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Severe Perinatal Thrombosis in Double and Triple Heterozygous Offspring
of a Family Segregating Two Independent Protein S Mutations
and a Protein C Mutation
By Caroline J. Formstone, Paula J. Hallam, Edward G.D. Tuddenham, Jennifer Voke, Mark Layton,
Kypros Nicolaides, Ian M. Hann, and David N. Cooper
Molecular genetic and phenotypic analyses were performed
in a highly unusual case of combined protein S and protein
C deficiency manifesting in a family in which a child had died
perinatally from renal vein thrombosis. Antenatal diagnosis
in a second pregnancy was initially performed by indirect
restriction fragment length polymorphism (RFLP) tracking
using a neutral dimorphism within the PROS gene and
served t o exclude severe protein S deficiency. An umbilical
vein blood sample at 22 weeks gestation showed isolated
protein C deficiency. This pregnancy proceeded t o a fullterm delivery without thrombotic complications. Molecular
genetic analysis of the PROC and PROS genes segregating
in the family then yielded one PROCgene lesion in the father
and two PROS gene lesions, one in each parent. These lesions were shown t o segregate with the respective deficiency states through the family pedigree. Analysis of DNA
from paraffin-embedded liver tissue taken from the deceased child showed the presence of both PROS mutations,
as well as the PROC mutation. Genotypic analysis of the
second child showed a PROC mutation, but neither PROS
mutation consistent with its possession of normal protein
S levels and a lowlborderline protein C level. Antenatal diagnosis was then performed in a third pregnancy by direct
mutation detection. However, although the fetus carried
only the paternal PROS and PROC gene lesions, the child
developed renal thrombosis in utero. It may be that a further
genetic lesion at a third locus still remains t o be defined.
Alternatively, the intrauterine development of thrombosis
in this infant could have been caused, at least in part, by a
transplacental thrombotic stimulus arising in the protein Sdeficient maternal circulation. This analysis may, therefore,
serve as a warning against extrapolating too readily from
genotype t o phenotype in families with a complex thrombotic disorder.
0 7996 by The American Society of Hematology.
T
Estimates of the prevalence of clinically symptomatic deficiencies of protein C in the general population lie between
1:16000 and 1:36000,’5.’6while the frequency of symptomatic protein S deficiency is 1:20000.’2Although most often
found in the heterozygous state, severe homozygous or compound heterozygous deficiencies of both protein C‘”“ and
protein SZ2-”have been reported and are usually associated
with severe early onset venous thrombosis. The characterization of several severe forms of both protein CZ5and protein
SZ6deficiencies at the DNA level has been initiated. We
describe here the clinical features together with the labora-
HE PROTEIN C anticoagulant pathway represents an
important control mechanism in hemostasis. Protein C,
a vitamin K-dependent glycoprotein and zymogen of a serine
proteinase, is activated at the endothelial cell surface by
the thrombidthrombomodulin complex and proteolytically
cleaves and inactivates procoagulant factors Va and VIIIa.
Protein S acts as a cofactor to activated protein C (APC) by
enhancing the rate of inactivation of these
Protein
S also serves to regulate hemostasis via an APC-independent
me~hanism.~.~
Approximately 60% of protein S in plasma is
noncovalently complexed with C4b binding protein
(C4bBP),’ while the remainder is unbound (‘free’). The interaction of functional free protein S with C4bBP abolishes the
anticoagulant activity of the former.’ The protein C (PROC)
gene resides on chromosome 2 and comprises 9 exons.9 The
protein S (PROS) gene is located on chromosome 3, comprises 15 exons, and is closely linked to a highly homologous
pseudogene.lo
Hereditary deficiencies of both protein C and protein S
are well-documented, and both are associated with an increased risk of venous thrombosis.” Together they account
for between 6% and 10% of families with hereditary thrombophilia.” Two distinct types of hereditary protein C deficiency are recognized’? type I, the most common, is characterized by a parallel reduction in protein C activity and
antigen levels, whereas type I1 exhibits a greater reduction
in activity than antigen. The classification of protein S deficiency is complicated by the presence of C4bBP-bound protein S in plasma. However, three types have been distinguished phen~typically’~:
type I deficiency is characterized
by reduced total and free antigen levels together with reduced
anticoagulant activity. In type I1 deficiency, protein S activity
is reduced, although total and free antigen levels are normal,
whereas in type 111 deficiency, the total protein S antigen
level is normal, but free protein S antigen and activity are
reduced.
’
Blood, Vol 87, No 9 (May 1). 1996: pp 3731-3737
From the Department of Biochemistry, Imperial College, London;
Academic Unit of Medical and Community Genetics, Kennedy Galton Centre, Harrow; Haemostasis Research Group, Clinical Sciences Centre, Hammersmith Hospital, London; Haematology Department, Luton and Dunstable Hospital, Luton; Haematology
Department, King’s College Hospital, London; Harris Birthright
Research Centre for Fetal Medicine, King’s College Hospital, London; Haematology Department, Great Ormond Street Hospital for
Children, London; and Institute of Medical Genetics, University oj
Wales College of Medicine, Card18 UK.
C.J.F., P.J.H., and D.N.C. were previously at Thrombosis Research Institute, London, UK.
Submitted July 18, 1995; accepted December 6, 1995.
Supported by the Thrombosis Research Trust, Charter plc, and
the Sun-Life Assurance Co Lid.
Address reprint requests to Paula J. Hallam, PhD, Academic Unit
of Medical and Community Genetics, Kennedy Galton Centre, Level
8V, Northwick Park Hospital, Watford Rd, Harrow, Middlesex HA1
3UJ, UK.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. section 1734 solely to
indicate this fact.
0 1996 by The American Society of Hematology.
0006-4971/96/8709-00$3.00/0
3731
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FORMSTONE ET AL
3732
Table 1. Phenotypic and Genotypic Data From Family Members
Family
Member
PC Activity
(NR 73-121)
PC Antigen
(NR 61-142)
PROC
Mutation
PS Total
(NR 77-100)
PS Free
(NR 81-113)
1.2
11.1
11.2
11.3
11.4
11.5
111.1
111.2
111.3
111.4
111.5
87
81
37
60
82
100
106
3t
85
76
59
128
88
60
64
109
125
76
16t
98
64
77
NT
NT
e
NT
NT
NT
NT
e
82
134
74
106
38
94
114
24t
44
58
39
15
58
54
120
30
127
82
<lot
13
NT
NT
31
4
PROS
Mutation
NT
-
+
NT
NT
+
+
+
+,
Abbreviations: 0. Arg 169 Gln (PROO;
D. Met 570 Thr (PROS); 2-bp deletion [codons 547/5481(PROS);
NR, normal range for healthy
adults; t, on warfarin treatment when tested; NT, not tested; -, no genetic lesion found.
+
+
tory and molecular genetic analysis of a highly unusual family in which one PROC and two nonidentical PROS gene
lesions are segregating.
MATERIALS AND METHODS
Family hisfory. The first child, 1V.I (Fig l), died at age 1 week
from bilateral renal vein thrombosis. Although she showed no evidence of purpura fulminans, one kidney was fibrotic at postmortem
with evidence of intrauterine thrombosis. The other kidney showed
evidence of recent infarction with renal vein thrombosis. Thrombosis
was also present in the major cerebral veins. Pretransfusion blood
samples from IV.1 were not available for analysis. Several members
of this English family had also experienced thrombotic manifestations and/or low levels of both protein C and protein S (Fig 1, Table
1). 1.2 experienced several episodes of phlebitis while 11.6 suffered
a deep vein thrombosis after surgery and has also experienced a
pulmonary embolism. 111.2, the propositus, experienced two ileofemoral thromboses as a teenager, the first at the age of 13 following
traumatic injury sustained while playing football, the second at the
age of 20, which was apparently spontaneous. He is currently on
long-term warfarin prophylaxis. The mother of the deceased child
(111.3) possessed levels of protein S consistent with an heterozygous
type I deficiency state (Table I). Measured protein C and S values
for the father (III.2) (Table 1) were not useful because this individual
had received anticoagulant therapy as he initially presented with
clinical symptoms. His father (11.2) exhibited both protein C and
protein S deficiency phenotypes (types I1 and 111, respectively) (Table l). Although matemal (111.3) protein C levels appeared normal,
several of her immediate relatives (11.3, III.4, and 111.5) possessed
borderline normal protein C activity and/or antigen levels (Table 1).
During a second pregnancy (IV.2), fetal blood sampling (cordocentesis) at 22 weeks gestation showed protein S activity and free
protein S antigen levels of 29 U/dL (normal range [NR], 23 to 30)
and 33 U/dL (NR, 35 to 41), respectively (Table 2), both of which
were normal for gestational age. Protein C activity, however, was
lower than normal for gestational age (5 U/dL, NR, 9.5 to 13.5).
The pregnancy was uneventful and a clinically normal baby was
born at 39 weeks gestation. At term, protein C activity was still low
(8 U/dL, NR, 21 to 65), while total protein S antigen remained
within the normal range (43 U/dL, NR, 20 to 64). Although protein
S values were unchanged by 16 months of age, protein C antigen
and activity levels were borderline normal (Table 2). It is unclear,
however, why protein C activity and antigen levels are very similar
in IV.2. This child is still healthy.
A third pregnancy (IV.3) was monitored very carefully. Chorionic
villus sampling was performed at 14 weeks gestation. Delivery was
by emergency Caesarean section at 38 weeks due to decreased fetal
movement. Cord blood taken at the time of delivery contained a
protein C activity level of 4 U/dL [NR, 24 to 38 weeks gestation,
fetuses, and neonates, 8.1 to 22.91;’ and a borderline normal protein
C antigen level of 17 U/dL [NR day 1 (full-term), 17 to 531. Total
and free protein S antigen levels were found to be 14 U/dL [NR
day 1 (full-term), 17 to 531 and 2 U/dL [NR day 1 (full-term), 32
to 841, respectively, while protein S activity was 24 U/dL (NR, 55
to 119). A palpable kidney at 12 hours of age was investigated
further by ultrasound imaging that showed left renal vein thrombosis,
left adrenal hemorrhage, thrombosis in the inferior vena cava, and
a thrombus in the bladder. Because protein C deficiency was felt to
be his major problem, initial treatment was with intravenous fresh
frozen plasma and heparin and then a few days later with protein C
concentrate. His plasma protein C levels increased accordingly. A
right atrial Hickman catheter was inserted at the age of 2 weeks for
the regular administration of protein C concentrate and heparin.
This was subsequently removed at the age of 6 weeks, protein C
concentrate and heparin were withdrawn, and warfarin therapy commenced. The Intemational Normalized Ratio is being maintained
between 3.0 and 3.5, and it is planned to continue this treatment
indefinitely. At the time of writing, at the age of 9 months, there
have been no further thrombotic episodes. The child i s thriving
despite the functional loss of about 90% of his left kidney.
Immunological assays. Protein C and protein S antigen levels
(total and free) were assayed by standard enzyme-linked immunosorbent assay (ELISA) (Dako, High Wycombe, UK) using a rabbit
polyclonal antibody. Protein S and protein C activities were measured using the Stago kit (Diagnostica Stago, Asnikres, France).
DNA extracfion from parafin-embedded tissue. Only paraffinembedded liver tissue was available for DNA analysis from the
deceased child, IV. 1. Under sterile conditions, sections were removed and transferred to 1.5 mL Eppendorf tubes, previously
washed in 10 mmol/L HC1 followed by oven-drying. DNA extraction
was performed using the procedure of L.S. Young (unpublished
method): briefly, paraffin sections were incubated at room temperature for 5 minutes in 1 mL xylene (BDH, Poole, Dorset) and then
centrifuged at 13,000 rpm for 10 minutes. The xylene was removed
and the above procedure repeated. The resulting pellet was washed
three times in ethanol and the final pellet dried overnight at 37°C.
A total of 400 pL 1x polymerase chain reaction (PCR) buffer (Promega, Southampton, UK) together with 100 pg/mL proteinase K
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3733
COMBINED PROTEIN S AND PROTEIN C DEFICIENCY
(GIBCO-BRL, Middlesex, UK) were added to the dried pellet, and
the mixture incubated at 55°C for 1 hour followed by 95°C for 10
minutes. A control tube containing no paraffin section was treated
in a similar manner. The resulting solutions were used as templates
for PCR amplification.
PROS gene: Pro626 polymorphism analysis. The alleles of the
exon XV Pro626 polymorphism were determined at the level of both
genomic DNA and mRNA (cDNA) by restriction fragment length
polymorphism (RFLP) analysis as described.”
PCWdirect sequencing analysis of exon-containing DNA fragments. Genomic DNA extraction from family members, DNA amplification, and PCWdirect sequencing were performed as des ~ r i b e d .The
~ ~ PROC
. ~ ~ gene exon VI1 and the PROS gene exon XIV
from the deceased child (IV. 1) were PCR amplified from paraffinembedded liver tissue DNA in tubes washed previously in 10 mmoV
L HCI and oven-dried. PCR amplification was performed as des ~ r i b e d , 2but
~ * following
~~
the recommendations of Woodward et a13’;
20 pL of a 1: 10 dilution of both control and paraffin section samples
were used for PCR amplification of PROC and PROS gene exons.
DNA samples from IV.2 and IV.3 were derived from chorionic villus
samples, cord blood, and venipuncture after birth.
Reverse-transcription (RT) PCR analysis for the PROS gene.
Platelets from 11.4 were separated from whole blood” and RNA
extracted.” The specific reverse transcription of PROS mRNA and
subsequent PCR amplification of cDNA fragments were performed
as described.28
Screening for APC resistance and factor V Leiden. Activated
protein C ( A X ) resistance assays and screening for the common
factor V Leiden lesion (CGA -+ CAA converting Arg 506 to Gln)
were performed essentially as d e ~ c r i b e d . ~ ~ . ~ ~
RESULTS
Studies on the segregation of PROS gene Lesions by RFLP
tracking and PCWdirect sequencing. Prior to the identification by PCWdirect sequencing analysis of the PROS gene
lesions segregating in this family, indirect PROS RFLP analysis was performed to attempt a rapid antenatal exclusion
of severe protein S deficiency in individual IV.2. Family
members spanning four generations were typed for the CCA/
CCG (henceforth termed A and G) alleles of the neutral
dimorphism at Pro626 in exon XV of the PROS gene.35
This dimorphism is detectable directly by restriction enzyme
(BstXZ) cleavage of PCR-amplified exon XV. The segregation pattern of the Pro626 RFLP alleles in this family is
shown in Fig 1. Both the maternal and paternal defective
PROS alleles cosegregated with the Pro626 A allele. The
possibility of severe protein S deficiency in IV.2 was excluded antenatally by the Pro626 RFLP analysis since the
maternal A allele was absent in the fetal material (Fig 1).
Pro626 RFLP data for individuals 1.2, 11.4, 11.6, III.2, and
111.3 have been presented previously.’8
This previous publication” also described the detection
and characterization of two different inherited PROS gene
lesions in exon XIV from certain members of this family.
PCWdirect sequencing of the 15 exons and splice junctions
of the PROS gene identified a Met570 Thr (ATG ACG)
substitution in 111.2 and a short deletion (1908 del AC) in
111.3. The Met570 Thr substitution and the 2bp deletion
were also detected in 11.2 and II.4, respectively.28For the
purposes of this study, the presence or absence of the two
PROS gene mutations in further family members was deter+
+
+
1
I
M
I
Adkkiw
IV
M
AG
AG
Fig 1. Family pedigree. Semishaded symbols denote clinicalty affected individuals. A diagonal line through a symbol denotes indvidual deceased. 0 denotes proven carrier of the Argl69 Gin mutation
and
respectively, denote
within exon VI1 of the PROC gene.
proven carriers of the Met570 Thr mutation and the 2-bp deletion
et position 1908, both of which lie within axon XIV of the PROSgene.
AA and GO, respectively, denote homozygority for the CCA and CCG
alleles at Pro626 of the PROS gene, while AG denotes heterozygosity.
NT denotes PROS gene not analyzed by PCR/dirat sequencing. Data
on allelic exclusion for PROS mRNA also provided for Individuals 11.4
and 111.3. Individual 111.2 was on warfarin prophylaxis.
-
+,
-
mined by PCWdirect sequencing of PROS exon XIV in these
individuals (Fig 1, Table 1). Additional evidence for the
Met570 Thr substitution being the pathological lesion in
the family was provided by sequence analysis of exon XIV
from 50 controls (of Caucasian origin with no history of
venous thrombosis), which failed to detect another example
of the Met570 Thr substitution. This mutation does not
appear to be a common polymorphism. Moreover, its presence is associated with the clinical phenotype in the family
through two generations (Fig 1). The maternal 2-bp deletion
was detected in individuals 111.4, III.5, and IV.1, but not in
1.2 or IV.2. Thus, the deceased child (IV.l) possessed both
paternal and maternal PROS gene lesions (Fig 1, Table 2).
Cord blood DNA was used to PCR amplify exon XIV from
the second child (IV.2). Neither PROS gene lesion was detected in IV.2, an observation consistent with (1) the normal
levels of total and free protein S noted in this child and (2)
the absence of the maternal A allele demonstrated previously
by antenatal Pro626 RFLP tracking analysis (see above). In
the case of the third child (IV.3), antenatal screening for the
presence of PROS gene mutations showed only the paternal
Met570 -+ Thr mutation, the maternal PROS gene lesion
being absent. Genotypic and phenotypic data obtained from
the siblings in generation IV are summarized in Table 2.
It was previously demonstrated that individual III.3, who
was heterozygous A/G for the Pro626 PROS RFLP at the
genomic DNA level, exhibited the loss of the A allele at the
mRNA level [“allelic e~clusion”].’~The A allele was in
phase with the 2-bp PROS gene deletion identified in this
individual. Loss of the PROS mRNA species bearing this
mutation was consistent with her type I protein S deficiency
state. The absence of the 2-bp deletion in II.4cDNA (Fig 1)
was also demonstrated by PCWdirect sequencing of plateletderived cDNA from this individual. The demonstration of
the loss of the A allele for the Pro626 RFLP by PCWdirect
-+
-+
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3734
FORMSTONE ET AL
Table 2. Phenotypic Data From Siblings IV.l, IV.2, and IV.3
Family
Member
IV.1
Weeks
GestationIAge
1 wk
IV.2
22/40
IV.3
38/40 (term)
16 m o
38/40 (term)
PC Activity
(U/dL) (NR)
PC Antigen
WdL) (NR)
PROC
Mutation
23 (8.1-22.91
23 (17-53)
a
a
5 (9.5-13.5)
8 (21-65)
66 (70-140)
4 (8.1-22.9)
PS Activity
(U/dL) (NRI
PS Antigen WdL)
(NR)
29 (23-30)
28++ (17-53) total
16++ (32-84) free
33 (35-41) free
PROS
Mutation
a+
-
43 (20-64) total
67 (61-142)
17 (17-53)
88 (55-119)
24 (55-119)
a
14 (17-53) total
2 (32-84) free
Abbreviations: ++, Phenotypic analysis performed posttransfusion of fresh plasma. No pretransfusion plasma available for phenotypic
analysis; NR, normal ranges for gestational age/age of child.
sequencing and RFLP analysis confirmed “allelic exclusion” in 11.4.
Direct detection of the pathological lesion in the PROC
gene. PCWdirect sequencing of all nine exons and splice
junctions of the PROC genes of individual 111.2 yielded only
one deviation from the wild-type sequence: a heterozygous
CGG CAG transition in exon VII, which predicts an arginine to glutamine substitution at amino acid residue 169.
The authenticity of this lesion was confirmed by sequencing
the antisense DNA strand. This substitution was also found
in individuals 11.2, IV.2, IV.3, and the deceased child 1V.I
(Fig 1) and, therefore, segregates with the protein C deficiency state through the family. By contrast, PCWdirect sequencing of all nine exons and splice junctions of the PROC
genes of individual 111.3 did not yield any deviation from
the wild-type sequence.
Factor V Leiden screening. For individuals 111.2, 111.3,
and IV.3, the presence of APC resistance3‘ was excluded by
direct screening for the common factor V Leiden mutation
and/or by means of the APC resistance assay.
-+
DISCUSSION
R E P tracking was partially successful in that severe protein S deficiency was rapidly excluded in individual IV.2
during the second trimester. PCWdirect sequencing of PROC
and PROS genes from family members was then employed to
detect and characterize the underlying pathological lesions.
Three mutations were found, one in the PROC gene and two
in the PROS gene. The PROC mutation identified (Arg169
Gln) has been reported at least three times previously in
individuals with type I1 protein C defi~iency.’~
Its location
within the Arg169-Leu170 activation site of protein C explains its association with a type I1 deficiency state. The
detection and characterization of the PROS gene lesions has
been reported previously.28In that study, we confirmed the
authenticity of both lesions by sequencing the antisense
DNA strand. In addition, no further deviations from the wildtype sequence were present in any of the other 14 exons and
splice junctions of the PROS gene.28The 2-bp PROS gene
deletion generated an in-frame TAA termination codon just
three amino acids C-terminal to the lesion, and this accounts
both for the type I protein S deficiency observed in maternal
family members and for the allelic exclusion noted in 11.4
and 111.3. The paternal Met570 Thr substitution occurred
in a residue that is conserved in both rabbita and bovine4’
protein S and in the homologous human protein, sex hormone binding globulin!’
The amino acid environment of
Met570 appears hydrophobic, which suggests that this region
is internal to the protein. The present study provided further
support for the conclusion that Met570 Thr is the pathological mutation by demonstrating its absence in 100 normal
alleles. In addition, the presence of this PROS gene lesion
is associated with the clinical phenotype in two generations
of this kindred. We have previously suggested that the
Met570 Thr substitution may be associated with type 111
protein S deficiency.28Total and free protein S antigen levels
from individual IV.3 are also consistent with a type 111 deficiency. However, the biochemical characterization of an in
vitro-expressed protein should establish whether the
Met570 -+ Thr substitution is indeed a disease-causing mutation and possibly the pathological mechanism underlying
this disorder.
The genotypic data from family members broadly confirmed our initial assumptions made regarding the number
and nature of the gene defects and their pattern of inheritance. The deceased child’s father (111.2) had correctly been
predicted to be doubly heterozygous for mutations in both
+
+
Few families with a thrombotic diathesis and two distinct
prothrombotic defects have been reported to date.37However, the recent discovery of the common factor V Leiden
variant (present in between 2% and 7% of individuals in
European populations) has shown that combined deficiency
states may not be as rare as was originally
Two
cases of a triple deficiency have been reported from a single
Canadian kindred: one individual was deficient in antithrombin 111, protein C, and heparin cofactor 11, and the other was
deficient in antithrombin 111, protein S, and heparin cofactor
II.38 Such cases are, however, exceptionally rare, and none
have been characterized at the DNA level. We report here
the molecular genetic analysis of, and antenatal diagnosis in,
a family with combined protein C and protein S deficiency in
which three different gene defects are segregating.
The phenotypic data collected from family members indicated the complexity of antenatal diagnosis and the difficulty
in counselling the parents of the deceased child in subsequent
pregnancies. Since phenotypic data may be considered to be
particularly unreliable in the case of fetal blood sampling
owing to the low plasma protein levels at early gestational
age,27molecular genetic analysis was employed in the hope
of improving diagnostic accuracy.
.--)
-+
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3735
COMBINED PROTEIN S AND PROTEIN C DEFICIENCY
PROC and PROS genes and her mother (111.3) to be heterozygous for a PROS mutation. The deceased child was shown
to have possessed all three gene lesions segregating in this
family and was thus compound heterozygous for protein S
deficiency and heterozygous for protein C deficiency. This
explanation for the clinical severity manifested by IV.1 suggested that predictions regarding clinical severity might be
possible on the basis of genotype in other family members.
This appears to be the case at least for the second child,
IV.2, who is asymptomatic. She possesses the PROC gene
lesion, but neither PROS mutation. Fetal and neonatal assays
for IV.2 are consistent with the genotypic data, but her protein C activity level at age 16 months fell just outside the
normal range for her age. Some considerable overlap between plasma protein C levels exhibited by heterozygotes
and normals has been
and IV.2 may represent
another example of this phenomenon.
Antenatal diagnosis was performed on a third child (IV.3)
who was found to be heterozygous for the paternal PROS
gene lesion, but lacked the maternal PROS gene lesion. That
IV.3 also possessed the PROC gene lesion was only established after birth owing to the time required to detect and
characterize this lesion. Since this child’s father (111.2) and
grandfather (11.2) possessed both PROC and PROS gene
lesions and were either clinically normal or only affected
after childhood, it was reasoned that there should not be
any thrombotic manifestations in the neonatal period. The
inaccuracy of this prognosis rapidly became apparent when
the child suffered severe early onset venous thrombosis.
Phenotypic data from individual IV.3, derived from cord
blood and taken at the time of delivery, were consistent with
his possession of the paternal PROS gene lesion (Table 2).
His protein S activity was reduced to below 50% of the
normal range for age (NR, 55 to 119). In addition, the protein
S antigen levels (total and free) of IV.3 suggested that he
manifested a similar type of protein S deficiency to his grandfather (11.2) [type 1111. The protein C phenotype of this child
(type I1 deficiency) was also similar to that of 11.2. The
presence of protein C deficiency in IV.3 was confirmed by
the detection of the Arg169 Gln mutation in his PROC
gene.
Despite the wealth of clinical, phenotypic, and genetic
data generated by this study, prognostic accuracy was still
limited. One possible explanation for our inability to predict
the clinical severity of individual IV.3 would be the presence
of a second PROC gene lesion in both 111.3 and IV.3. Although 111.3 possessed normal levels of protein C activity
and antigen, several of her relatives exhibited protein C levels, which were borderline normal (11.3, 111.4, and 111.5).
However, sequencing of the PROC genes from both 111.3
and IV.3 failed to detect another lesion.
The low protein C values apparent in 11.3, 111.4 and 111.5
could in principle be attributable to another lesion in the
PROC gene. There is no evidence to suggest, however, that
the low protein C levels in 11.3 are heritable. 11.3 is unrelated
to 111.4, and 111.5 who are full sisters and the remaining
maternal family members (1.2, 11.4, 11.6, and 111.3) exhibit
normal protein C levels. No information is available on indi-
-
vidual 11.5 (Fig 1: father of 111.4 and 111.5). Since no PROC
gene lesion was identified in 111.3, further analysis of the
PROC gene from individuals 11.3,111.4,and 111.5 was deemed
unnecessary for the purposes of this diagnostic analysis. Alternatively their protein C deficiency phenotype may have
been acquired.
The severe clinical phenotype manifested by IV.3 could
also be explained by a further genetic lesion at a third locus
(possibly as yet unknown); the presence of the relatively
common factor V Leiden lesion was, however, excluded.
Another possible explanation is that the doubly heterozygous
fetus, IV.3, was being carried by a mother (111.3) who was
herself known to be predisposed to thrombotic events by
virtue of her possession of a PROS gene lesion. Exposure
of the fetus to a potentially prothrombotic intrauterine environment might have served as a trigger for the early development of thrombotic symptoms.
Two further anomalies were encountered in our attempts
to correlate genotype with phenotype in this family. Firstly,
individual 11.6 is clinically symptomatic yet her protein C
and protein S levels are well within the normal range. This
could conceivably be explained by the presence of another
lesion in an as yet unidentified gene acting as an additional
risk factor. Alternatively, her thrombosis may have been
triggered by the acquired deficiency of an as yet unidentified
anticoagulant factor. Secondly, while individual 1.2 is clinically symptomatic (she also possesses a very low free protein
S level and bears the PROS Pro626 A allele associated with
the type I protein S deficiency state), we were unable to
detect the 2-bp deletion within a sample of her lymphocyte
DNA. The absence of the PROS gene deletion in 1.2 may
indicate de novo mutation in her germline giving rise to the
lesion carried by her daughter 11.4 and subsequently transmitted through the pedigree. Because neither phenotypic data
nor blood samples were available from her father (Ll), the
possibility that the lesion was passed down from him cannot
be excluded. An intragenic recombination event occurring
between the mutation and the polymorphic marker at codon
626 is unlikely, however, as these two sites are separated by
only 556 bp.
Although highly unusual in terms of its complexity, this
analysis and diagnosis should thus serve as a warning against
extrapolating too readily from genotype to phenotype in kindreds with a multifactorial disorder, such as venous thrombosis. Clearly, the considerable clinical and phenotypic variability evident from this analysis must be taken into account
in counselling families as complex as the one illustrated
here.
ACKNOWLEDGMENT
We thank Lawrence S. Young for provision of the protocol for
DNA extraction from paraffin-embedded tissue, Elaine Jenkins and
Jane Clark for performing fetal coagulation studies, and Dan Thompson and Michael Chapple for clinical referral and liaison.
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1996 87: 3731-3737
Severe perinatal thrombosis in double and triple heterozygous
offspring of a family segregating two independent protein S
mutations and a protein C mutation
CJ Formstone, PJ Hallam, EG Tuddenham, J Voke, M Layton, K Nicolaides, IM Hann and DN
Cooper
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