	O	O
)	NN	O	O
.	NN	O	O

It	NN	O	O
occurred	NN	O	O
in	NN	O	O
15	NN	O	O
.	NN	O	O

4	NN	O	O
%	NN	O	O
of	NN	O	O
52	NN	O	O
Ashkenazi	NN	O	O
Israelis	NN	O	O
with	NN	O	O
familial	NN	O	O
cancer	NN	O	B-Disease
(	NN	O	O
P	NN	O	O
=	NN	O	O
0	NN	O	O
.	NN	O	O
02	NN	O	O
)	NN	O	O
and	NN	O	O
was	NN	O	O
not	NN	O	O
detected	NN	O	O
in	NN	O	O
51	NN	O	O
non	NN	O	O
-	NN	O	O
European	NN	O	O
Jews	NN	O	O
at	NN	O	O
increased	NN	O	O
cancer	NN	O	B-Disease
risk	NN	O	O
.	NN	O	O

Colorectal	NN	O	B-Disease
neoplasia	NN	O	I-Disease
occurred	NN	O	O
personally	NN	O	O
or	NN	O	O
in	NN	O	O
the	NN	O	O
families	NN	O	O
of	NN	O	O
13	NN	O	O
of	NN	O	O
20	NN	O	O
Ashkenazi	NN	O	O
I1307K	NN	O	O
carriers	NN	O	O
,	NN	O	O
8	NN	O	O
of	NN	O	O
whom	NN	O	O
also	NN	O	O
had	NN	O	O
a	NN	O	O
personal	NN	O	O
or	NN	O	O
family	NN	O	O
history	NN	O	O
of	NN	O	O
noncolonic	NN	O	O
neoplasia	NN	O	B-Disease
.	NN	O	O

CONCLUSIONS	NN	O	O
The	NN	O	O
I1307K	NN	O	O
APC	NN	O	O
variant	NN	O	O
may	NN	O	O
represent	NN	O	O
a	NN	O	O
susceptibility	NN	O	O
gene	NN	O	O
for	NN	O	O
colorectal	NN	O	B-Disease
,	NN	O	I-Disease
or	NN	O	I-Disease
other	NN	O	I-Disease
,	NN	O	I-Disease
cancers	NN	O	I-Disease
in	NN	O	O
Ashkenazi	NN	O	O
Jews	NN	O	O
,	NN	O	O
and	NN	O	O
partially	NN	O	O
explains	NN	O	O
the	NN	O	O
higher	NN	O	O
incidence	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
in	NN	O	O
European	NN	O	O
Israelis	NN	O	O
.	NN	O	O

Systematic	NN	O	O
analysis	NN	O	O
of	NN	O	O
coproporphyrinogen	NN	O	O
oxidase	NN	O	O
gene	NN	O	O
defects	NN	O	O
in	NN	O	O
hereditary	NN	O	B-Disease
coproporphyria	NN	O	I-Disease
and	NN	O	O
mutation	NN	O	O
update	NN	O	O
.	NN	O	O

Hereditary	NN	O	B-Disease
coproporphyria	NN	O	I-Disease
(	NN	O	O
HC	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
an	NN	O	O
acute	NN	O	O
hepatic	NN	O	B-Disease
porphyria	NN	O	I-Disease
with	NN	O	O
autosomal	NN	O	O
dominant	NN	O	O
inheritance	NN	O	O
caused	NN	O	O
by	NN	O	O
deficient	NN	O	B-Disease
activity	NN	O	I-Disease
of	NN	O	I-Disease
coproporphyrinogen	NN	O	I-Disease
III	NN	O	I-Disease
oxidase	NN	O	I-Disease
(	NN	O	O
CPO	NN	O	O
)	NN	O	O
.	NN	O	O

Clinical	NN	O	O
manifestations	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
are	NN	O	O
characterized	NN	O	O
by	NN	O	O
acute	NN	O	O
attacks	NN	O	O
of	NN	O	O
neurological	NN	O	B-Disease
dysfunction	NN	O	I-Disease
often	NN	O	O
precipitated	NN	O	O
by	NN	O	O
drugs	NN	O	O
,	NN	O	O
fasting	NN	O	O
,	NN	O	O
cyclical	NN	O	O
hormonal	NN	O	O
changes	NN	O	O
,	NN	O	O
or	NN	O	O
infectious	NN	O	B-Disease
diseases	NN	O	I-Disease
.	NN	O	O

Skin	NN	O	O
photosensitivity	NN	O	O
may	NN	O	O
also	NN	O	O
be	NN	O	O
present	NN	O	O
.	NN	O	O

The	NN	O	O
seven	NN	O	O
exons	NN	O	O
,	NN	O	O
the	NN	O	O
exon	NN	O	O
/	NN	O	O
intron	NN	O	O
boundaries	NN	O	O
and	NN	O	O
part	NN	O	O
of	NN	O	O
3	NN	O	O
noncoding	NN	O	O
sequence	NN	O	O
of	NN	O	O
the	NN	O	O
CPO	NN	O	O
gene	NN	O	O
were	NN	O	O
systematically	NN	O	O
analyzed	NN	O	O
by	NN	O	O
an	NN	O	O
exon	NN	O	O
-	NN	O	O
by	NN	O	O
-	NN	O	O
exon	NN	O	O
denaturing	NN	O	O
gradient	NN	O	O
gel	NN	O	O
electrophoresis	NN	O	O
(	NN	O	O
DGGE	NN	O	O
)	NN	O	O
strategy	NN	O	O
followed	NN	O	O
by	NN	O	O
direct	NN	O	O
sequencing	NN	O	O
in	NN	O	O
seven	NN	O	O
unrelated	NN	O	O
heterozygous	NN	O	O
HC	NN	O	B-Disease
patients	NN	O	O
from	NN	O	O
France	NN	O	O
,	NN	O	O
Holland	NN	O	O
,	NN	O	O
and	NN	O	O
Czech	NN	O	O
Republic	NN	O	O
.	NN	O	O

Seven	NN	O	O
novel	NN	O	O
mutations	NN	O	O
and	NN	O	O
two	NN	O	O
new	NN	O	O
polymorphisms	NN	O	O
were	NN	O	O
detected	NN	O	O
.	NN	O	O

Among	NN	O	O
these	NN	O	O
mutations	NN	O	O
two	NN	O	O
are	NN	O	O
missense	NN	O	O
(	NN	O	O
G197W	NN	O	O
,	NN	O	O
W427R	NN	O	O
)	NN	O	O
,	NN	O	O
two	NN	O	O
are	NN	O	O
nonsense	NN	O	O
(	NN	O	O
Q306X	NN	O	O
,	NN	O	O
Q385X	NN	O	O
)	NN	O	O
,	NN	O	O
two	NN	O	O
are	NN	O	O
small	NN	O	O
deletions	NN	O	O
(	NN	O	O
662de14bp	NN	O	O
;	NN	O	O
1168del3bp	NN	O	O
removing	NN	O	O
a	NN	O	O
glycine	NN	O	O
at	NN	O	O
position	NN	O	O
390	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
one	NN	O	O
is	NN	O	O
a	NN	O	O
splicing	NN	O	O
mutation	NN	O	O
(	NN	O	O
IVS1	NN	O	O
-	NN	O	O
15c	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
g	NN	O	O
)	NN	O	O
which	NN	O	O
creates	NN	O	O
a	NN	O	O
new	NN	O	O
acceptor	NN	O	O
splice	NN	O	O
site	NN	O	O
.	NN	O	O

The	NN	O	O
pathological	NN	O	O
significance	NN	O	O
of	NN	O	O
the	NN	O	O
point	NN	O	O
mutations	NN	O	O
G197W	NN	O	O
,	NN	O	O
W427R	NN	O	O
,	NN	O	O
and	NN	O	O
the	NN	O	O
in	NN	O	O
-	NN	O	O
frame	NN	O	O
deletion	NN	O	O
390delGly	NN	O	O
were	NN	O	O
assessed	NN	O	O
by	NN	O	O
their	NN	O	O
respective	NN	O	O
expression	NN	O	O
in	NN	O	O
a	NN	O	O
prokaryotic	NN	O	O
system	NN	O	O
using	NN	O	O
site	NN	O	O
-	NN	O	O
directed	NN	O	O
mutagenesis	NN	O	O
.	NN	O	O

These	NN	O	O
mutations	NN	O	O
resulted	NN	O	O
in	NN	O	O
the	NN	O	O
absence	NN	O	O
or	NN	O	O
a	NN	O	O
dramatic	NN	O	O
decrease	NN	O	O
of	NN	O	O
CPO	NN	O	O
activity	NN	O	O
.	NN	O	O

The	NN	O	O
two	NN	O	O
polymorphisms	NN	O	O
were	NN	O	O
localized	NN	O	O
in	NN	O	O
noncoding	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
gene	NN	O	O
1	NN	O	O
)	NN	O	O
a	NN	O	O
C	NN	O	O
/	NN	O	O
G	NN	O	O
polymorphism	NN	O	O
in	NN	O	O
the	NN	O	O
promotor	NN	O	O
region	NN	O	O
,	NN	O	O
142	NN	O	O
bp	NN	O	O
upstream	NN	O	O
from	NN	O	O
the	NN	O	O
transcriptional	NN	O	O
initiation	NN	O	O
site	NN	O	O
(	NN	O	O
-	NN	O	O
142C	NN	O	O
/	NN	O	O
G	NN	O	O
)	NN	O	O
,	NN	O	O
and	NN	O	O
2	NN	O	O
)	NN	O	O
a	NN	O	O
6	NN	O	O
bp	NN	O	O
deletion	NN	O	O
polymorphism	NN	O	O
in	NN	O	O
the	NN	O	O
3	NN	O	O
noncoding	NN	O	O
part	NN	O	O
of	NN	O	O
the	NN	O	O
CPO	NN	O	O
gene	NN	O	O
,	NN	O	O
574	NN	O	O
bp	NN	O	O
downstream	NN	O	O
of	NN	O	O
the	NN	O	O
last	NN	O	O
base	NN	O	O
of	NN	O	O
the	NN	O	O
normal	NN	O	O
termination	NN	O	O
codon	NN	O	O
(	NN	O	O
+	NN	O	O
574	NN	O	O
delATTCTT	NN	O	O
)	NN	O	O
.	NN	O	O

Five	NN	O	O
intragenic	NN	O	O
dimorphisms	NN	O	O
are	NN	O	O
now	NN	O	O
well	NN	O	O
characterized	NN	O	O
and	NN	O	O
the	NN	O	O
high	NN	O	O
degree	NN	O	O
of	NN	O	O
allelic	NN	O	O
heterogeneity	NN	O	O
in	NN	O	O
HC	NN	O	B-Disease
is	NN	O	O
demonstrated	NN	O	O
with	NN	O	O
seven	NN	O	O
new	NN	O	O
different	NN	O	O
mutations	NN	O	O
making	NN	O	O
a	NN	O	O
total	NN	O	O
of	NN	O	O
nineteen	NN	O	O
CPO	NN	O	O
gene	NN	O	B-Disease
defects	NN	O	I-Disease
reported	NN	O	O
so	NN	O	O
far	NN	O	O
.	NN	O	O
.	NN	O	O

Coincidence	NN	O	O
of	NN	O	O
two	NN	O	O
novel	NN	O	O
arylsulfatase	NN	O	O
A	NN	O	O
alleles	NN	O	O
and	NN	O	O
mutation	NN	O	O
459	NN	O	O
+	NN	O	O
1G	NN	O	O
>	NN	O	O
A	NN	O	O
within	NN	O	O
a	NN	O	O
family	NN	O	O
with	NN	O	O
metachromatic	NN	O	B-Disease
leukodystrophy	NN	O	I-Disease
:	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
phenotypic	NN	O	O
heterogeneity	NN	O	O
.	NN	O	O

In	NN	O	O
a	NN	O	O
family	NN	O	O
with	NN	O	O
three	NN	O	O
siblings	NN	O	O
,	NN	O	O
one	NN	O	O
developed	NN	O	O
classical	NN	O	O
late	NN	O	O
infantile	NN	O	O
metachromatic	NN	O	B-Disease
leukodystrophy	NN	O	I-Disease
(	NN	O	O
MLD	NN	O	B-Disease
)	NN	O	O
,	NN	O	O
fatal	NN	O	O
at	NN	O	O
age	NN	O	O
5	NN	O	O
years	NN	O	O
,	NN	O	O
with	NN	O	O
deficient	NN	O	O
arylsulfatase	NN	O	O
A	NN	O	O
(	NN	O	O
ARSA	NN	O	O
)	NN	O	O
activity	NN	O	O
and	NN	O	O
increased	NN	O	O
galactosylsulfatide	NN	O	O
(	NN	O	O
GS	NN	O	O
)	NN	O	O
excretion	NN	O	O
.	NN	O	O

The	NN	O	O
two	NN	O	O
other	NN	O	O
siblings	NN	O	O
,	NN	O	O
apparently	NN	O	O
healthy	NN	O	O
at	NN	O	O
12	NN	O	O
(	NN	O	O
1	NN	O	O
/	NN	O	O
2	NN	O	O
)	NN	O	O
and	NN	O	O
15	NN	O	O
years	NN	O	O
,	NN	O	O
respectively	NN	O	O
,	NN	O	O
and	NN	O	O
their	NN	O	O
father	NN	O	O
,	NN	O	O
apparently	NN	O	O
healthy	NN	O	O
as	NN	O	O
well	NN	O	O
,	NN	O	O
presented	NN	O	O
ARSA	NN	O	O
and	NN	O	O
GS	NN	O	O
values	NN	O	O
within	NN	O	O
the	NN	O	O
range	NN	O	O
of	NN	O	O
MLD	NN	O	B-Disease
patients	NN	O	O
.	NN	O	O

Mutation	NN	O	O
screening	NN	O	O
and	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
disclosed	NN	O	O
the	NN	O	O
involvement	NN	O	O
of	NN	O	O
three	NN	O	O
different	NN	O	O
ARSA	NN	O	O
mutations	NN	O	O
being	NN	O	O
the	NN	O	O
molecular	NN	O	O
basis	NN	O	O
of	NN	O	O
intrafamilial	NN	O	O
phenotypic	NN	O	O
heterogeneity	NN	O	O
.	NN	O	O

The	NN	O	O
late	NN	O	O
infantile	NN	O	O
patient	NN	O	O
inherited	NN	O	O
from	NN	O	O
his	NN	O	O
mother	NN	O	O
the	NN	O	O
frequent	NN	O	O
0	NN	O	O
-	NN	O	O
type	NN	O	O
mutation	NN	O	O
459	NN	O	O
+	NN	O	O
1G	NN	O	O
>	NN	O	O
A	NN	O	O
,	NN	O	O
and	NN	O	O
from	NN	O	O
his	NN	O	O
father	NN	O	O
a	NN	O	O
novel	NN	O	O
,	NN	O	O
single	NN	O	O
basepair	NN	O	O
microdeletion	NN	O	O
of	NN	O	O
guanine	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
7	NN	O	O
in	NN	O	O
exon	NN	O	O
1	NN	O	O
(	NN	O	O
7delG	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
two	NN	O	O
clinically	NN	O	O
unaffected	NN	O	O
siblings	NN	O	O
carried	NN	O	O
the	NN	O	O
maternal	NN	O	O
mutation	NN	O	O
459	NN	O	O
+	NN	O	O
1G	NN	O	O
>	NN	O	O
A	NN	O	O
and	NN	O	O
,	NN	O	O
on	NN	O	O
their	NN	O	O
paternal	NN	O	O
allele	NN	O	O
,	NN	O	O
a	NN	O	O
novel	NN	O	O
cytosine	NN	O	O
to	NN	O	O
thymidine	NN	O	O
transition	NN	O	O
at	NN	O	O
nucleotide	NN	O	O
2435	NN	O	O
in	NN	O	O
exon	NN	O	O
8	NN	O	O
,	NN	O	O
resulting	NN	O	O
in	NN	O	O
substitution	NN	O	O
of	NN	O	O
alanine	NN	O	O
464	NN	O	O
by	NN	O	O
valine	NN	O	O
(	NN	O	O
A464V	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
fathers	NN	O	O
genotype	NN	O	O
thus	NN	O	O
was	NN	O	O
7delG	NN	O	O
/	NN	O	O
A464V	NN	O	O
.	NN	O	O

Mutation	NN	O	O
A464V	NN	O	O
was	NN	O	O
not	NN	O	O
found	NN	O	O
in	NN	O	O
18	NN	O	O
unrelated	NN	O	O
MLD	NN	O	B-Disease
patients	NN	O	O
and	NN	O	O
50	NN	O	O
controls	NN	O	O
.	NN	O	O

A464V	NN	O	O
,	NN	O	O
although	NN	O	O
clearly	NN	O	O
modifying	NN	O	O
ARSA	NN	O	O
and	NN	O	O
GS	NN	O	O
levels	NN	O	O
,	NN	O	O
apparently	NN	O	O
bears	NN	O	O
little	NN	O	O
significance	NN	O	O
for	NN	O	O
clinical	NN	O	O
manifestation	NN	O	O
of	NN	O	O
MLD	NN	O	B-Disease
,	NN	O	O
mimicking	NN	O	O
the	NN	O	O
frequent	NN	O	O
ARSA	NN	O	O
pseudodeficiency	NN	O	O
allele	NN	O	O
.	NN	O	O

Our	NN	O	O
results	NN	O	O
demonstrate	NN	O	O
that	NN	O	O
in	NN	O	O
certain	NN	O	O
genetic	NN	O	O
conditions	NN	O	O
MLD	NN	O	B-Disease
-	NN	O	O
like	NN	O	O
ARSA	NN	O	O
and	NN	O	O
GS	NN	O	O
values	NN	O	O
need	NN	O	O
not	NN	O	O
be	NN	O	O
paralleled	NN	O	O
by	NN	O	O
clinical	NN	O	O
disease	NN	O	O
,	NN	O	O
a	NN	O	O
finding	NN	O	O
with	NN	O	O
serious	NN	O	O
diagnostic	NN	O	O
and	NN	O	O
prognostic	NN	O	O
implications	NN	O	O
.	NN	O	O

Moreover	NN	O	O
,	NN	O	O
further	NN	O	O
ARSA	NN	O	O
alleles	NN	O	O
functionally	NN	O	O
similar	NN	O	O
to	NN	O	O
A464V	NN	O	O
might	NN	O	O
exist	NN	O	O
which	NN	O	O
,	NN	O	O
together	NN	O	O
with	NN	O	O
0	NN	O	O
-	NN	O	O
type	NN	O	O
mutations	NN	O	O
,	NN	O	O
may	NN	O	O
cause	NN	O	O
pathological	NN	O	O
ARSA	NN	O	O
and	NN	O	O
GS	NN	O	O
levels	NN	O	O
,	NN	O	O
but	NN	O	O
not	NN	O	O
clinical	NN	O	O
outbreak	NN	O	O
of	NN	O	O
the	NN	O	O
disease	NN	O	O
.	NN	O	O
.	NN	O	O

Human	NN	O	O
MLH1	NN	O	O
deficiency	NN	O	O
predisposes	NN	O	O
to	NN	O	O
hematological	NN	O	B-Disease
malignancy	NN	O	I-Disease
and	NN	O	O
neurofibromatosis	NN	O	B-Disease
type	NN	O	I-Disease
1	NN	O	I-Disease
.	NN	O	O

Heterozygous	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
DNA	NN	O	O
mismatch	NN	O	O
repair	NN	O	O
genes	NN	O	O
lead	NN	O	O
to	NN	O	O
hereditary	NN	O	B-Disease
nonpolyposis	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
disease	NN	O	O
susceptibility	NN	O	O
of	NN	O	O
individuals	NN	O	O
who	NN	O	O
constitutionally	NN	O	O
lack	NN	O	O
both	NN	O	O
wild	NN	O	O
-	NN	O	O
type	NN	O	O
alleles	NN	O	O
is	NN	O	O
unknown	NN	O	O
.	NN	O	O

We	NN	O	O
have	NN	O	O
identified	NN	O	O
three	NN	O	O
offspring	NN	O	O
in	NN	O	O
a	NN	O	O
hereditary	NN	O	B-Disease
nonpolyposis	NN	O	I-Disease
colorectal	NN	O	I-Disease
cancer	NN	O	I-Disease
family	NN	O	O
who	NN	O	O
developed	NN	O	O
hematological	NN	O	B-Disease
malignancy	NN	O	I-Disease
at	NN	O	O
a	NN	O	O
very	NN	O	O
early	NN	O	O
age	NN	O	O
,	NN	O	O
and	NN	O	O
at	NN	O	O
least	NN	O	O
two	NN	O	O
of	NN	O	O
them	NN	O	O
displayed	NN	O	O
signs	NN	O	O
of	NN	O	O
neurofibromatosis	NN	O	B-Disease
type	NN	O	I-Disease
1	NN	O	I-Disease
(	NN	O	O
NF1	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

DNA	NN	O	O
sequence	NN	O	O
analysis	NN	O	O
and	NN	O	O
allele	NN	O	O
-	NN	O	O
specific	NN	O	O
amplification	NN	O	O
in	NN	O	O
two	NN	O	O
siblings	NN	O	O
revealed	NN	O	O
a	NN	O	O
homozygous	NN	O	O
MLH1	NN	O	O
mutation	NN	O	O
(	NN	O	O
C676T	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
Arg226Stop	NN	O	O
)	NN	O	O
.	NN	O	O

Thus	NN	O	O
,	NN	O	O
a	NN	O	O
homozygous	NN	O	O
germ	NN	O	O
-	NN	O	O
line	NN	O	O
MLH1	NN	O	O
mutation	NN	O	O
and	NN	O	O
consequent	NN	O	O
mismatch	NN	O	O
repair	NN	O	O
deficiency	NN	O	O
results	NN	O	O
in	NN	O	O
a	NN	O	O
mutator	NN	O	O
phenotype	NN	O	O
characterized	NN	O	O
by	NN	O	O
leukemia	NN	O	B-Disease
and	NN	O	O
/	NN	O	O
or	NN	O	O
lymphoma	NN	O	B-Disease
associated	NN	O	O
with	NN	O	O
neurofibromatosis	NN	O	B-Disease
type	NN	O	I-Disease
1	NN	O	I-Disease
.	NN	O	O
.	NN	O	O

Missense	NN	O	O
mutations	NN	O	O
in	NN	O	O
the	NN	O	O
most	NN	O	O
ancient	NN	O	O
residues	NN	O	O
of	NN	O	O
the	NN	O	O
PAX6	NN	O	O
paired	NN	O	O
domain	NN	O	O
underlie	NN	O	O
a	NN	O	O
spectrum	NN	O	O
of	NN	O	O
human	NN	O	O
congenital	NN	O	B-Disease
eye	NN	O	I-Disease
malformations	NN	O	I-Disease
.	NN	O	O

Mutations	NN	O	O
of	NN	O	O
the	NN	O	O
human	NN	O	O
PAX6	NN	O	O
gene	NN	O	O
underlie	NN	O	O
aniridia	NN	O	B-Disease
(	NN	O	O
congenital	NN	O	B-Disease
absence	NN	O	I-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
iris	NN	O	I-Disease
)	NN	O	O
,	NN	O	O
a	NN	O	O
rare	NN	O	O
dominant	NN	O	O
malformation	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
eye	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
spectrum	NN	O	O
of	NN	O	O
PAX6	NN	O	O
mutations	NN	O	O
in	NN	O	O
aniridia	NN	O	B-Disease
patients	NN	O	O
is	NN	O	O
highly	NN	O	O
biased	NN	O	O
,	NN	O	O
with	NN	O	O
92	NN	O	O
%	NN	O	O
of	NN	O	O
all	NN	O	O
reported	NN	O	O
mutations	NN	O	O
leading	NN	O	O
to	NN	O	O
premature	NN	O	O
truncation	NN	O	O
of	NN	O	O
the	NN	O	O
protein	NN	O	O
(	NN	O	O
nonsense	NN	O	O
,	NN	O	O
splicing	NN	O	O
,	NN	O	O
insertions	NN	O	O
and	NN	O	O
deletions	NN	O	O
)	NN	O	O
and	NN	O	O
just	NN	O	O
2	NN	O	O
%	NN	O	O
leading	NN	O	O
to	NN	O	O
substitution	NN	O	O
of	NN	O	O
one	NN	O	O
amino	NN	O	O
acid	NN	O	O
by	NN	O	O
another	NN	O	O
(	NN	O	O
missense	NN	O	O
)	NN	O	O
.	NN	O	O

The	NN	O	O
extraordinary	NN	O	O
conservation	NN	O	O
of	NN	O	O
the	NN	O	O
PAX6	NN	O	O
protein	NN	O	O
at	NN	O	O
the	NN	O	O
amino	NN	O	O
acid	NN	O	O
level	NN	O	O
amongst	NN	O	O
vertebrates	NN	O	O
predicts	NN	O	O
that	NN	O	O
pathological	NN	O	O
missense	NN	O	O
mutations	NN	O	O
should	NN	O	O
in	NN	O	O
fact	NN	O	O
be	NN	O	O
common	NN	O	O
even	NN	O	O
though	NN	O	O
they	NN	O	O
are	NN	O	O
hardly	NN	O	O
ever	NN	O	O
seen	NN	O	O
in	NN	O	O
aniridia	NN	O	B-Disease
patients	NN	O	O
.	NN	O	O

This	NN	O	O
indicates	NN	O	O
that	NN	O	O
there	NN	O	O
is	NN	O	O
a	NN	O	O
heavy	NN	O	O
ascertainment	NN	O	O
bias	NN	O	O
in	NN	O	O
the	NN	O	O
selection	NN	O	O
of	NN	O	O
patients	NN	O	O
for	NN	O	O
PAX6	NN	O	O
mutation	NN	O	O
analysis	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
missing	NN	O	O
PAX6	NN	O	O
missense	NN	O	O
mutations	NN	O	O
frequently	NN	O	O
may	NN	O	O
underlie	NN	O	O
phenotypes	NN	O	O
distinct	NN	O	O
from	NN	O	O
textbook	NN	O	O
aniridia	NN	O	B-Disease
.	NN	O	O

Here	NN	O	O
we	NN	O	O
present	NN	O	O
four	NN	O	O
novel	NN	O	O
PAX6	NN	O	O
missense	NN	O	O
mutations	NN	O	O
,	NN	O	O
two	NN	O	O
in	NN	O	O
association	NN	O	O
with	NN	O	O
atypical	NN	O	O
phenotypes	NN	O	O
ectopia	NN	O	B-Disease
pupillae	NN	O	I-Disease
(	NN	O	O
displaced	NN	O	B-Disease
pupils	NN	O	I-Disease
)	NN	O	O
and	NN	O	O
congenital	NN	O	B-Disease
nystagmus	NN	O	I-Disease
(	NN	O	O
searching	NN	O	B-Disease
gaze	NN	O	I-Disease
)	NN	O	O
,	NN	O	O
and	NN	O	O
two	NN	O	O
in	NN	O	O
association	NN	O	O
with	NN	O	O
more	NN	O	O
recognizable	NN	O	O
aniridia	NN	O	B-Disease
phenotypes	NN	O	O
.	NN	O	O

Strikingly	NN	O	O
,	NN	O	O
all	NN	O	O
four	NN	O	O
mutations	NN	O	O
are	NN	O	O
located	NN	O	O
within	NN	O	O
the	NN	O	O
PAX6	NN	O	O
paired	NN	O	O
domain	NN	O	O
and	NN	O	O
affect	NN	O	O
amino	NN	O	O
acids	NN	O	O
which	NN	O	O
are	NN	O	O
highly	NN	O	O
conserved	NN	O	O
in	NN	O	O
all	NN	O	O
known	NN	O	O
paired	NN	O	O
domain	NN	O	O
proteins	NN	O	O
.	NN	O	O

Our	NN	O	O
results	NN	O	O
support	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
the	NN	O	O
under	NN	O	O
-	NN	O	O
representation	NN	O	O
of	NN	O	O
missense	NN	O	O
mutations	NN	O	O
is	NN	O	O
caused	NN	O	O
by	NN	O	O
ascertainment	NN	O	O
bias	NN	O	O
and	NN	O	O
suggest	NN	O	O
that	NN	O	O
a	NN	O	O
substantial	NN	O	O
burden	NN	O	O
of	NN	O	O
PAX6	NN	O	B-Disease
-	NN	O	I-Disease
related	NN	O	I-Disease
disease	NN	O	I-Disease
remains	NN	O	O
to	NN	O	O
be	NN	O	O
uncovered	NN	O	O
.	NN	O	O
.	NN	O	O

The	NN	O	O
chromosomal	NN	O	O
order	NN	O	O
of	NN	O	O
genes	NN	O	O
controlling	NN	O	O
the	NN	O	O
major	NN	O	O
histocompatibility	NN	O	O
complex	NN	O	O
,	NN	O	O
properdin	NN	O	O
factor	NN	O	O
B	NN	O	O
,	NN	O	O
and	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
second	NN	O	I-Disease
component	NN	O	I-Disease
of	NN	O	I-Disease
complement	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
relationship	NN	O	O
of	NN	O	O
the	NN	O	O
genes	NN	O	O
coding	NN	O	O
for	NN	O	O
HLA	NN	O	O
to	NN	O	O
those	NN	O	O
coding	NN	O	O
for	NN	O	O
properdin	NN	O	O
Factor	NN	O	O
B	NN	O	O
allotypes	NN	O	O
and	NN	O	O
for	NN	O	O
deficiency	NN	O	B-Disease
of	NN	O	I-Disease
the	NN	O	I-Disease
second	NN	O	I-Disease
component	NN	O	I-Disease
of	NN	O	I-Disease
complement	NN	O	I-Disease
(	NN	O	O
C2	NN	O	O
)	NN	O	O
was	NN	O	O
studied	NN	O	O
in	NN	O	O
families	NN	O	O
of	NN	O	O
patients	NN	O	O
with	NN	O	O
connective	NN	O	O
tissue	NN	O	O
disorders	NN	O	O
.	NN	O	O

Patients	NN	O	O
were	NN	O	O
selected	NN	O	O
because	NN	O	O
they	NN	O	O
were	NN	O	O
heterozygous	NN	O	O
or	NN	O	O
homozygous	NN	O	O
for	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
.	NN	O	O

12	NN	O	O
families	NN	O	O
with	NN	O	O
15	NN	O	O
matings	NN	O	O
informative	NN	O	O
for	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
were	NN	O	O
found	NN	O	O
.	NN	O	O

Of	NN	O	O
57	NN	O	O
informative	NN	O	O
meioses	NN	O	O
,	NN	O	O
two	NN	O	O
crossovers	NN	O	O
were	NN	O	O
noted	NN	O	O
between	NN	O	O
the	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
gene	NN	O	O
and	NN	O	O
the	NN	O	O
HLA	NN	O	O
-	NN	O	O
B	NN	O	O
gene	NN	O	O
,	NN	O	O
with	NN	O	O
a	NN	O	O
recombinant	NN	O	O
fraction	NN	O	O
of	NN	O	O
0	NN	O	O
.	NN	O	O

035	NN	O	O
.	NN	O	O

A	NN	O	O
lod	NN	O	O
score	NN	O	O
of	NN	O	O
13	NN	O	O
was	NN	O	O
calculated	NN	O	O
for	NN	O	O
linkage	NN	O	O
between	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
and	NN	O	O
HLA	NN	O	O
-	NN	O	O
B	NN	O	O
at	NN	O	O
a	NN	O	O
maximum	NN	O	O
likelihood	NN	O	O
value	NN	O	O
of	NN	O	O
the	NN	O	O
recombinant	NN	O	O
fraction	NN	O	O
of	NN	O	O
0	NN	O	O
.	NN	O	O

04	NN	O	O
.	NN	O	O

18	NN	O	O
families	NN	O	O
with	NN	O	O
21	NN	O	O
informative	NN	O	O
matings	NN	O	O
for	NN	O	O
both	NN	O	O
properdin	NN	O	O
Factor	NN	O	O
B	NN	O	O
allotype	NN	O	O
and	NN	O	O
HLA	NN	O	O
-	NN	O	O
B	NN	O	O
were	NN	O	O
found	NN	O	O
.	NN	O	O

Of	NN	O	O
72	NN	O	O
informative	NN	O	O
meioses	NN	O	O
,	NN	O	O
three	NN	O	O
recombinants	NN	O	O
were	NN	O	O
found	NN	O	O
,	NN	O	O
giving	NN	O	O
a	NN	O	O
recombinant	NN	O	O
fraction	NN	O	O
of	NN	O	O
0	NN	O	O
.	NN	O	O

042	NN	O	O
.	NN	O	O

A	NN	O	O
lod	NN	O	O
score	NN	O	O
of	NN	O	O
16	NN	O	O
between	NN	O	O
HLA	NN	O	O
-	NN	O	O
B	NN	O	O
and	NN	O	O
Factor	NN	O	O
B	NN	O	O
allotypes	NN	O	O
was	NN	O	O
calculated	NN	O	O
at	NN	O	O
a	NN	O	O
maximum	NN	O	O
likelihood	NN	O	O
value	NN	O	O
of	NN	O	O
the	NN	O	O
recombinant	NN	O	O
fraction	NN	O	O
of	NN	O	O
0	NN	O	O
.	NN	O	O

04	NN	O	O
.	NN	O	O

A	NN	O	O
crossover	NN	O	O
was	NN	O	O
shown	NN	O	O
to	NN	O	O
have	NN	O	O
occurred	NN	O	O
between	NN	O	O
genes	NN	O	O
for	NN	O	O
Factor	NN	O	O
B	NN	O	O
and	NN	O	O
HLA	NN	O	O
-	NN	O	O
D	NN	O	O
,	NN	O	O
in	NN	O	O
which	NN	O	O
HLA	NN	O	O
-	NN	O	O
D	NN	O	O
segregared	NN	O	O
with	NN	O	O
HLA	NN	O	O
-	NN	O	O
A	NN	O	O
and	NN	O	O
B	NN	O	O
.	NN	O	O

These	NN	O	O
studies	NN	O	O
suggest	NN	O	O
that	NN	O	O
the	NN	O	O
genes	NN	O	O
for	NN	O	O
Factor	NN	O	O
B	NN	O	O
and	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
are	NN	O	O
located	NN	O	O
outside	NN	O	O
those	NN	O	O
for	NN	O	O
HLA	NN	O	O
,	NN	O	O
that	NN	O	O
the	NN	O	O
order	NN	O	O
of	NN	O	O
genese	NN	O	O
is	NN	O	O
HLA	NN	O	O
-	NN	O	O
A	NN	O	O
,	NN	O	O
-	NN	O	O
B	NN	O	O
,	NN	O	O
-	NN	O	O
D	NN	O	O
,	NN	O	O
Factor	NN	O	O
B	NN	O	O
allotype	NN	O	O
,	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
,	NN	O	O
that	NN	O	O
the	NN	O	O
genes	NN	O	O
coding	NN	O	O
for	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
and	NN	O	O
Factor	NN	O	O
B	NN	O	O
allotypes	NN	O	O
are	NN	O	O
approximately	NN	O	O
3	NN	O	O
-	NN	O	O
-	NN	O	O
5	NN	O	O
centimorgans	NN	O	O
from	NN	O	O
the	NN	O	O
HLA	NN	O	O
-	NN	O	O
A	NN	O	O
and	NN	O	O
HLA	NN	O	O
-	NN	O	O
B	NN	O	O
loci	NN	O	O
,	NN	O	O
and	NN	O	O
that	NN	O	O
the	NN	O	O
apparent	NN	O	O
lack	NN	O	O
of	NN	O	O
recombinants	NN	O	O
between	NN	O	O
the	NN	O	O
Factor	NN	O	O
B	NN	O	O
gene	NN	O	O
and	NN	O	O
C2	NN	O	B-Disease
deficiency	NN	O	I-Disease
gene	NN	O	O
suggests	NN	O	O
that	NN	O	O
these	NN	O	O
two	NN	O	O
genes	NN	O	O
lie	NN	O	O
in	NN	O	O
close	NN	O	O
proximity	NN	O	O
to	NN	O	O
one	NN	O	O
another	NN	O	O
.	NN	O	O

Distribution	NN	O	O
of	NN	O	O
emerin	NN	O	O
and	NN	O	O
lamins	NN	O	O
in	NN	O	O
the	NN	O	O
heart	NN	O	O
and	NN	O	O
implications	NN	O	O
for	NN	O	O
Emery	NN	O	B-Disease
-	NN	O	I-Disease
Dreifuss	NN	O	I-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
.	NN	O	O

Emerin	NN	O	O
is	NN	O	O
a	NN	O	O
nuclear	NN	O	O
membrane	NN	O	O
protein	NN	O	O
which	NN	O	O
is	NN	O	O
missing	NN	O	O
or	NN	O	O
defective	NN	O	O
in	NN	O	O
Emery	NN	O	B-Disease
-	NN	O	I-Disease
Dreifuss	NN	O	I-Disease
muscular	NN	O	I-Disease
dystrophy	NN	O	I-Disease
(	NN	O	O
EDMD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

It	NN	O	O
is	NN	O	O
one	NN	O	O
member	NN	O	O
of	NN	O	O
a	NN	O	O
family	NN	O	O
of	NN	O	O
lamina	NN	O	O
-	NN	O	O
associated	NN	O	O
proteins	NN	O	O
which	NN	O	O
includes	NN	O	O
LAP1	NN	O	O
,	NN	O	O
LAP2	NN	O	O
and	NN	O	O
lamin	NN	O	O
B	NN	O	O
receptor	NN	O	O
(	NN	O	O
LBR	NN	O	O
)	NN	O	O
.	NN	O	O

A	NN	O	O
panel	NN	O	O
of	NN	O	O
16	NN	O	O
monoclonal	NN	O	O
antibodies	NN	O	O
(	NN	O	O
mAbs	NN	O	O
)	NN	O	O
has	NN	O	O
been	NN	O	O
mapped	NN	O	O
to	NN	O	O
six	NN	O	O
specific	NN	O	O
sites	NN	O	O
throughout	NN	O	O
the	NN	O	O
emerin	NN	O	O
molecule	NN	O	O
using	NN	O	O
phage	NN	O	O
-	NN	O	O
displayed	NN	O	O
peptide	NN	O	O
libraries	NN	O	O
and	NN	O	O
has	NN	O	O
been	NN	O	O
used	NN	O	O
to	NN	O	O
localize	NN	O	O
emerin	NN	O	O
in	NN	O	O
human	NN	O	O
and	NN	O	O
rabbit	NN	O	O
heart	NN	O	O
.	NN	O	O

Several	NN	O	O
mAbs	NN	O	O
against	NN	O	O
different	NN	O	O
emerin	NN	O	O
epitopes	NN	O	O
did	NN	O	O
not	NN	O	O
recognize	NN	O	O
intercalated	NN	O	O
discs	NN	O	O
in	NN	O	O
the	NN	O	O
heart	NN	O	O
,	NN	O	O
though	NN	O	O
they	NN	O	O
recognized	NN	O	O
cardiomyocyte	NN	O	O
nuclei	NN	O	O
strongly	NN	O	O
,	NN	O	O
both	NN	O	O
at	NN	O	O
the	NN	O	O
rim	NN	O	O
and	NN	O	O
in	NN	O	O
intranuclear	NN	O	O
spots	NN	O	O
or	NN	O	O
channels	NN	O	O
.	NN	O	O

A	NN	O	O
polyclonal	NN	O	O
rabbit	NN	O	O
antiserum	NN	O	O
against	NN	O	O
emerin	NN	O	O
did	NN	O	O
recognize	NN	O	O
both	NN	O	O
nuclear	NN	O	O
membrane	NN	O	O
and	NN	O	O
intercalated	NN	O	O
discs	NN	O	O
but	NN	O	O
,	NN	O	O
after	NN	O	O
affinity	NN	O	O
purification	NN	O	O
against	NN	O	O
a	NN	O	O
pure	NN	O	O
-	NN	O	O
emerin	NN	O	O
band	NN	O	O
on	NN	O	O
a	NN	O	O
western	NN	O	O
blot	NN	O	O
,	NN	O	O
it	NN	O	O
stained	NN	O	O
only	NN	O	O
the	NN	O	O
nuclear	NN	O	O
membrane	NN	O	O
.	NN	O	O

These	NN	O	O
results	NN	O	O
would	NN	O	O
not	NN	O	O
be	NN	O	O
expected	NN	O	O
if	NN	O	O
immunostaining	NN	O	O
at	NN	O	O
intercalated	NN	O	O
discs	NN	O	O
were	NN	O	O
due	NN	O	O
to	NN	O	O
a	NN	O	O
product	NN	O	O
of	NN	O	O
the	NN	O	O
emerin	NN	O	O
gene	NN	O	O
and	NN	O	O
,	NN	O	O
therefore	NN	O	O
,	NN	O	O
cast	NN	O	O
some	NN	O	O
doubt	NN	O	O
upon	NN	O	O
the	NN	O	O
hypothesis	NN	O	O
that	NN	O	O
cardiac	NN	O	B-Disease
defects	NN	O	I-Disease
in	NN	O	O
EDMD	NN	O	B-Disease
are	NN	O	O
caused	NN	O	O
by	NN	O	O
absence	NN	O	O
of	NN	O	O
emerin	NN	O	O
from	NN	O	O
intercalated	NN	O	O
discs	NN	O	O
.	NN	O	O

Although	NN	O	O
emerin	NN	O	O
was	NN	O	O
abundant	NN	O	O
in	NN	O	O
the	NN	O	O
membranes	NN	O	O
of	NN	O	O
cardiomyocyte	NN	O	O
nuclei	NN	O	O
,	NN	O	O
it	NN	O	O
was	NN	O	O
absent	NN	O	O
from	NN	O	O
many	NN	O	O
non	NN	O	O
-	NN	O	O
myocyte	NN	O	O
cells	NN	O	O
in	NN	O	O
the	NN	O	O
heart	NN	O	O
.	NN	O	O

This	NN	O	O
distribution	NN	O	O
of	NN	O	O
emerin	NN	O	O
was	NN	O	O
similar	NN	O	O
to	NN	O	O
that	NN	O	O
of	NN	O	O
lamin	NN	O	O
A	NN	O	O
,	NN	O	O
a	NN	O	O
candidate	NN	O	O
gene	NN	O	O
for	NN	O	O
an	NN	O	O
autosomal	NN	O	O
form	NN	O	O
of	NN	O	O
EDMD	NN	O	B-Disease
.	NN	O	O

In	NN	O	O
contrast	NN	O	O
,	NN	O	O
lamin	NN	O	O
B1	NN	O	O
was	NN	O	O
absent	NN	O	O
from	NN	O	O
cardiomyocyte	NN	O	O
nuclei	NN	O	O
,	NN	O	O
showing	NN	O	O
that	NN	O	O
lamin	NN	O	O
B1	NN	O	O
is	NN	O	O
not	NN	O	O
essential	NN	O	O
for	NN	O	O
localization	NN	O	O
of	NN	O	O
emerin	NN	O	O
to	NN	O	O
the	NN	O	O
nuclear	NN	O	O
lamina	NN	O	O
.	NN	O	O

Lamin	NN	O	O
B1	NN	O	O
is	NN	O	O
also	NN	O	O
almost	NN	O	O
completely	NN	O	O
absent	NN	O	O
from	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
nuclei	NN	O	O
.	NN	O	O

In	NN	O	O
EDMD	NN	O	B-Disease
,	NN	O	O
the	NN	O	O
additional	NN	O	O
absence	NN	O	O
of	NN	O	O
lamin	NN	O	O
B1	NN	O	O
from	NN	O	O
heart	NN	O	O
and	NN	O	O
skeletal	NN	O	O
muscle	NN	O	O
nuclei	NN	O	O
which	NN	O	O
already	NN	O	O
lack	NN	O	O
emerin	NN	O	O
may	NN	O	O
offer	NN	O	O
an	NN	O	O
alternative	NN	O	O
explanation	NN	O	O
of	NN	O	O
why	NN	O	O
these	NN	O	O
tissues	NN	O	O
are	NN	O	O
particularly	NN	O	O
affected	NN	O	O
.	NN	O	O
.	NN	O	O

Genetic	NN	O	O
mapping	NN	O	O
of	NN	O	O
the	NN	O	O
copper	NN	O	B-Disease
toxicosis	NN	O	I-Disease
locus	NN	O	O
in	NN	O	O
Bedlington	NN	O	O
terriers	NN	O	O
to	NN	O	O
dog	NN	O	O
chromosome	NN	O	O
10	NN	O	O
,	NN	O	O
in	NN	O	O
a	NN	O	O
region	NN	O	O
syntenic	NN	O	O
to	NN	O	O
human	NN	O	O
chromosome	NN	O	O
region	NN	O	O
2p13	NN	O	O
-	NN	O	O
p16	NN	O	O
.	NN	O	O

Abnormal	NN	O	O
hepatic	NN	O	B-Disease
copper	NN	O	I-Disease
accumulation	NN	O	I-Disease
is	NN	O	O
recognized	NN	O	O
as	NN	O	O
an	NN	O	O
inherited	NN	O	B-Disease
disorder	NN	O	I-Disease
in	NN	O	O
man	NN	O	O
,	NN	O	O
mouse	NN	O	O
,	NN	O	O
rat	NN	O	O
and	NN	O	O
dog	NN	O	O
.	NN	O	O

The	NN	O	O
major	NN	O	O
cause	NN	O	O
of	NN	O	O
hepatic	NN	O	B-Disease
copper	NN	O	I-Disease
accumulation	NN	O	I-Disease
in	NN	O	O
man	NN	O	O
is	NN	O	O
a	NN	O	O
dysfunctional	NN	O	O
ATP7B	NN	O	O
gene	NN	O	O
,	NN	O	O
causing	NN	O	O
Wilson	NN	O	B-Disease
disease	NN	O	I-Disease
(	NN	O	O
WD	NN	O	B-Disease
)	NN	O	O
.	NN	O	O

Mutations	NN	O	O
in	NN	O	O
the	NN	O	O
ATP7B	NN	O	O
genes	NN	O	O
have	NN	O	O
also	NN	O	O
been	NN	O	O
demonstrated	NN	O	O
in	NN	O	O
mouse	NN	O	O
and	NN	O	O
rat	NN	O	O
.	NN	O	O

The	NN	O	O
ATP7B	NN	O	O
gene	NN	O	O
has	NN	O	O
been	NN	O	O
excluded	NN	O	O
in	NN	O	O
the	NN	O	O
much	NN	O	O
rarer	NN	O	O
human	NN	O	O
copper	NN	O	B-Disease
overload	NN	O	I-Disease
disease	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
Indian	NN	O	I-Disease
childhood	NN	O	I-Disease
cirrhosis	NN	O	I-Disease
,	NN	O	O
indicating	NN	O	O
genetic	NN	O	O
heterogeneity	NN	O	O
.	NN	O	O

By	NN	O	O
investigating	NN	O	O
the	NN	O	O
common	NN	O	O
autosomal	NN	O	O
recessive	NN	O	O
copper	NN	O	B-Disease
toxicosis	NN	O	I-Disease
(	NN	O	O
CT	NN	O	B-Disease
)	NN	O	O
in	NN	O	O
Bedlington	NN	O	O
terriers	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
identified	NN	O	O
a	NN	O	O
new	NN	O	O
locus	NN	O	O
involved	NN	O	O
in	NN	O	O
progressive	NN	O	O
liver	NN	O	B-Disease
disease	NN	O	I-Disease
.	NN	O	O

We	NN	O	O
examined	NN	O	O
whether	NN	O	O
the	NN	O	O
WD	NN	O	B-Disease
gene	NN	O	O
ATP7B	NN	O	O
was	NN	O	O
also	NN	O	O
causative	NN	O	O
for	NN	O	O
CT	NN	O	B-Disease
by	NN	O	O
investigating	NN	O	O
the	NN	O	O
chromosomal	NN	O	O
co	NN	O	O
-	NN	O	O
localization	NN	O	O
of	NN	O	O
ATP7B	NN	O	O
and	NN	O	O
C04107	NN	O	O
,	NN	O	O
using	NN	O	O
fluorescence	NN	O	O
in	NN	O	O
situ	NN	O	O
hybridization	NN	O	O
(	NN	O	O
FISH	NN	O	O
)	NN	O	O
.	NN	O	O

C04107	NN	O	O
is	NN	O	O
an	NN	O	O
anonymous	NN	O	O
microsatellite	NN	O	O
marker	NN	O	O
closely	NN	O	O
linked	NN	O	O
to	NN	O	O
CT	NN	O	B-Disease
.	NN	O	O

However	NN	O	O
,	NN	O	O
BAC	NN	O	O
clones	NN	O	O
containing	NN	O	O
ATP7B	NN	O	O
and	NN	O	O
C04107	NN	O	O
mapped	NN	O	O
to	NN	O	O
the	NN	O	O
canine	NN	O	O
chromosome	NN	O	O
regions	NN	O	O
CFA22q11	NN	O	O
and	NN	O	O
CFA10q26	NN	O	O
,	NN	O	O
respectively	NN	O	O
,	NN	O	O
demonstrating	NN	O	O
that	NN	O	O
WD	NN	O	B-Disease
cannot	NN	O	O
be	NN	O	O
homologous	NN	O	O
to	NN	O	O
CT	NN	O	B-Disease
.	NN	O	O

The	NN	O	O
copper	NN	O	O
transport	NN	O	O
genes	NN	O	O
CTR1	NN	O	O
and	NN	O	O
CTR2	NN	O	O
were	NN	O	O
also	NN	O	O
excluded	NN	O	O
as	NN	O	O
candidate	NN	O	O
genes	NN	O	O
for	NN	O	O
CT	NN	O	B-Disease
since	NN	O	O
they	NN	O	O
both	NN	O	O
mapped	NN	O	O
to	NN	O	O
canine	NN	O	O
chromosome	NN	O	O
region	NN	O	O
CFA11q22	NN	O	O
.	NN	O	O

2	NN	O	O
-	NN	O	O
22	NN	O	O
.	NN	O	O

5	NN	O	O
.	NN	O	O

A	NN	O	O
transcribed	NN	O	O
sequence	NN	O	O
identified	NN	O	O
from	NN	O	O
the	NN	O	O
C04107	NN	O	O
-	NN	O	O
containing	NN	O	O
BAC	NN	O	O
was	NN	O	O
found	NN	O	O
to	NN	O	O
be	NN	O	O
homologous	NN	O	O
to	NN	O	O
a	NN	O	O
gene	NN	O	O
expressed	NN	O	O
from	NN	O	O
human	NN	O	O
chromosome	NN	O	O
2p13	NN	O	O
-	NN	O	O
p16	NN	O	O
,	NN	O	O
a	NN	O	O
region	NN	O	O
devoid	NN	O	O
of	NN	O	O
any	NN	O	O
positional	NN	O	O
candidate	NN	O	O
genes	NN	O	O
.	NN	O	O

Molecular	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
in	NN	O	O
205	NN	O	O
families	NN	O	O
:	NN	O	O
extended	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
in	NN	O	O
FAP	NN	O	B-Disease
and	NN	O	O
evidence	NN	O	O
for	NN	O	O
the	NN	O	O
role	NN	O	O
of	NN	O	O
APC	NN	O	B-Disease
amino	NN	O	O
acid	NN	O	O
changes	NN	O	O
in	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
predisposition	NN	O	O
.	NN	O	O

BACKGROUND	NN	O	O
/	NN	O	O
AIMS	NN	O	O
The	NN	O	O
development	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
and	NN	O	O
a	NN	O	O
variable	NN	O	O
range	NN	O	O
of	NN	O	O
extracolonic	NN	O	O
manifestations	NN	O	O
in	NN	O	O
familial	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
(	NN	O	O
FAP	NN	O	B-Disease
)	NN	O	O
is	NN	O	O
the	NN	O	O
result	NN	O	O
of	NN	O	O
the	NN	O	O
dominant	NN	O	O
inheritance	NN	O	O
of	NN	O	O
adenomatous	NN	O	B-Disease
polyposis	NN	O	I-Disease
coli	NN	O	I-Disease
(	NN	O	O
APC	NN	O	B-Disease
)	NN	O	O
gene	NN	O	O
mutations	NN	O	O
.	NN	O	O

In	NN	O	O
this	NN	O	O
study	NN	O	O
,	NN	O	O
direct	NN	O	O
mutation	NN	O	O
analysis	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
was	NN	O	O
performed	NN	O	O
to	NN	O	O
determine	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
for	NN	O	O
nine	NN	O	O
extracolonic	NN	O	O
manifestations	NN	O	O
and	NN	O	O
to	NN	O	O
investigate	NN	O	O
the	NN	O	O
incidence	NN	O	O
of	NN	O	O
APC	NN	O	B-Disease
mutations	NN	O	O
in	NN	O	O
non	NN	O	O
-	NN	O	O
FAP	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

METHODS	NN	O	O
The	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
was	NN	O	O
analysed	NN	O	O
in	NN	O	O
190	NN	O	O
unrelated	NN	O	O
FAP	NN	O	B-Disease
and	NN	O	O
15	NN	O	O
non	NN	O	O
-	NN	O	O
FAP	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
using	NN	O	O
denaturing	NN	O	O
gradient	NN	O	O
gel	NN	O	O
electrophoresis	NN	O	O
,	NN	O	O
the	NN	O	O
protein	NN	O	O
truncation	NN	O	O
test	NN	O	O
,	NN	O	O
and	NN	O	O
direct	NN	O	O
sequencing	NN	O	O
.	NN	O	O

RESULTS	NN	O	O
Chain	NN	O	O
terminating	NN	O	O
signals	NN	O	O
were	NN	O	O
only	NN	O	O
identified	NN	O	O
in	NN	O	O
patients	NN	O	O
belonging	NN	O	O
to	NN	O	O
the	NN	O	O
FAP	NN	O	B-Disease
group	NN	O	O
(	NN	O	O
105	NN	O	O
patients	NN	O	O
)	NN	O	O
.	NN	O	O

Amino	NN	O	O
acid	NN	O	O
changes	NN	O	O
were	NN	O	O
identified	NN	O	O
in	NN	O	O
four	NN	O	O
patients	NN	O	O
,	NN	O	O
three	NN	O	O
of	NN	O	O
whom	NN	O	O
belonged	NN	O	O
to	NN	O	O
the	NN	O	O
non	NN	O	O
-	NN	O	O
FAP	NN	O	O
group	NN	O	O
of	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O

Genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
identified	NN	O	O
significant	NN	O	O
differences	NN	O	O
in	NN	O	O
the	NN	O	O
nature	NN	O	O
of	NN	O	O
certain	NN	O	O
extracolonic	NN	O	O
manifestations	NN	O	O
in	NN	O	O
FAP	NN	O	B-Disease
patients	NN	O	O
belonging	NN	O	O
to	NN	O	O
three	NN	O	O
mutation	NN	O	O
subgroups	NN	O	O
.	NN	O	O

CONCLUSIONS	NN	O	O
Extended	NN	O	O
genotype	NN	O	O
-	NN	O	O
phenotype	NN	O	O
correlations	NN	O	O
made	NN	O	O
in	NN	O	O
this	NN	O	O
study	NN	O	O
may	NN	O	O
have	NN	O	O
the	NN	O	O
potential	NN	O	O
to	NN	O	O
determine	NN	O	O
the	NN	O	O
most	NN	O	O
appropriate	NN	O	O
surveillance	NN	O	O
and	NN	O	O
prophylactic	NN	O	O
treatment	NN	O	O
regimens	NN	O	O
for	NN	O	O
those	NN	O	O
patients	NN	O	O
with	NN	O	O
mutations	NN	O	O
associated	NN	O	O
with	NN	O	O
life	NN	O	O
threatening	NN	O	O
conditions	NN	O	O
.	NN	O	O

This	NN	O	O
study	NN	O	O
also	NN	O	O
provided	NN	O	O
evidence	NN	O	O
for	NN	O	O
the	NN	O	O
pathological	NN	O	O
nature	NN	O	O
of	NN	O	O
amino	NN	O	O
acid	NN	O	O
changes	NN	O	O
in	NN	O	O
APC	NN	O	O
associated	NN	O	O
with	NN	O	O
both	NN	O	O
FAP	NN	O	B-Disease
and	NN	O	O
non	NN	O	O
-	NN	O	O
FAP	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
patients	NN	O	O
.	NN	O	O
.	NN	O	O

Inherited	NN	O	B-Disease
colorectal	NN	O	I-Disease
polyposis	NN	O	I-Disease
and	NN	O	O
cancer	NN	O	B-Disease
risk	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
polymorphism	NN	O	O
.	NN	O	O

Germ	NN	O	O
-	NN	O	O
line	NN	O	O
and	NN	O	O
somatic	NN	O	O
truncating	NN	O	O
mutations	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
are	NN	O	O
thought	NN	O	O
to	NN	O	O
initiate	NN	O	O
colorectal	NN	O	B-Disease
tumor	NN	O	I-Disease
formation	NN	O	O
in	NN	O	O
familial	NN	O	B-Disease
adenomatous	NN	O	I-Disease
polyposis	NN	O	I-Disease
syndrome	NN	O	I-Disease
and	NN	O	O
sporadic	NN	O	O
colorectal	NN	O	O
carcinogenesis	NN	O	O
,	NN	O	O
respectively	NN	O	O
.	NN	O	O

Recently	NN	O	O
,	NN	O	O
an	NN	O	O
isoleucine	NN	O	O
-	NN	O	O
-	NN	O	O
>	NN	O	O
lysine	NN	O	O
polymorphism	NN	O	O
at	NN	O	O
codon	NN	O	O
1307	NN	O	O
(	NN	O	O
I1307K	NN	O	O
)	NN	O	O
of	NN	O	O
the	NN	O	O
APC	NN	O	B-Disease
gene	NN	O	O
has	NN	O	O
been	NN	O	O
identified	NN	O	O
in	NN	O	O
6	NN	O	O
%	NN	O	O
-	NN	O	O
7	NN	O	O
%	NN	O	O
of	NN	O	O
the	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
population	NN	O	O
.	NN	O	O

To	NN	O	O
assess	NN	O	O
the	NN	O	O
risk	NN	O	O
of	NN	O	O
this	NN	O	O
common	NN	O	O
APC	NN	O	B-Disease
allelic	NN	O	O
variant	NN	O	O
in	NN	O	O
colorectal	NN	O	O
carcinogenesis	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
analyzed	NN	O	O
a	NN	O	O
large	NN	O	O
cohort	NN	O	O
of	NN	O	O
unselected	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
subjects	NN	O	O
with	NN	O	O
adenomatous	NN	O	B-Disease
polyps	NN	O	I-Disease
and	NN	O	O
.	NN	O	O
or	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
,	NN	O	I-Disease
for	NN	O	O
the	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
polymorphism	NN	O	O
.	NN	O	O

The	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
allele	NN	O	O
was	NN	O	O
identified	NN	O	O
in	NN	O	O
48	NN	O	O
(	NN	O	O
10	NN	O	O
.	NN	O	O
1	NN	O	O
%	NN	O	O
)	NN	O	O
of	NN	O	O
476	NN	O	O
patients	NN	O	O
.	NN	O	O

Compared	NN	O	O
with	NN	O	O
the	NN	O	O
frequency	NN	O	O
in	NN	O	O
two	NN	O	O
separate	NN	O	O
population	NN	O	O
control	NN	O	O
groups	NN	O	O
,	NN	O	O
the	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
allele	NN	O	O
is	NN	O	O
associated	NN	O	O
with	NN	O	O
an	NN	O	O
estimated	NN	O	O
relative	NN	O	O
risk	NN	O	O
of	NN	O	O
1	NN	O	O
.	NN	O	O

5	NN	O	O
-	NN	O	O
1	NN	O	O
.	NN	O	O

7	NN	O	O
for	NN	O	O
colorectal	NN	O	B-Disease
neoplasia	NN	O	I-Disease
(	NN	O	O
both	NN	O	O
P	NN	O	O
=	NN	O	O
.	NN	O	O
01	NN	O	O
)	NN	O	O
.	NN	O	O

Furthermore	NN	O	O
,	NN	O	O
compared	NN	O	O
with	NN	O	O
noncarriers	NN	O	O
,	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
carriers	NN	O	O
had	NN	O	O
increased	NN	O	O
numbers	NN	O	O
of	NN	O	O
adenomas	NN	O	B-Disease
and	NN	O	O
colorectal	NN	O	B-Disease
cancers	NN	O	I-Disease
per	NN	O	O
patient	NN	O	O
(	NN	O	O
P	NN	O	O
=	NN	O	O
.	NN	O	O
03	NN	O	O
)	NN	O	O
,	NN	O	O
as	NN	O	O
well	NN	O	O
as	NN	O	O
a	NN	O	O
younger	NN	O	O
age	NN	O	O
at	NN	O	O
diagnosis	NN	O	O
.	NN	O	O

We	NN	O	O
conclude	NN	O	O
that	NN	O	O
the	NN	O	O
APC	NN	O	O
I1307K	NN	O	O
variant	NN	O	O
leads	NN	O	O
to	NN	O	O
increased	NN	O	O
adenoma	NN	O	B-Disease
formation	NN	O	O
and	NN	O	O
directly	NN	O	O
contributes	NN	O	O
to	NN	O	O
3	NN	O	O
%	NN	O	O
-	NN	O	O
4	NN	O	O
%	NN	O	O
of	NN	O	O
all	NN	O	O
Ashkenazi	NN	O	O
Jewish	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
.	NN	O	O

The	NN	O	O
estimated	NN	O	O
relative	NN	O	O
risk	NN	O	O
for	NN	O	O
carriers	NN	O	O
may	NN	O	O
justify	NN	O	O
specific	NN	O	O
clinical	NN	O	O
screening	NN	O	O
for	NN	O	O
the	NN	O	O
360	NN	O	O
,	NN	O	O
000	NN	O	O
Americans	NN	O	O
expected	NN	O	O
to	NN	O	O
harbor	NN	O	O
this	NN	O	O
allele	NN	O	O
,	NN	O	O
and	NN	O	O
genetic	NN	O	O
testing	NN	O	O
in	NN	O	O
the	NN	O	O
setting	NN	O	O
of	NN	O	O
long	NN	O	O
-	NN	O	O
term	NN	O	O
-	NN	O	O
outcome	NN	O	O
studies	NN	O	O
may	NN	O	O
impact	NN	O	O
significantly	NN	O	O
on	NN	O	O
colorectal	NN	O	B-Disease
cancer	NN	O	I-Disease
prevention	NN	O	O
in	NN	O	O
this	NN	O	O
population	NN	O	O
.	NN	O	O

Localization	NN	O	O
of	NN	O	O
human	NN	O	O
BRCA1	NN	O	O
and	NN	O	O
its	NN	O	O
loss	NN	O	O
in	NN	O	O
high	NN	O	O
-	NN	O	O
grade	NN	O	O
,	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
inherited	NN	O	I-Disease
breast	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

Although	NN	O	O
the	NN	O	O
link	NN	O	O
between	NN	O	O
the	NN	O	O
BRCA1	NN	O	O
tumour	NN	O	B-Disease
-	NN	O	O
suppressor	NN	O	O
gene	NN	O	O
and	NN	O	O
hereditary	NN	O	B-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancer	NN	O	I-Disease
is	NN	O	O
established	NN	O	O
,	NN	O	O
the	NN	O	O
role	NN	O	O
,	NN	O	O
if	NN	O	O
any	NN	O	O
,	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
in	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
familial	NN	O	I-Disease
cancers	NN	O	I-Disease
is	NN	O	O
unclear	NN	O	O
.	NN	O	O

BRCA1	NN	O	O
mutations	NN	O	O
are	NN	O	O
rare	NN	O	O
in	NN	O	O
sporadic	NN	O	B-Disease
cancers	NN	O	I-Disease
,	NN	O	O
but	NN	O	O
loss	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
resulting	NN	O	O
from	NN	O	O
reduced	NN	O	O
expression	NN	O	O
or	NN	O	O
incorrect	NN	O	O
subcellular	NN	O	O
localization	NN	O	O
is	NN	O	O
postulated	NN	O	O
to	NN	O	O
be	NN	O	O
important	NN	O	O
in	NN	O	O
non	NN	O	B-Disease
-	NN	O	I-Disease
familial	NN	O	I-Disease
breast	NN	O	I-Disease
and	NN	O	I-Disease
ovarian	NN	O	I-Disease
cancers	NN	O	I-Disease
.	NN	O	O

Epigenetic	NN	O	O
loss	NN	O	O
,	NN	O	O
however	NN	O	O
,	NN	O	O
has	NN	O	O
not	NN	O	O
received	NN	O	O
general	NN	O	O
acceptance	NN	O	O
due	NN	O	O
to	NN	O	O
controversy	NN	O	O
regarding	NN	O	O
the	NN	O	O
subcellular	NN	O	O
localization	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
proteins	NN	O	O
,	NN	O	O
reports	NN	O	O
of	NN	O	O
which	NN	O	O
have	NN	O	O
ranged	NN	O	O
from	NN	O	O
exclusively	NN	O	O
nuclear	NN	O	O
,	NN	O	O
to	NN	O	O
conditionally	NN	O	O
nuclear	NN	O	O
,	NN	O	O
to	NN	O	O
the	NN	O	O
ER	NN	O	O
/	NN	O	O
golgi	NN	O	O
,	NN	O	O
to	NN	O	O
cytoplasmic	NN	O	O
invaginations	NN	O	O
into	NN	O	O
the	NN	O	O
nucleus	NN	O	O
.	NN	O	O

In	NN	O	O
an	NN	O	O
attempt	NN	O	O
to	NN	O	O
resolve	NN	O	O
this	NN	O	O
issue	NN	O	O
,	NN	O	O
we	NN	O	O
have	NN	O	O
comprehensively	NN	O	O
characterized	NN	O	O
19	NN	O	O
anti	NN	O	O
-	NN	O	O
BRCA1	NN	O	O
antibodies	NN	O	O
.	NN	O	O

These	NN	O	O
reagents	NN	O	O
detect	NN	O	O
a	NN	O	O
220	NN	O	O
-	NN	O	O
kD	NN	O	O
protein	NN	O	O
localized	NN	O	O
in	NN	O	O
discrete	NN	O	O
nuclear	NN	O	O
foci	NN	O	O
in	NN	O	O
all	NN	O	O
epithelial	NN	O	O
cell	NN	O	O
lines	NN	O	O
,	NN	O	O
including	NN	O	O
those	NN	O	O
derived	NN	O	O
from	NN	O	O
breast	NN	O	B-Disease
malignancies	NN	O	I-Disease
.	NN	O	O

Immunohistochemical	NN	O	O
staining	NN	O	O
of	NN	O	O
human	NN	O	O
breast	NN	O	O
specimens	NN	O	O
also	NN	O	O
revealed	NN	O	O
BRCA1	NN	O	O
nuclear	NN	O	O
foci	NN	O	O
in	NN	O	O
benign	NN	O	O
breast	NN	O	O
,	NN	O	O
invasive	NN	O	B-Disease
lobular	NN	O	I-Disease
cancers	NN	O	I-Disease
and	NN	O	O
low	NN	O	B-Disease
-	NN	O	I-Disease
grade	NN	O	I-Disease
ductal	NN	O	I-Disease
carcinomas	NN	O	I-Disease
.	NN	O	O

Conversely	NN	O	O
,	NN	O	O
BRCA1	NN	O	O
expression	NN	O	O
was	NN	O	O
reduced	NN	O	O
or	NN	O	O
undetectable	NN	O	O
in	NN	O	O
the	NN	O	O
majority	NN	O	O
of	NN	O	O
high	NN	O	O
-	NN	O	O
grade	NN	O	O
,	NN	O	O
ductal	NN	O	B-Disease
carcinomas	NN	O	I-Disease
,	NN	O	O
suggesting	NN	O	O
that	NN	O	O
absence	NN	O	O
of	NN	O	O
BRCA1	NN	O	O
may	NN	O	O
contribute	NN	O	O
to	NN	O	O
the	NN	O	O
pathogenesis	NN	O	O
of	NN	O	O
a	NN	O	O
significant	NN	O	O
percentage	NN	O	O
of	NN	O	O
sporadic	NN	O	B-Disease
breast	NN	O	I-Disease
cancers	NN	O	I-Disease
.	NN	O	O
.	NN	O	O