Dominant
Inheritance
in Three
Generations
By John
William
B. Graham,
P. Webster,
Philip
J. P. AIlain,
of Hemophilia
of Women
Emily
S. Barrow,
M. Blatt,
Philip
D. A. Barrett,
VIDENCE
ACQUIRED
genetic
control
tion geneticists
have
shown
within
the normal
population
tem
are
the
and
that
the
levels
consistent
with
X chromosome.
activity
is reduced
autosomal
that
VIII
within
and
between
kindred
(von
are
implies
Barrow
that
and
From
the School
Submitted
June
Supported
the General
Address
University
1975
of Medicine,
10. 1974;
in part
Clinical
existence
for
reprint
of North
by Grune
University
by USPHS
requests.Carolina,
& Stratton,
Vol. 46, No. 2 (August),
Grant
Centers
HL-06350
Dr.
John
Hill,
Popula-
the
levels
sys-
of hemophilia
A families
at the hemophilia
that
coagulant
locus
on
factor
VIII
mutation
at
conclusive
a locus
genetic
having
in the
genetic
Chapel
control
all
an
others,
recessive
of factor
situation
Hill,
on
evidence
a combined
different
from
as an autosomal
that
Carolina,
that
factor
VIII
of a polygenic
of persons
the
suggests
complex.
VIII.
is probably
N. C. 27514.
11. 1975.
Branch
Chapel
of plasma
activity
is involved
of North
March
is very
radically
inherited
emphasized
accepted
Research
locus
of
disease),
The
a third
have
alleles
shown
as a result
Willebrand’s
involved.
at least
Graham
the
sources
members
of factors
V and VIII,
a phenotype
to a single
sibship
and apparently
trait,
Blood,
loci
of
factor
that
the distribution
is consistent
with
in some
two
a variety
coagulation
existence
of multiple
Family
studies
have
chromosome
at least
defect
limited
©
the
A. I. Cederbaum,
H. R. Gralnick
when
a chymotryptic
digest
of cryoprecipitate
of the prebend
is examined
by
SDS-polyacrylamide
gel
electrophoresis.
Six possible
genetic
explanations
are
entertained.
Balanced
X-autosomal
translocation
of hemophilia
A heterozygotes
has been excluded
by cytogenetic
analysis
of metaphase
chromosomes.
Classic
von
Willebrand’s
disease
(vWd)
is probably
excluded
on the basis of the laboratory
data,
and extreme
lyonization
of hemophilia
A heterozygotes
on probabilistic
grounds.
The genetic
possibilities
which
cannot
be excluded
include
a previously
unrecognized
variant
mutation
at the vWd
locus, a dominant
mutation
at the hemophilia A locus on the X chromosome,
and
dominant
mutation
at
a hypothetical
fourth
locus involved
in factor
VIII synthesis and control.
FROM
of blood
R. Roberts,
Buchanan,
and
A bleeding
diathesis
is described
which
is
phenotypically
indistinguishable
from
hemophilia
A and which
has been transmitted
as a dominant
trait
in three generations
of women
in a North
Carolina
kindred.
The abnormal
phenotype
is characterized
by clinical
mildness
and slightly
abnormal
clotting
time,
prothrombin
consumption,
and
partial
thromboplastin
time.
Bleeding
time,
platelet
count,
clot
retraction,
tourniquet
test, and prothrombin time
are normal.
Concentrations
of
factors I, II, V, VII, IX, X, and XII are normal, while
factor VIII activity
is reduced
to 2%-5%
of control
values.
Dc nova
synthesis
of factor VIII does not occur after
transfusion;
factor VIll-related
antigen
is
normal;
patients’
plasmas
aggregate
platelets
normally
in the presence
of nstocetin,
and a typical
protein
pattern
is seen
E
Harold
A
and USPHS
(NIHL)
of the Division
B. Graham,
Research
of Research
Professor
of
Grant
RR-46
from
Resources.
Pathology,
School
of
Medicine,
N. C.
Inc.
1975
175
1 76GRAHAM
ET AL.
even
more
terns
of
complex,
because
inheritance
kindred
differ
from
with
the
factor
patterns
VIII
defects
described
exist
above.’
complexities,
plus the evidence
concerning
molecular
variants
IX, and X have
forced
the blood
coagulation
establishment
tional
genetic
nomenclature
nomenclaturists
whom
abnormal
to
accomodate
have recommended
products
are
garded
as phenotypes
types (i.e., CRM)
from
hemophilia
dominant
has
We
shall
sponsible
lemmas
for
posed
ambiguous
this hereditary
by this family,
phenotypes
been
one
was
obtained
occasion
and
family.
syringe
of 8:
The
earlier
was
discarded
0.5
ml
mediately
of
and
years,
3.8%
I hr
shipped
or
centrifugation
The
and
days
using
after
3 days
the
for
with
Tests
of hemostasis
the North
Carolina
levels
were
factors
in which
pooled
normal
control
was also
assessed
assessed
the
this
family.
(Fenwal
Laboratories,
the plasma
frozen
and
cipitate
at
-
30’C.
After
dissolved
in
was
tested
and
XII
of the
thawing
supernatant
immediately.
disuch
were
test
plasma
the
overnight
It was
im-
either
tested
Plasma
with
at
samples
70’C
-
solid
after
CO2.
and
approximately
frozen
at
-
70’C,
then
using
from
sy-
37’C
the
for
3
cultures
chromosomes
plasma
with
a standard
from
as
containing
after
1/9
(240
the
cryoprecipitate
volume
of
at
with
24’C
±
VII
time
and
control
volume
at 5000
ml.
plasma
plasmaphereses
the
4%
sodium
g at 4’C
for
The
solution
of
citrate
15 mm,
was
centrifuged,
alpha-chymotrypsin
per-
proband
20 ml per I U of blood)
11-12
VIII
X were
test.4
and
was
for
a standard,
Factor
normal
single-unit
centrifugation
of
of factors
hemophilia,
plasma
that
substrate.
a pooled
of
of
Assays
thromboplastin-type
with
prothrombin
plasmas
Laboratory
elsewhere.4
partial
Concentrations
bags
a final
at
Coagulation
plasma
plastic
digested
heparinized
of the
described
compared
defective
at 4’C,
in
examination
out
was
X-linked
and
using
incubated
harvested
Clinical
with
cell-free
were
been
carried
plasma
using
NIH
Ill.),
The
plasma
method
centrifuged
frozen
were
for
patients
in
Grove,
obtained
her
first
in a ratio
plasma
system,
was
of
in the
citrate
analysis.
quickly
at least
vacutainer
the
filled
on
blood
was
quickly
was
Cells
have
a specifically
three
respun.
the
the
and
Fenwal
in use at the
of them
of the test
and
re-
technique.3
effect
Morton
was removed
most
collected
the
prepared
the corrective
was
blood
immediately
were
methods
at
citrated
citrate
and
sodium
by
tubes,
cultures
procedure.5
prepared
3.2%
were
using
Seabright
substrate
with
subsequent
technique.
slides
using
individuals,
blood
the
members
wherein
in containers
sodium
TGT
were
13 normal
The
IX,
ability
1974
by a modified
deficient
Cryoprecipitates
on
VIII,
for
proband
available
collected
glass
lower
Moorhead
by the
plasma
by comparing
a specifically
formed
V,
9-mm
established
Hospital;
corrective
the
lymphocyte
were
air-dried
done
a
be
of her cryoprecipitate.
a modified
Memorial
assays
against
and
instances,
transported
July
of
which
using
as
might
to resolve
of reporting
the
other
determination
of 4%
study
of the usual
Giemsa
of
in
preparation
of incubation,
by staining
were
50 ml
microcultures
a modification
both
antigen
plasmapheresed
for
been
or
at
containing
mixed
-20’C
blood
to the NIH
pheno-
generations
which
technique
was
into
of the citrated
CO2
three
from
and
have
blood
pipetted
for
blood
and
In
stored
in solid
Peripheral
citrate.
the plasma
occasions
second
of
Sweden
was
ringes,
in the
and
plasma
deficient
in
re-
METHODS
numerous
by a two-syringe
samples
and
of her
shipped
blood
sodium
frozen
proband
ml
plasma
the
the
kindred
of women
is indistinguish-
mechanism
granddaughter,
obtained
4.5-mI
to Connecticut
AND
on
daughter,
were
at l000-2000g,
within
level
mother,
samples
1. In recent
into
250
her
for
Thus
kindred.
by venipuncture
from
the
I, VIII,
addi-
phenotypes
CRM)
be
purely
etc.
pattern,
describe
our efforts
and emphasize
the importance
MATERIALS
Blood
knowledge.2
a unique
phenotype
transmitted
indicate
patgenetic
of factors
to develop
deficiency
which
are
is to describe
Their
abnormal
A and
characteristic.
new
by rs alleles,
while
the
as the fruit of aa
alleles,
produced
be regarded
The purpose
of this communication
transmitting
factor
VIII deficiency.
able
the
that coagulation
demonstrated
(i.e.,
whose
These
then
recovered,
of
cryopre-
(Worthington
HEMOPHILIA
A
177
Biochemical
Corp.,
Freehold,
N.J.)
chloric
at a concentration
of
the
acid
cryoprecipitate
addition
time
to
of0.03
of
ml,
25 g/ml
40-cm
The
of 4’C,
with
HC1
pH
to
collected.
sured
column
was
and
the collected
fractions
levels
ability
by the method
precipitate
Lars
with
Holmberg
pared
with
The
ability
antigen
of the
whole
of
at 280
nm,
and
protein
volume
of
the
2.5 x 40-cm
receiving
digested
each
a 2.5 x
Inc.,
and
Uppsala,
temperature
NaCl
adjusted
l.6-1.8-ml
with
fractions
concentration
were
was
column
was
cryoprecipitates
was
mea48
from
electrophoresis
to
subsequent
through
H2O
and 0.1 M
and
hydro-
added
concentration
filtered
Chemicals,
20 cm
M
was
enzyme
Fine
14 ml/hr,
0.001
Each
was
ultra-pure)
were
Laurell
plasma
The
by Dr.
of the
ability
was
of two
of the
Hoyer
proband
laboratories
was
compared
plasmas
Leon
three
inhibitor
The
antibody
method.9
in
VIII
Graham.8
VIII
controls
assessed
anti-factor
and
anti-factor
the
to a final
Polyacrylamide-gel
a human
by Barrow
of normal
void
in
cryoprecipitate.
(Pharmacia
averaged
use
chymotrypsin
ml.
normal,
performed
on
described.7
VIII-related
using
plasmas
The
plasmas.
to neutralize
a rabbit
4B
columns
as previously
described
column
al.6
from
proband’s
of factor
The
et
of
of
cryoprecipitate
(Mann
Tris
before
ml
of approximately
by absorbance
Lowry
collected
the
M
of the
g/ml
amounted
Sepharose
0.05
rate
just
0.250
treatment,
at a pressure
monitored
of
were
hemophilic,
with
eluted,
flow
method
eluates
The
packed
dissolved
205
intervals,
After
containing
The
been
Initially,
of
15-30-mm
were
a buffer
7.35.
Protein
niques.
at
had
concentration
cryoprecipitate.
columns
by the
Similar
made
of
Pharmacia
Sweden).
a final
which
10 mg/mI.
using
and
her
of the
with
three
by
different
determined
abnormal
normal
affected
tech-
in Chapel
Hill
plasmas
controls
females
by
were
to
Dr.
com-
a radioimmunoassay.’0
daughter
to
cause
the
aggregation
of
fixed, washed human platelets in the presence of ristocetin
was examined
by a method
recently
described.11’12
The location
of the ristocetin-aggregating
factor
in the plasma
of the proband
was further defined by gel filtration.
Ten milliliters
of plasma from a normal
control,
a patient
with hemophilia
A, and the proband
were filtered separately
through
a Bio-Gel
A-iS M column
(2.5 x 40 cm) using a Tris-HC1
buffer, 0.05 M, pH 6.8, containing
0.15 M NaCl.
Three protein
peaks were eluted in each instance. Samples from each peak of each plasma were pooled and tested
separately
in the
as the end
point.
ristocetin
Medical
Histories
platelet
aggregation
test
using
agglutination
of fixed,
washed
platelets
RESULTS
of Pertinent
Members
The proband,
VIII-2,
was born
Dr. Jessica
Lewis in January
1954
lowing
tooth
extraction.
She was
Graham
hematuria.
Dr.
Harold
while
being
Since
1960
Roberts,
in the outpatient
sary by Dr. W.
lem
was
who
clinic.
Her
P. Webster.
The patient
has
was first noted
characterized
bled excessively
at age 15. While
treated
she has
of the “Be”
in 1927 and was first seen in Chapel
Hill by
at age 27 because
of prolonged
bleeding
folnext seen in 1960,
at age 33, by Dr. John
at the North
Carolina
Memorial
been treated
in our hospital
on four
has
also
dental
followed
her
problems
suffered
since’ childhood
when
she began
cutting
by numerous
bruises
and
when
she
Kindred
condition
have
been
Hospital
occasions
between
dealt
with
for
by
admissions
when
neces-
from
excessive
bleeding.
This probdeciduous
teeth,
and her childhood
free bleeding
from
minor
cuts.
She
teeth were extracted
and
did not bleed
excessively
bled for 4 wk
at childbirth,
following
dilation
and curettage,
an episode
which
blood
and 1 U of plasma.
Following
the extraction
required
of a third
after
tonsillectomy
she bled
for 3 mo
17 U of whole
molar
at age 27,
she required
several
blood
transfusions.
On May
23, 1967,
she was
North
Carolina
Memorial
Hospital
for relief of a nasal
obstruction
been caused
by a blow
to the nose 5 mo previously.
After
preparation
gery with 2 U of cryoprecipitate,
her left turbinate
was electrocoagulated
treated
which
for
at
had
surwith-
178
GRAHAM
out
difficulty.
She
hematuria,
3-day
period,
January
bags
most
4,
readmitted
ceased
of it on
1971,
to the
after
again
receipt
the
for
hospital
of
day
on
17 bags
of admission.
hematuria
which
November
12,
1968,
of cryoprecipitate
Her
ceased
latest
after
with
during
admission
a
was
administration
on
of
80
of cryoprecipitate.
The
menses
proband
has been
doing
well since
her
with
Ovral
and only
rarely
experiencing
has never
limitation
The
She
was
which
ET AL.
had
hemarthrosis
nor
of motion
of her joints.
mother
has
of the
no history
several
accidents
traumatic
both
and
events
bones
the
VII-l,
of the
and
have
lower
uterus
in
was
even
has
1942,
in 1898
though
never
included
leg,
and
and
she
required
childbirth
three
of a small
and
been
involved
well.
in
fracture
removal
of
no
Physically
times,
1940,
tumor
multiple
dental
extractions.
The father
of the proband,
VII-2,
was born
in
carcinoma
of the prostate.
He was admitted
several
Veterans
Administration
at Salisbury
and
Durham,
is
is living
has
in
her
She
there
transfusion.
cholecystectomy
removal
regulating
bleeding.
bleeding,
born
bleeding,
life
right
admission,
breakthrough
gastrointestinal
operations,
in her
of
myomata
proband,
of excessive
last
one
of
of
hip
in
leio1966,
and
of impaired
observed.
hemostasis
A prothrombin
control,
and
cystography
cleared
in
bleeding
in 1962,
he
was
had
tendency
though
her
of the
excessive
bleeding,
VIII
granddaughter
infancy
and
Laboratory
level
noted
battery
many
test
that
her
of the
effective
2% to as high
transfusion,
and
born
The
surgically
for therapy
but
as low.
1946
and
much
less
than
She
repaired
of the
bleeding.
in
has
underwent
9 without
difficulty
and
replacement
therapy.
was
typical
whole-blood
other
clotting
evidenced
her
a
mother,
al-
tonsillectomy
gave
birth
at
to a daughter
in 1972.
tendency.
She
has
out
time
on
are
was
slightly
factors
normal
have
showed
on
been
values
retraction.
Factor
VIII
levels
as 4%
of
during
the
20 yr
it does
control
not
proband,
V 111-2,
in Table
prolonged,
seem
to
in the
normal
all of many
occasions,
and
present
in normal
numbers
in clot
and
the
recorded
had
a
matter
whether
have
of
ranged
observation,
the
substrate
several
I. It will
and
be
prothrom-
impaired.
The partial
thromboplastin
due solely
to reduction
of factor
coagulant
time was
platelets
carried
observations
was slightly
presumably
The bleeding
was negative;
fully
hernia
was
in 1963
of the proband,
X-l, was born
shown
no evidence
of a bleeding
of tests
times,
bin consumption
clearly
prolonged,
assays
hemorrhage.
by excessive
was
is almost
at age
requiring
has
without
was never
100%
of
as
Data
A complete
of them
1958
accompanied
IX-l,
toward
factor
were
proband,
age 6 and appendectomy
(X-1) at age 26 without
The
normal
in
bleeding
recorded
by transurethral
prostatectomy,
bleeding
from
which
the catheter
having
been
removed
without
excessive
Neither
daughter
slight
cystography
and excessive
1958 was
day 6. A right inguinal
orchidectomy
was performed
carcinoma.
The
in
bladder
was followed
a few days,
on postoperative
and bilateral
prostatic
never
obtained
time
performed
1892 and
died
in 1964 of
times
to hospitals
of the
N. C., where
a history
time
was
VIII,
since
range.
the
and
tourniquet
have been
from
less
except
plasma
than
after
for
HEMOPHILIA
A
179
Table
1. Laboratory
Data
on Pertinent
Members
Normal
Test
Whole
Blood
Proband
time (mm),
Granddaughter
(IX-1)
(X-1)
14/41.5
13.5/45;
9;
>80
73
50-80
157/77/86
121/57/62
2-4
2; 2; 2.5
3.5; 4.5
Negative
Neg.
x3
150-450
312;
178
Good
Good
12; 10.5/69
consumption
consumed
Partial
Proband’s
Daughter
8-10/25-30
glass/silicone
Prothrombin
Kindred
Proband’s
(Vlll-2)
Range
clotting
of the “B.”
(%
in 1 hr)
thpln.
time
(secs),
subect/control/mixture
Bleeding
time (min)(Duke)
Tourniquet
Platelet
Clot
test
count
x 10
retraction
Prothrombin
11.5-14
time (sec),
subject/control
11.5/12
12.2/12
12.1/11.2
12.7/12
Clotting factor assays
Factor
I (fibrinogen)(mg/100
Factor
II (prothrombin)
(Iowa
ml)
200-400
400
350-500
450
50- 150
90
50-150
66
units)
Factor
V (proaccelerin)
Factor
VII (proconvertin)(%)
(%)*
Factor
VIII (AHF)
one-stage
(%)
50-150
<2
two-stage
(%)
50- 150
1.9
Factor
IX (Christmas)(%)
50-150
110
Factor
X (Stuart)(%)
50-150
92
Factor
XII (Hageman)(%)
50-150
130
Factor
VIII-related
Neut.
inhibitor
anti-VIlI
(%)t
(%)
50-150
100
100
50-150
76
93
Radioimmune,
rabbit Ab. (%)
50-150
72
46
Ristocetin aggregation
of
50-150
66
60
normal
Unless
platelets
otherwise
*per
indicated,
Laboratory
normal
data
and
E. S. Barrow.
Malmo,
VIII
VIII
Allain,
assay
values
Chapel
in the
Clinical
Coagulation
Laboratory
Highly
potent
inhibitor
from
male
with
hemophilia
Conn.
Hill and Paris.
has been activated
with kaolin
prior
to use. Almost
identiwere obtained
on the same sample
with assays
of the one-
types.
The
as assessed
normally
collected
Sweden.
of 1. Hoyer, Farmington,
of i-P.
were
Hill.
control.
of L. Holmberg,
and two-stage
in full amount,
platelets
Chapel
of J. B. Graham
§ Laboratory
II Laboratory
the factor
cal factor
laboratory
Hospital,
cent of pooled
67
II
(%)
of the N. C. Memorial
tLaboratory
used.
3;2
antigen
of human
Ppt. of rabbit
4;5
in the
subject’s
in three
presence
plasma
different
contains
systems,
factor
VIII-related
and her plasma
antigen
aggregates
of ristocetin.
Filtration
of the proband’s
plasma
through
Bio-Gel
A-l5
M showed
that her
ristocetin-aggregating
capacity
is equal
to that of a normal
control
and a man
with hemophilia
A (Table
2). In all three
subjects,
more
than
85% of the activity
Since
was
present
the
proband
in the
(and
void
volume.
her
daughter
and
granddaughter)
can
be
presumed
A
180
GRAHAM
Table 2.
of Rist ocetin-aggregati
Recovery
ng Factor
Units’
Bio-GeI
From
Peak
lyp,
of Plasma
Hemophilia
A
Proband
*One
unit is amount
on genetic
tempt
was
made
to
be
to detect
an
Experiments
showed
on Sepharose
4B
cipitates
prepared
from
peak
tein
protein
content
column.
volume
in the
as
the
A very
fraction
20%-40%).
factor
from
the
proband’s
did
not
an
or
of the
patients
volume
of
gene
factor
with
VIII
0.169
of the
(see
the
with
were
enter
X-linked
in her
A.
cryoprecipitate
of the
Spe-
being
0.113.
eluted
of
in pro-
from
the
was found
in the void
activity
of 3% (normal,
When
pooled,
was
concentration
abrupt
rise
X-linked
to 4%.)
variety
the
found
in her
gel
in the
presence
with
had
fractions
and
was
observed
have
column
concentrated,
a protein
pattern
protein
hemophilia
proteins
a 5% polyacrylamide
gel
at-
pattern
of her
cryofrom
that
of cryopre-
activity
factor
VIII
hemophilia
1%
an
VIII-like
cryoprecipitate
from
below),
activity
eluted
at the void volume
± 0.058,
mean
±
1 SD, n = 13).
proband’s
of procoagulant
with a peak
varied
identical
CRYOPRECI
55
a mutant
the protein
elution
not be distinguished
electrophoresis,
2 BAGS
-J
for
cryoprecipitate
to polyacrylamide-gel
sulfate,
0.00
in 1 ml of plasma.
with factor
(O.D.
280,
majority
patients
dodecyl
0.03
factor
small
amount
of the proband,
which
which
0.00
0.17
was followed
by return
of the protein
nearly
to base line, followed
by an
levels
fraction
0.00
0.02
that
could
void
(Male
VIII
0.02
normals
This small
protein
peak
ensuing
column
fractions
3
0.23
abnormality
cifically,
a small
protein
peak
of the normal
cryoprecipitate
Peak
2
0.22
heterozygous
plasma.
precipitate
The
Peak
of ristocetin-aggregating
grounds
Factor
Peak
1
V0
Normal
A-15 M EIua tes of Plasma
of Ristocetin-aggregating
Protein
ET AL.
subjected
void
volume
of sodium
cryoprecipitates
OF
PITATE
0
50
z
0
40
O
35
30
25
20
0
IU
0
4
Ls.
5
I
0
10
20
30
40
TIME
FIg. 1.
Factor VIII levels
cryoprecipitate
from normal
of the
plasma.
prebend
Infusions
IN
I
I
I
I
I
50
60
70
80
90
twice
of two
I
100
HOURS
following
administration
were done 10 hr apart.
bags
of
the
HEMOPHILIA
A
of normals
181
and
each
genotype,
band
had
X-linked
and
hemophiliacs.
After
electrophoresis,
an estimated
molecular
The response
of
examined
carefully
the
to
might
of
be a variant
weight
proband
assess
von
reduction
a single
to
band
seen
fractions
in each,
from
and
each
of 235,000.
transfusion
possibility
the
of the
was
Willebrand’s
of
that
disease.
normal
cryoprecipitate
was
the
hemophilic
phenotype
In
preparation
for
electroco-
agulation
of her left turbinate
followed
by two additional
each transfusion
are shown
in 1967, she was given two bags of cryoprecipitate
bags
10 hr later.
The factor
VIII
levels
following
in Fig. 1. It will be noted
that each
time there
was
an immediate,
in plasma
maximal
diminished
rapidly
rise
after
factor
transfusion,
with
VIII
and
a half-time
that
the
Only
selected
laboratory
(IX-l)
and her granddaughter
will be noted
that there
are
tests
were carried
out on the
(X-1).
These
are also shown
no exceptions
to the conclusion
and
same
tor
granddaughter
VIII
have
the
phenotype
factor
of about
as the
VIII
level
8 hr.
proband’s
in Table
that
the
proband,
i.e.,
daughter
I, and
daughter
CRM
,
it
fac-
deficiency.
Genetic
Studies
History
of the “Be”
kindred.
The
German
origin,
and the family
Rowan,
North
Carolina,
since
from the Rhineland
Palatinate.
segment
of the
kindred
are
kindred
“Be”
(Fig.
2) is of predominantly
has resided
in the counties
of Cabarrus
and
at least
1727 when
Johannes
“Pe”
emigrated
The origins
and dates of emigration
of the “Be”
less
certain,
but
the
surname
has
been
listed
in the
NUMBER
OF
MEMBERS
GENERATION
I
(2)
JOHANNES
It
(4)
2
MARGARET
I
MICHAEL
‘PE’
:iir
(II)
2
LEWIS
NE
IX
V
REGINA
LL
ELIAS
“BE”
(10)
(26)
VIE
(58)
RACHEL
6
SUSAN
3
FO
5
JOE
‘BE”
JANE
“PE”
ISSAC
BE
7
JOHN
HENRY
‘BE”
4
MARTHA
“BE”
8
LEAH
3
TRAVIS
‘DR’
2
“BE”
CLIFFORD
“BE”
“BE’
RUBY’S
“NA’
(97)
GEORGE
“SM’
\
2
SANDRA
“HA”
X
6
SOLOMON
“OR”
2
FRED
‘NA’
IX
8
BARBARA
4
CARRIE
(87)
“BE”
7
ELIZA
2
SOLOMON
‘PE’
MARY
XIII
CHRISTOPHER
“DR’
5
PAUL’PE”
CATHY
I
SUSAN
“BE’
XE
DANIEL
PE
“HA”
(3)
ASHLEY
‘HA”
TOTAL
=
(307)
Fig. 2.
The “Be”
kindred
transmitting
dominant
hemophilia
A. Females
are represented
as
circles, males as squares;
abnormal
females
are shown
as solid circles.
Only the portions
of the
pedigree
pertinent
to the question
of consanguinity
of the parents
of the proband
and her
descendants are shown. The prebend
is indicated
by the arrow.
182
GRAHAM
censuses
of these counties
now been anglicized,
the
certainly
of Germanic
since
at least
1820. Although
surnames,
“Pe,”
“Be,”
“Dr,”
was no history
of the family
band
(VIII-2)
has
A systematic
of the proband
tion
clearly
ancestors
three
her
coincidence
of both
of
the
made
to test
ing the three
nant
fertile
had
a hemorrhagic
recessive
parents
small
VIII
be
all men.
affected
proband
levels.
Cytogenetic
specific
from
of the
number
of
the
the
the
father
surname
is a possible
proband
a common
ancestor
shared
a common
men
or the
female
pro-
diathesis.
whether
both bore
trait
the
the
mode
were
was
ancestor
original
and mother
“Be,”
and
of inheritance.
identified
not
for
the
discovered.
in an earlier
German
settlers
pre-
Nevergeneraand
the
of surnames.
Coagulant
factor
proband
who could
cept the
tor VIII
suffered
generations,
and
parents
probably
because
of the kindred
distributed
the sex of five being
unsince x2 = 0.94,
p > 0.30.
of a bleeding
tendency
among
either
prior
to the eighth
generation,
and only
for an autosomal
All of the
ceding
theless,
307 members
140 females,
from
unity
search
was made
to discover
were genetically
related,
since
homozygosity
have
almost
origin.
Some
information
was obtained
on
over
ten generations-l62
males
and
known.
This
sex ratio
is not different
There
women
all the surnames
and “Fo”
are
ET AL.
reason
hemophilia
females
transmitted
and
assays
found
Factor
women,
her
studies.
bearing
were
done
and would
VIII levels
are shown
descendants,
Detailed
upon
A might
heterozygous
a balanced
on all of the
cooperate,
an
close
relatives
especial
effort
of the
being
on 12 members
of the kindred,
in Fig. 3. It will be noted
that
includall ex-
including
four
chromosome
the
inheritance
males,
analyses
of
were
hemophilia
conceivably
for X-linked
have
occurred
hemophilia
translocation
between
have
performed
in women.
in three
A if they
an
normal
generations
possessed
X chromosome
facfor
a
Domiof
and
bearing
GENERATION
Ix
x
2%
Fig. 3.
numbers
affected
and the
thos.
in
The plasma factor VIII levels of the close relatives of the proband
are indicated
by the
below
the pedigree
symbols.
Females
are represented
by circles, males by squares, and
females
by solid circles.
The left marginal
Roman
numerals
representing
generations
Arabic
numerals
within
the solid circles and representing
individuals
are identical
with
Fig. 2. The proband
is again
indicated
by the arrow.
HEMOPHILIA
A
183
X-CHROMOSOMES
HEMOPHILIC
OF 3
WOMEN
1)
Ii
VIII-2
tX-I
X-1
Fig. 4.
The X chromosomes
of the proband
(Vlll-2),
her daughter
(lX-1),
and
her granddaughter
(X-1),
all of whom
have factor
VIII levels
less than
5% of normal.
The chromosomes
have been stained
with
Giemsa,
and a single
G-band
is present
in the short arms,
and
two
G-bands
are present
in the long arms of all of them.
All three women
were 46, XX, and no abnormal
chromosomes
or bands were present.
the hemophilia
which
would
allele
and
an autosomal
chromosome,
appear
to be dominant
for reasons
which
Chromosome
preparations
were made
from
band’s
mother
(VII-l),
the proband
(VIII-2),
and her granddaughter
(X-1).
Air-dried
slides
the chromosomes
were counted
and karyotyped.
XX chromosome
X chromosome
tain
two.
females
are
Normal
can
complement.
expected
banding
be seen
in Fig.
described
our
With
to contain
will
a genetic
mechanism
be discussed
later.
cultured
lymphocytes
of the prothe proband’s
daughter
(IX-l),
were
stained
with
Giemsa,
and
All four females
showed
a 46,
Giemsa
staining,
the short
one G-band
and the long
patterns
for the
4; there
is no evidence
X chromosomes
of the
arms
arms
three
of the
to conaffected
of a translocation.
DISCUSSION
We
have
in three
generations
questions:
hemophila
(1)
A?
mechanism
The
philia
the
of the
women
Do the studies
(2) If the women
really
are
dominant
in one
transmission
family.
show
that
transmitting
Our
of hemophilia
data
the women
hemophilia
raise
are
A,
two
A
major
transmitting
what
genetic
is responsible?
phenotypes
A in several
proband
impairment
of
studies
of the proband
and her descendants
respects.
From
the laboratory
shows
slight
prolongation
of
of prothrombin
consumption.
whole-blood
Bleeding
closely
standpoint
resemble
(Table
clotting
time
time, tourniquet
hemo1), the
and
test,
slight
plate-
184
GRAHAM
let numbers,
and
mal, partial
gestion
ofan
IX, X, and
XII
not
a delayed
tate
(Fig.
clot
retraction
thromboplastin
inhibitor.
are
time
entirely
Specific
show that only factor
VIII
rise of factor
VIII following
1). When
the
normal.
Prothrombin
is moderately
prolonged,
coagulant
assays
for factors
proband’s
plasma
ET AL.
time
is nor-
and there
is no sugI, II, V, VII, VIII,
is significantly
transfusion
with
depressed,
normal
and there
cryoprecipi-
is examined
by SDS-polyacrylamide-
is
gel electrophoresis
of a purified
void
volume
fraction
of cryoprecipitate,
it
shows
bands
not different
from
normal
controls
and hemophilic
men.
Factor
VIII-related
antigen
is present
in full amount.
Ristocetin
aggregation
of platelets is effected
successfully
by the plasma
of the proband
and her daughter,
and
the Ristocetin-aggregating
ume just as it is in controls
From
the clinical
thesis
with
that
of
factor
which
activity
is found
and hemophiliacs.
standpoint,
hemophilia
the
A
is
almost
correspondence
less perfect.
of
in the
similar
factor
VIII
fact that
her daughter
though
their factor
VIII
clusion-admittedly
clinical
phenotype
based
symptoms
is not
does not
responsible
levels.
Probably
of even
and
granddaughter
levels are consistently
largely
that
A.
From
the
has occurred
continue
to
genetic
standpoint,
with
it is clear
that
in her large kindred
and that the trait
referred
to as dominant.
Furthermore,
in a germ cell of one of her parents.
the term
“dominant”
are discussed
we do not know
whether
the trait
must
be
of the
outbreeding
considered
have
been
significance
related
were
for
generation.
in the
normal
the
discrepancy
past,
were
the
which
proband
between
the
make
a distinction
is the
first
II of Reference
or X linked,
the
factor
though
unrelated
to
alcon-
that
their
conclusion
at a locus other
than
refer
to the phenotype
abnormal
Even
distant
and
an
is the
that
as
affected
is being
transmitted
in the fashion
it is assumed
that
a mutation
(Qualifications
concerning
use of
in Appendix
is autosomal
heterozygous
in each
granddaughter
greater
that
hemophilia
A until we discover
a parameter
which
is more objective
than intuition.
person
usually
occurred
diabasal
are essentially
asymptomatic,
less than 5% of normal.
Our
on intuition-is
mutation
We shall
vol-
not have
symptoms
for hemophilia
and
and clotting
factor
level in these
women
indicates
exactly
the same
as hemophilia
A, although
this
necessarily
imply
for hemophilia
void
the hemorrhagic
proband,
whose
The
VIII level appears
to be about
2% of normal,
does
are as severe
as those
usually
seen in men hemizygous
having
may
exclusively
fathers
the
2.) Although
affected
women
VIII
allele
parents
of both
to the “Be”
because
of the
the
proband
daughter
and
kindred.
As to the genetic
mechanism
responsible,
the six most
likely
(1) Our subjects
have von Willebrand’s
disease
(vWd),
(2) Our
variant
of vWd due to an unusual
mutation
at the vWd
locus,
possibilities
are
subjects
have a
(3) A balanced
X-autosomal
and has been
hemophilia
lyonization
produced
translocation
transmitted
a hemophilic
phenotype
zygotes
for hemophilia
philia
A locus
on the
mutation
at a previously
mal
It is quite
bleeding
plasmas
has occurred
in a heterozygote
for three
generations,
(4) Extreme
unlikely
times
aggregate
in three
successive
for
generations
of
A
has
hetero-
A, (5) A dominant
mutation
has occurred
at the hemoX-chromosome,
and (6) We are observing
a dominant
unrecognized
locus,
the fourth
factor
VIII locus.
that our subjects
and are CRM
platelets
normally
+
have the vWd
for the factor
in the
presence
phenotype.
V Ill-related
of
They
have norantigen.
Their
Ristocentin,
and
this
HEMOPHILIA
A
activity
185
is located
fused
would
in the
with
cryoprecipitate,
have,
i.e., there
Linkage
studies
we cannot
imagine
from
The
most
anced,
in
yet
heterozygotes
both
of the
adult
reported
the
showed
that
fertile
the
and
et al.,’4
in
short
that
who
X-autosomal
females
with
reviewed
all
active
in
replicator.
in the
cellular
chromosome
has
been
Barr
a reciprocal
on
the
our women
pedigree
these
marker
might
translocation,
are heterozygous
pattern
further
of
the
The
pattern
of
be
who
bal-
of
that
that
They
balanced
X
were
late.
Cohen
carriers
cytologically
is
if
chromoonly
that
possesses
structurally
have
that
because
body,
not
individual
X is
normal
is expected,
of the
of
chromosome
the morphologically
the translocated
the
translocated
one
14.
complement
normal
the
and
described
chromosome
replicated
cases
and
requires
the
intelligence,
the
that
for
transloca-
usually
produces
translocation
et al.,’3
both
vWd
of normal
of
but
bearing
account
entered
the Barr
balance
implies
that
is in
to the
imbalance
X-autosomal
circumstances
preferentially
Chromosomal
X chromosome
genetic
chromosomal
Sequestration
re-
positive
kindred
A.
human
under
have
in our
a translocation
This
finding
indicates
Barr
body,
which
implies
body
has
such
arm
that
which
one
trait
X chromosome
out
of the translocated
chromosomes
and
chromosomes
function
in cell metabolism.
reside
long
reported
chromosome
would
result.
Assuming
that
tion, the dominant
Buckton
the
suffering
X-autosomal
and
by
studies,
are
hemophilia
that
A
to succeed
in demonhad
the same
linkage
which
balanced
pointed
metabolism.
in the
the translocated
somal
imbalance
the
the
other
fertile
normal
translocations,
the late
X resides
are
X and
cytologically
any
trans-
linkage
we should
the
(Chromosomal
A balanced
generations
of the
the
always
normal
there
two
arm
locus,
vWd,
that
require
women
has
been
classic
X-linked
would
also appears
to be normal.
and/or
mental
retardation.)
for
women
vWd
was
for hemophilia
of factor
V III.
these
A is a balanced
for
kindred
proband
Except
If we were
phenotype
mechanism
hemophilia
the
that
certain
yet linked
the baby
infertility
involving
helpful.
at the
chromosomal
in our
since
very
possibility
as does
not
has
of dominant
occurring
When
as a hemizygote
of de novo synthesis
mutation
no one
plausible
transmission
the
we are
and
transmission
be
test
marker
Unfortunately,
fact autosomal,
locus.
fraction.
hemophilia
A phenotype.
locus
responsible
for this
to a vWd-linked
proof.
tion
would
to
undescribed
in a CRM
that
the
relation
vWd
how
plasma
she responded
no suggestion
was
of
a previously
sulted
strating
usual
both
abnormal
a balanced
translocathe hemophilia
allele
autosomal
DNA,
while
the normal
allele at this locus resides
on the cytologically
normal
chromosome.
The transmission
of the hemophilia
phenotype
under
this cytogenetic
hypothesis will be dominant,
even though
the allele is one which
is recessive
in heterozygotes
having
morphologically
inheritance
results
because,
for
normal
reasons
chromosomes.
stated
above,
are sequestered
in the Barr bodies.
(A moment’s
iso-X
chromosome
in a hemophilia
heterozyote
tern since it is known’
that the iso-X
preferentially
beauty
of the balanced
vides an explanation
X-autosomal
for this pattern
translocation
of inheritance,
finding
chromosomes
in the
of 46 normal
three
The dominant
mode
of
all of the normal
alleles
reflection
will reveal
that
an
would
follow
a recessive
patgoes to the Barr body.)
The
hypothesis
but that
affected
is not that it proit is testable.
Our
females
excludes
this
186
GRAHAM
chromosomal
mechanism
as that
responsible
for dominant
hemophilia
ET AL.
A in this
family.
The
almost
occurrence
all of the
bodies
has
conditions:
now
been
hemophilia
dystrophy,’7
extreme
kindred,
of extreme
normal
alleles
and
unambiguously
B,’5 deutan
has
lyonization
the probability
in a single
kindred
out why extreme
philia
treme
lyonization,
i.e.,
in a heterozygote
probably
might
demonstrated
color
blindness,’6
been
have
that this
is very
lyonization
random
inactivation
by sequestration
in at
and
demonstrated
produced
rare event
Barrow,
to occur
that
a single
kindred
would
be encountered
in three
successive
generations
probability,
or iO-iO’,
A.”8
this
probability
of exThe probability
event
is, therefore,
the
a possibility
which
While
in this
by chance
Elston’9
have
pointed
frequently
in hemo-
that
the
iO-iO.
in which
X-linked
muscular
of inheritance
three
times
and
more
B than
in hemophilia
A and have
estimated
lyonization
in hemophilia
is of the order
of
chance
vidual
least
three
Duchenne
in hemophilia
the pattern
has occurred
slight.
Graham,
is expected
of all or
in the Barr
third
can
had
occurred
by
power
of the indireasonably
be dis-
missed.
The possibility
that a dominant
locus on the X chromosome
cannot
not
been
reported
tural
gene
since
heterozygous
previously.
coding
mutation
has occurred
be excluded.
However,
Since
for a peptide
women
it is believed
which
enters
necessarily
that
into
the
the
produce
at the hemophilia
such a mutation
two
X locus
factor
bears
VIII
products,
of normal
peptides
produced
this possibility
under
a variety
in mind
we have
of circumstances
normal
:9-9:
is mixed
in
change
in the
plasma
1, the
obtained
hemophilic
when
normal
man.
Also,
concentration
human,
inhibits
rabbit,
A more
uct
the
or dog
strenuous
resembling
the
phenomenon
the
various
factor
time
philia
be tested
could
blindness,
the
loci
attempt
was
made
VIII.
The
factor
subunit
possibility
by studying
on the
kindred.
phenotypes
the
X chromosome
lacking
the
cells.
proband’s
different
plasma
proband’s
of normal
An
be
plasma
from
abacWith
plasma,
from
that
an unrelated
plasma
at any
when
abnormal
tested
with
molecular
protein
identical
with
individuals.2’23
in
factor
VIII
mutation
linkage
adjacent
existence
numbers
alternative
test
of
males
of
trait
to the
X locus
to G-6-P-D
hemophilia
of compound
of progeny,
dominant
and
females,
prodproband’s
in patients
a less than
volume
of the paamount
of protein
electrophoresis,
protein.
at the
of the
the
that
seen
Although
eluted
at the void
with
a normal
to polyacrylamide-gel
require
the
and large
affected
an
VIII-related
of the human
of a dominant
such studies
proper
types,
in our
with
the
values
is not
to detect
appeared
to
and in normal
Unfortunately,
matings
of the
to compare
of other
from
the
of the proband
repeatedly
inhibitory
product.
When
is similarly
mixed
with
is no evidence
that
the
amount
of procoagulant
activity
cryoprecipitate,
this was associated
when
concentrated
and subjected
typical
the
alleles
plasma
of an
proportions
VIII assay
prothrombin
however,
hemophilia
revealed
the
In principle,
normal
with
thromboplastin.
factor
cryoprecipitate,
with X-linked
normal
tient’s
which,
plasma
there
from
examined
the
for evidence
a struc-
complex2#{176} and
of genetic
dominance
might
be observed
if the peptides
produced
normal
alleles
by some
of the cells of a heterozygote
interfered
tivity
A
has
for
hemo-
or colorA locus.24
heterozygotes,
all of which
X linkage
would
since
in X-linked,
are
be
HEMOPHILIA
187
A
dominantly
inherited
hypophosphatemia
hemizygous
males
males.25
Unfortunately,
the
occurrence
by itself
excluded
Finally,
a previously
there
hint
whether
to
and
vitamin
usually
more
severely
there
are no affected
of male-to-male
have
as
are
D-resistant
rickets,
the
affected
than
heterozygous
males
in the kindred.
Of
transmission
of the
abnormal
fecourse,
phenotype
would
X linkage.
is the possibility
that we are observing
a dominant
unrecognized
factor
VIII
locus.
The segregational
such
a mutant
gene
is located
on
the
mutation
data
give
X or
an
at
no
autosomal
chromosome.
The latter
is more
likely
a priori
simply
because
DNA
is present
in autosomal
chromosomes
than
X chromosomes.
more
of
There
nothing
are
different
in the
from
men
gard
with
the
phenotype,
with
however,
hemophilia
such phenotypic
a genetic
mutation
cluded
of the
transmission
VIII
ates27’28
reported
transfusion.
that
de novo
is probably
We
shall
transmitting
continue
synthesis
because
objective
find it very easy
new
rules
in blood
from
the
pedigree
pattern,
we
infer
that
certain
We
of the locus involved,
we record
have been studying
this kindred
good,
and
we
diagnostic
plan
test
as new
characteristic
workers
who
publish
their
produce
the
to
examine
tests
become
data,
because
the
which
indeed
re-
dealing
a finding
which
and
occur
ex-
because
synthesis
his
associ-
in these
subjects
the affected
persons
have
concluded
that
have
their
of vWd.
of our
subjects
representation
of the
as hemophilia
A,
is lacking.
accurately
We
using
phenotype
subjects
of
uncertainty
phenotypes
about
the
and
chromo-
of our subjects
can be recorded
as
CRM
factor
VIII deficiency.
Since,
,
they
are
heterozygous
but
we
are
not
their genotypes
as VIII-O-aa/rs.
for almost
20 yr. Our relationships
the
which
distinguishes
may
have
studied
clarification
does
symbolic
somal
location
of the locus.
The phenotypes
F. VIII rs, since we know
that they have
We
are
have described
is
et al. in 1965.26
Veltkamp
not have this
state
of our
in spite
we
at first not to be vWd
absence
of de novo
phenotypes
for
coagulation,2
that
present,
our kindred,
antigen.
They
evidence
that they do
to represent
the genetic
international
genotypes
to the
symptoms.
one we
Hensen
recently,
a variant
to refer
the
by
appeared
apparent
More
subjects
conclude
locus.
A was
the
our
of their
resembling
reported
first
transfusion
and that,
unlike
levels
of factor
VIII-related
kindred
the
nearly
the phenotype
times
and
following
have
following
reduced
that
mildness
of hemophilia
X linkage,
and
normal
bleeding
of factor
suggest
the
evidence
as insufficient
to
at a previously
unrecognized
The published
kindred
most
family
of Dutch
hemophiliacs
Father-to-son
to
A except
the
is
affected
available,
them
similarly
collating
members
with
hoping
each
to discover
from
hemophilia
A.
ambiguous
kindred
and
comparison
are
additional
a phenotypic
We
or
urge
other
patients
to
of such
material
Thromb
Diath
might
is needed.
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