© 2000 American Heart Association, Inc.
Thrombosis |
Familial Thrombophilia Associated With Homozygosity for the Cystathionine ß-Synthase 833T
C Mutation
From the Department of Clinical Biochemistry (M.G., N.R., K.R) and the Center for Hemophilia and Thrombosis (J.I.), Skejby University Hospital, Aarhus, Denmark.
Correspondence to Jørgen Ingerslev, MD, Center for Hemophilia and Thrombosis, Department of Clinical Immunology, Skejby University Hospital, DK-8200 Aarhus N, Denmark. E-mail j-ing{at}post3.tele.dk
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Abstract |
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Abstract—Severe hyperhomocysteinemia due to cystathionine ß-synthase (CBS) deficiency is a strong risk factor for premature cardiovascular disease. Among untreated patients,
50% have suffered a
thromboembolic event by 30 years of age. We report on 3 sisters with
severe hyperhomocysteinemia due to homozygosity for the CBS 833T
C
mutation. These patients, who displayed no other known thrombophilic
predisposition, had suffered single or multiple venous thrombosis
before CBS deficiency was diagnosed relatively late in life. In this
family, homozygosity for the 833TC mutation was associated with a
mild phenotype with respect to other sequelae of CBS
deficiency. Consequently, our results indicate that most cases with
this genotype may remain undiagnosed. Investigated family
members heterozygous for the 833TC mutation displayed normal total
homocysteine in plasma (tHcy) levels, even when they were homozygous
for the methylenetetrahydrofolate
reductase 677CT polymorphism. The prevalence of homozygosity for
the 833TC mutation has previously been estimated at no less than
1:20 500 in our population. Because a reduction of the severely
elevated levels of tHcy in CBS deficiency reduces
cardiovascular risk and because homozygosity for the
833TC mutation is more prevalent than previously thought, our
results emphasize the importance of measuring tHcy routinely in
thrombophilia screening.
Key Words: venous thrombosis • severe hyperhomocysteinemia • cystathionine ß-synthase deficiency • family history • mutation analysis
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Introduction |
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TopAbstractIntroductionMethodsResultsDiscussionReferences |
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An elevated plasma level of homocysteine is an independent risk factor for arterial as well as venous thrombosis.1 2 Mild or moderate hyperhomocysteinemia (an increased level of total homocysteine in plasma [tHcy] up to 30 µmol/L and 30 to 100 µmol/L, respectively) may result from a relative deficiency of folic acid, vitamin B12, or vitamin B63 or from homozygosity for a common polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene (677C
T).4 Severe hyperhomocysteinemia
(tHcy >100 µmol/L) is most often caused by cystathionine
ß-synthase (CBS, EC 4.2.1.22) deficiency.5 This
autosomal recessive disease is the most common inborn error of sulfur
amino acid metabolism, with an estimated incidence of
1:200 000 worldwide, ranging from 1:20 500 to 1:1 000 000 in
different populations.6 7 The clinical manifestations
include premature atherosclerosis and early
thromboembolism, ectopia lentis, mental retardation, other
neuropsychiatric manifestations, and skeletal abnormalities, including
osteoporosis. Vascular complications constitute the major cause of
death.5 Overall, 50% of patients with untreated CBS
deficiency have suffered a thromboembolic event by 30 years of
age.8 Vascular occlusion can occur in any vessel at any
age, with the majority of such occurrences involving
peripheral veins (51%) and complicated by
pulmonary embolism in one fourth of those cases.5
Because most countries do not systematically screen newborns for
homocystinuria, the majority of cases of CBS deficiency have been
diagnosed on the basis of the patients’ phenotypical features. In transsulfuration of homocysteine, CBS catalyzes the condensation of homocysteine with serine, forming cystathionine in a pyridoxal 5'-phosphate (the active form of vitamin B6)–dependent reaction. High-dose administration of vitamin B6 in CBS deficiency may lower homocysteine, and patients are classified as pyridoxine responsive or nonresponsive according to their response pattern after pyridoxine supplementation. Alternatively, remethylation of homocysteine is stimulated by folic acid and betaine treatment. Early detection and lowering of a severely increased tHcy may prevent complications.5 In CBS-deficient patients, a total of 92 mutations have been detected in the CBS gene so far, with the majority being missense mutations.9
We have recently shown that 1.4% of Danish newborns (n=500) are
heterozygous for the geographically widespread 833TC
mutation,10 indicating a prevalence of homozygosity for
this mutation at 1 in 20 500.7 In the present study,
we report on 3 sisters with CBS deficiency and severe
hyperhomocysteinemia due to homozygosity for this mutation. CBS
deficiency was diagnosed late in life (ages 54 to 58 years) after at
least 1 thromboembolic event.
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Methods |
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Patients
The proposita, family pattern II:4 (Figure
), a female aged 58 years, was referred for
thrombophilia investigation with a history of deep venous thrombosis
(DVT) and pulmonary embolism at the age of 23 years occurring
at labor. She had 4 spontaneous recurrences (ages 27, 39, 49
and 53 years). Patient II:6, a female aged 56 years, suffered DVT at
the age of 21 years after 1 month of oral contraceptive use.
Additionally, she had recently experienced an episode of transient
ischemic attacks. Patient II:7, a female aged 55 years,
suffered DVT at the age of 21 years during oral contraceptive use. Only
patient II:4 is maintained on oral anticoagulation because of
thrombotic recurrences. Early DVT incidents were detected by
phlebography, which previously constituted the standard procedure for
documentation of venous thrombosis in Denmark. Documentation of recent
episodes of DVT was performed by Doppler sonography and
phlebography. Pulmonary embolisms were detected by ventilation
perfusion scintigraphy.
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Thrombophilia Screening Methods
Determinations of antithrombin, protein C, protein S, and
plasminogen activities were performed by use of citrated
platelet-poor plasma as previously described.11 Other
analyses consisted of determinations of activated
partial thromboplastin time, prothrombin time, P-fibrinogen, and
thrombin time and assays for anticardiolipin antibodies and lupus
anticoagulant.
Determination of tHcy
tHcy was measured after a protein-free morning meal by using gas
chromatography–mass spectrometry and stable isotope
dilution. Samples were collected in tubes containing heparin as
anticoagulant and sodium fluoride, which was added to a final
concentration of 4g/L,12 and plasma was separated by
centrifugation within 2 hours. To exclude
hyperhomocysteinemia due to cobalamin deficiency, methylmalonic acid in
plasma was measured by gas chromatography–mass
spectrometry.13 Furthermore, creatinine was
determined in plasma with a Vitros 700 RC analyzer.
Genetic Analyses
Genomic DNA was isolated from EDTA-stabilized blood by using a
Puregene DNA Isolation Kit (Gentra Systems, Inc). CBS genotypes
and haplotypes were analyzed by polymerase chain reaction
amplification and direct sequencing of genomic DNA containing the
entire coding region of the CBS gene, including adjacent intron-exon
boundaries as previously described.14 Analyses of
MTHFR 677 genotypes, factor V 1691 genotypes, and
prothrombin 20210 genotypes were carried out by polymerase
chain reaction amplification and mutation-specific restriction enzyme
cleavage assays as described.11 15
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Results |
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Thrombophilia Investigation and Genetic Analysis
Routine thrombophilia investigation in patient II:4 (Figure
) revealed no deficiency of antithrombin III, protein C,
protein S, or plasminogen. Neither the factor V Leiden
mutation (1691GA) nor the prothrombin 20210GA variant was
present. The MTHFR 677 genotype was C/T. No laboratory
result pointed to autoimmune thrombosis. Analysis of tHcy
revealed severe hyperhomocysteinemia (tHcy 181 µmol/L). Genetic
analysis revealed homozygosity for the 833TC mutation in
exon 8, resulting in the amino acid substitution I278T. This result was
confirmed by BsrI cleavage of genomic DNA. The common 68-bp
insertion in exon 8 at base 844 was not
present.16 This variant occurs on the same
allele as the 833TC mutation in 5% of Danish
alleles.7 It is a neutral splice variant that does not
appear to cause hyperhomocysteinemia.17 No other
nongenetic causes of hyperhomocysteinemia were identified in this
patient. Additionally, all siblings of the proposita (patient II:4), 5
sisters and 2 brothers, were investigated (Figure). Two sisters
were homozygous for the 833TC mutation (patients II:6 and II:7).
Both had severe hyperhomocysteinemia (tHcy 246 and 206 µmol/L,
respectively) and had suffered DVT. No other tested thrombophilic risk
factor was present. The 2 brothers and 3 of the sisters (aged 53 to
63 years) were heterozygous for the 833TC mutation. None had
suffered a thromboembolic event. All 5 unaffected siblings had tHcy
levels within the normal range for their age and sex according to
Rasmussen et al.18 Two had the MTHFR T/T genotype
(Figure). The 4 children of patients II:4 and II:7 were also
investigated, although 1 had his genotype determined but
otherwise abstained from participation in the study. Plasma tHcy was
normal in each of the children (aged 27 to 37 years) in whom it was
measured. None of the probands displayed other known risk factors.
Haplotypes of the CBS 833T allele and the 833C allele were
determined with respect to 2 common polymorphisms in exons 6
(699CT) and 10 (1080TC), respectively. The 833T allele was
always associated with the 699C variant and the 1080T variant, whereas
the 833C allele cosegregated with the 699T variant and the 1080C
variant, in agreement with the haplotypes determined in 2 other Danish
CBS-deficient patients with the 833TC mutation.14
The proposita’s mother had died of cancer at 68 years of age. Terminally, a thromboembolic event had occurred. The father died from pancreatitis at 73 years of age with no history of thromboembolic events. The parents were unrelated. No material was available for genetic analysis.
Further Clinical Investigations
None of the 3 patients had reduced bone mineral content as
determined by dual-energy x-ray absorptiometric scanning.
Ophthalmologic examination revealed no ectopia lentis or other visual
problems. The mental states of the patients were normal.
Pyridoxine Responsiveness
tHcy levels decreased from 181.0 to 63.7 µmol/L,
from 246.0 to 42.1 µmol/L, and from 206.0 to 23.9 µmol/L
on folic acid (5 mg/wk) plus pyridoxine (250 mg/d) treatment for 6
weeks. By increasing the dose of pyridoxine to 500 mg/d, tHcy levels
further decreased to 19.0, 14.4, and 17.7 µmol/L, respectively.
Betaine (3 g/d) was also tested in the treatment. Only patient II:6,
who had the highest initial level of tHcy, is at present treated
with betaine. The pyridoxine responsiveness of all 3 sisters is in
agreement with previous reports on patients homozygous for the 833TC
mutation.10 19
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Discussion |
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CBS deficiency is detected by means of either newborn screening or, most often, its clinical features. The severity of the disease varies strongly; generally, an early diagnosis and treatment may prohibit the development of severe clinical manifestations or the deterioration of preexisting sequelae.20 The 833T
C
mutation is one of the most prevalent mutations causing CBS deficiency,
accounting for almost 25% of all homocystinuric alleles detected
so far.9 It is present in Caucasian populations
as well as among North Americans.9 21
In the present study, we report on a family in which 3 sisters were
homozygous for the 833TC mutation. The phenotype conferred
by this genotype was mild with respect to known sequelae of CBS
deficiency, with thromboembolisms considered separately. Because of the
lack of a typical homocystinuric phenotype, CBS deficiency was
diagnosed relatively late in life in these patients. Considering the
mild phenotype reported in the present study for the
833TC mutation and because of the surprisingly high prevalence of
the mutation among Danish newborns, we anticipate that numerous
individuals homozygous for this, or other, mild CBS mutations remain
undiagnosed.
One patient (II:6) had the MTHFR T/T genotype. It is noteworthy that her tHcy level was considerably more elevated than those of her sisters who had the MTHFR C/T genotype. We speculate that this may be explained by a synergistic effect of the MTHFR T/T genotype and a deficiency in CBS, with the latter resulting in an elevated level of S-adenosylmethionine,22 which further results in an inhibition of MTHFR. The reduced specific activity of MTHFR caused by the T/T genotype may further decrease the amount of homocysteine subjected to remethylation, thus resulting in a substantially higher accumulation of homocysteine. A similar influence of the MTHFR T/T genotype on tHcy levels in CBS-deficient patients was recently reported by Kluijtmans et al.23 However, the number of patients was too small to reach statistical significance.
No other known thrombophilic risk factor was present in the 3 patients. Hence, our data do not support the hypothesis of Mandel et al24 that the factor V Leiden mutation or another thrombotic risk factor should be present for thrombosis to occur in CBS-deficient patients. This postulate has also been refuted by other investigators.23 25 26
Our study of this limited number of individuals does not point to
heterozygosity for the 833TC mutation as being a risk factor for
hyperhomocysteinemia or thrombosis, even in the presence of the MTHFR
T/T genotype. However, post–methionine-loading tHcy levels
were not recorded.
In this family, there were no signs of deleterious effects involving
the central nervous system that were due to CBS deficiency, nor did the
affected members have skeletal abnormalities or eye problems. This is
an unexpected result because the majority of untreated patients have
developed dislocated lenses by that age.5 Shih et
al19 previously reported on mild clinical features in 2
patients homozygous for the 833TC mutation. The patients, who were
diagnosed in early adulthood, had ectopia lentis and reduced bone
mineralization but no thromboembolic events. Expression studies of the
833TC mutation performed in Escherichia coli revealed a
low specific activity (<10% of normal values) and instability of the
polypeptide.10
In CBS deficiency and in a number of other monogenic diseases,
variation in phenotypes between patients with the same
genotype is observed. No well-defined correlation between
genotype and phenotype has been established in CBS
deficiency.9 27 However, it appears that homozygosity for
the 919GA (G307S) mutation correlates with pyridoxine
nonresponsiveness,28 whereas homozygosity for the 833TC
mutation correlates with pyridoxine responsiveness, in agreement with
treatment results in our study family.10 19 Yet exceptions
from this rule exist.29 Genetic and/or environmental
susceptibility factors not yet identified may influence the phenotypic
expression of a given genotype. Defects in, for example, the
"protein quality control" system, which is responsible for the
cellular handling of mutated and misfolded proteins,30
might be factors influencing the observed phenotypical differences in
CBS deficiency. Defects in these control systems (eg, in proteases or
chaperones) may alter the buffering capacity of the systems, thereby
modulating the phenotype caused by other, in this case CBS,
mutations.
The present data indicate that individuals with untreated CBS
deficiency due to homozygosity for the 833TC mutation are at high
risk of DVT. Our findings strongly emphasize the importance of
including tHcy in the routine thrombophilia program. Furthermore,
hyperhomocysteinemia should be suspected in cases of severe myopia in
childhood and in other clinical conditions associated with known
sequelae of homocystinuria to ensure an early detection of this
relatively frequent, and treatable, inborn error of
metabolism. Eventually, considerations on the perspective
of national screening programs are advocated.
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Acknowledgments |
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This study was supported by grants from the Danish Hearth Foundation (98-1-5-74A-22579) and the Institute for Experimental Clinical Research, University of Aarhus. The authors greatly acknowledge Jette Jensen, Mie Madsen, and the staff of the Coagulation Laboratory for skillful technical assistance.
Received October 12, 1999; accepted January 18, 2000.
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