Risk of venous thrombosis in carriers of a common mutation in the homocysteine regulatory enzyme methylenetetrahydrofolate reductase

Molecular DiagnosisVol. 2 No. 1 1997

Risk of Venous Thrombosis in Carriers of a Common Mutation in the Homocysteine Regulatory Enzyme Methylenetetrahydrofolate Reductase I D R I S T. O C A L , A B B A S S A D E G H I , R I C H A R D D. P R E S S Portland, Oregon

Background: Elevated levels of homocysteine are an independent risk factor for venous thrombosis. A common mutation in methylenetetrahydrofolate reductase (MTHFR), an enzyme required for efficient homocysteine metabolism, creates a thermolabile (tl-) enzyme with reduced activity that may predispose to hyperhomocysteinemia. Methods and Results: To assess whether this common mutation is a risk factor for venous thromboembolism, a polymerase chain reaction-based genotyping assay was used to compare the prevalence of this mutation in a group with thrombosis versus several control groups. Of the 331 thrombosis subjects, 47% were heterozygous and 11% homozygous for tI-MTHFR. In comparison, heterozygotes constituted 42-47% and homozygotes 15-16% of each of three control groups (totaling 593 subjects). There was no significant difference in the tI-MTHFR homozygote frequency or allele frequency between the thrombosis and control study groups. Although the prevalence of the factor V R506Q (Leiden) mutation causing activated protein C resistance was significantly higher in the thrombosis (19%) than in the control groups (4-9%), the concomitant presence of tl-MTHFR with factor V R506Q did not contribute to any excess thrombotic risk. Conclusions: Although the tl-MTHFR mutation may predispose to hyperhomocysteinemia, a known risk factor for venous thrombosis, this common genotype is not a direct genetic risk factor for venous thrombosis, either alone or in combination with the factor V R506Q mutation. Key words: methylenetetrahydrofolate reductase (MTHFR), thrombosis, homocysteine, risk factors.

acutely die [1]. In those who survive, serious medical complications such as recurrent thrombosis and the postphlebitic syndrome may cause significant morbidity. Although venous thromboembolism is a significant public health problem, the complex etiology of this condition has only recently begun to be clarified. Acquired risk factors predisposing to this common disease include both conditions promoting venous stasis (prolonged immobility, obesity, the postoperative state) and conditions promoting excessive activation of the coagulation cascade (trauma, malignancy, pregnancy, estrogen excess,

Venous thromboembolism is a common and potentially fatal syndrome afflicting up to 600,000 Americans annually, up to a third of whom may

From the Department of Pathology, Oregon Health Sciences University, Portland, Oregon. Supported by an American Heart Association (Oregon affiliate) grant to R. D. E and by developmental funds provided by the Department of Pathology, Oregon Health Sciences University. Reprint requests: Richard D. Press, MD, PhD, Department of Pathology, L113, Oregon Health Sciences University, 3181 SW Sam Jackson Park Road, Portland, OR 97201-3098.

61

62

Molecular Diagnosis Vol. 2 No. 1 March 1997

lupus anticoagulants). In addition to these acquired risk factors, a significant fraction of venous thrombosis cases appear to involve a genetic or familial component, particularly those with early-onset or recurrent disease [2]. More than half of those with familial thrombosis have recently been found to have a conserved missense mutation in coagulation factor V (factor V Leiden or factor V R506Q) that prevents efficient degradation of activated factor V by activated protein C [3]. The resulting prothrombotic condition, resistance to activated protein C, results from the excess procoagulant activity of this degradation-resistant factor V. In comparison, only -5 to 10% of these thrombophilic families carry deficiencies of other antithrombotic proteins, typically protein C, protein S, or antithrombin III [2]. Recently, a number of independent reports have confirmed elevated levels of homocysteine as an additional risk factor for venous thromboembolic events [4-8]. An initial indication of the link between hyperhomocysteinemia and venous thrombosis was the premature, often severe thrombotic episodes in children with classic homocystinuria due to inherited deficiencies in cystathionine betasynthase (CBS) or methylenetetrahydrofolate reductase (MTHFR) [9]. As homocysteine can be metabolized by both remethylation (to methionine) and transsulfuration (to cystathionine), and each of these pathways requires different enzymes and vitamin cofactors, a number of different genetic or nutritional deficiencies can affect homocysteine levels. One such genetic defect, a variant thermolabile (tl-) MTHFR with decreased enzymatic activity, is extremely common. The alteration creating this tl-MTHFR, a C to T point mutation at nucleotide 677 (changing Ala to Val), has been found in homozygous form in 5 to 16% of several normal American, Canadian, and Dutch cohorts [10-14]. Homozygotes for this common tl-MTHFR have also been shown to have both higher homocysteine levels [10-12] and an increased risk of cardiovascular disease [12] in selected populations. As tI-MTHFR mutation carriers may be predisposed to hyperhomocysteinemia, a known risk factor for venous thrombosis, we now present a retrospective study specifically addressing whether this mutation is a direct genetic risk factor for venous thrombosis. As both tl-MTHFR and venous thrombosis are common conditions, any association between the two would have significant public health consequences. In particular, as folic acid

therapy may lower homocysteine-associated vascular risk [15], genetically high-risk individuals might be good candidates for folate therapy.

Materials and Methods Thrombosis Group During the past 2 years, blood specimens from 685 different patients have been sent to our diagnostic molecular pathology laboratory for clinical evaluation of suspected thrombotic predisposition associated with the factor V R506Q (Leiden) mutation (Table 1). These 685 specimens represent subjects of average age 44 + 16 years, 44% of whom are men. Of these 685 subjects, 21% were either heterozygous or homozygous for the prevalent factor V R506Q mutation (Table 1). The 331 of these subjects randomly chosen for MTHFR genotype analysis (the thrombosis group) had a similar 19% frequency of the factor V mutation (Table 1). The approximate 20% prevalence rate of the factor V R506Q mutation in our thrombosis cohort is not significantly different (P > .1) than the 18 to 26% prevalence rate among previously reported cohorts with well-documented venous thrombotic episodes [3,16,17]. Brief clinical histories accompanied 212 (31%) of the 685 thrombosis specimens referred to our laboratory. These 212 subjects with available histories were subclassified into one of three clinical subgroups: a venous thrombosis group, an arterial thrombosis group, and another disorders group (Table 1). Most of the subjects (74%, 157 of 212) could be subclassified into the venous thrombosis subgroup based on documentation from the referring clinician that the patient was being evaluated for either a nonarterial thrombotic episode or a familial predisposition to thrombosis. Common clinical descriptors of those in this venous thrombosis subgroup included "venous thrombosis," "pulmonary embolism," "hypercoagulable state," "APC resistance," or similar syndromes in a family member. The 32 subjects (15%) assigned to the arterial thrombosis subgroup, in comparison, had accompanying clinical descriptors such as "stroke," "myocardial infarction," "arterial insufficiency," or "arm or leg ischemia." The 23 remaining subjects in the other disorders subgroup (11%) had vague, nonspecific histories that we could not clearly associate with a thrombotic event despite their clinical evaluation for thrombophilia.

MTHFR Mutation in Venous Thrombosis

•

Ocal et al.

performed on only 90 of these neonate specimens. The 54 healthy elderly control subjects (60% men, age 66 + 8) were the control group from our recent study examining the risk of stroke in factor V R506Q mutation carriers [19]. The factor V R506Q mutation was present at a significantly lower frequency (~4-9%) in each of these three healthy control groups as compared with our thrombosis cohort (P < .005) (Table 1). The 133 adult hospital control subjects (average age 54 + 13) were healthy volunteers recruited from hospital and laboratory person-

Control Groups Four different healthy control groups were also studied (Tables 1, 2). The blood donor group was composed of 197 healthy American Red Cross (of Portland, OR) volunteer blood donors (50% men, average age 46 + 14), 167 of whom were MTHFR genotyped. The Canadian neonate samples were from 293 healthy newborns randomly collected in 1 month through a genetic screening program in Quebec [18]. The factor V R506Q mutation assay was

Table 1. S t u d y Group Characteristics Study Group

n (% total)

Age + SD (years)

% Men

Factor V R506Q Frequency (%)*

685 331 (48)

44 + 16 45 + 16

44 45

147/685 (21)t 64/331 (19)t

473 (69) 212 (31) 157 (74) 32 (15) 23 (11) 544 197 293 54

44 + 15 44 + 16

44 44

46 + 14 0 66 + 8

50

100/473 47/212 41/157 3/32 3/23 25/341 15/197 8/90 2/54

Thrombosis (clinical request for factor V genotype) MTHFR genotype performed Clinical history None available Brief clinical description available:~ Venous thrombosis~ Arterial thrombosis$ Other disorders$ Healthy control subjects (total)§ Volunteer blood donors Neonates Healthy elderly subjects

60

(21)t (22) t (26)t (9) (13) (7.3)// (7.6) (8.9)// (3.7)

*Heterozygotes and homozygotes combined. tMore prevalent than the healthy control groups (P < .005). ~:Clinical subgroups assigned as described under Materials and Methods. §Data previously reported [ 14,19,21 ]. //Only 90 neonates had factor V genotype determinations.

Table 2. Prevalence of the Thermolabile Methylenetetrahydrofolate Reductase ( t I - M T H F R ) Mutation in Venous Thrombosis Subjects and Control Subjects MTHFR Genotype*t Study Group Venous thrombosis Factor V mutation (%)5; Healthy volunteer blood donors§ Factor V mutation (%)~ Canadian neonates§ Hospital/laboratory control subjects§ Combined control subjects

n 331 19 167 7.6 293 133 593

63

WT (%)

Het (%)

Homo (%)

tl-Allele (%)t

139 (42) 19 62 (37) 6.5 125 (43) 50 (38) 237 (40)

154 (47) 22 79 (47) 10 122 (42) 63 (47) 264 (44)

38 (11) 11 26 (16) 3.8 46 (15) 20 (15) 92 (16)

35 39 36 39 38

*Wild type (WT) = Ala/Ala; heterozygous (Het) = Ala/Val; homozygous (Homo) = Val/Val at nucleotide 677, the site of the tl-MTHFR mutation (677 C to T). tNo significant difference in either MTHFR genotype distribution, MTHFR homozygote frequency, or MTHFR mutant allele frequency between the four study groups. SNo significant difference in the factor V R506Q mutation frequency between the MTHFR genotype groups. §MTHFR data previously reported [14].

64

Molecular Diagnosis Vol. 2 No. 1 March 1997

gel electrophoresis and ethidium bromide staining (Fig. 1). The detection of the causative nucleotide substitution of factor V Leiden (R506Q, a G to A substitution at nucleotide 1691) was performed by MnlI digestion of PCR-amplified products as previously described [21 ].

nel at Oregon Health Sciences University [14]. None of these 133 hospital control subjects were factor V genotyped. The study was approved by the Oregon Health Sciences University human studies institutional review board.

MTHFR and Factor V Genotyping DNA was extracted from fresh, frozen, or dried spots of peripheral blood by one of two different standard methods: a rapid boiling method using Chelex resin or a silica resin affinity chromatography method. For the Chelex method, we followed the whole blood procedure of Walsh et al. [20] with some minor modifications [21 ]. For the silica column method, we used a commercially available DNA preparation kit (the QIAamp blood kit from Qiagen, Santa Clarita, CA). The region surrounding the nucleotide 677 MTHFR mutation site was polymerase chain reaction (PCR) amplified (with the primers 5'-TGAAGGAGAAGGTGTCTGCGGGA3' and 5'-AGGACGGTGCGGTGAGAGTG-3') as originally described [10]. As the nucleotide 677 mutation creates a restriction site for either TaqI or Hinfl, the genotype was determined by digestion with either of these enzymes followed by agarose

Statistics Genotype or allele frequency differences between the study groups were assessed with a chi-square or Fisher's exact test. Odds ratios with 95% confidence intervals were calculated to estimate the relative risk of thrombosis for the genotype groups. Statistical tests were performed using the InStat software package from GraphPad software (San Diego, CA).

Results The subjects in the thrombosis group and three control groups were each genotyped for the presence of the thermolabile (tl-) MTHFR mutation. A representative genotype experiment is shown in Fig. 1. Within the thrombosis group, the tI-MTHFR

PCR BB~ql BII . ~ l

I

198 bp MTHFR amplicon

J

t

677 C to T mutation (creates new Hinfl or Taql site)

Hinfl orTaql

I PA -.v-. -"

175 bp

= mutant wild type

198 bp

bp bp 1

2

3

4

5

6

7

M

Fig. 1. Direct detection of the thermolabile (tl-) methylenetetrahydrofolate reductase (MTHFR) mutation by a polymerase chain reaction (PCR)-based assay. Peripheral blood DNA from seven representative subjects was PCR-amplified with MTHFR-specific primers, digested with TaqI, and separated by agarose gel electrophoresis. As the tl-MTHFR mutation (C to T at nucleotide 677) creates a restriction site for either HinfI or TaqI, wild-type alleles yield a 198-bp product, whereas mutant alleles yield a smaller 175-bp product. Lanes 1 and 4-7 show tI-MTHFR heterozygotes, lane 3 shows a wild-type individual, and lane 2 shows a tl-MTHFR homozygote. Lane 8 (M) is a DNA size marker.

MTHFR Mutation in Venous Thrombosis

mutation was quite common: 47% of the 331 subjects were heterozygous for this mutation and 11% were homozygous (Table 2). The 35% allele frequency in the thrombosis group was similar to the 36 to 38% allele frequency reported in other North American groups [10,13]. The MTHFR genotype distributions did not significantly vary from those predicted by the Hardy-Weinberg equilibrium. We also determined the MTHFR genotype status of those in three healthy control groups: 167 volunteer blood donors, 293 Canadian newborns, and 133 hospital/laboratory control subjects (Table 2) [14]. In these control groups, the frequencies of tlMTHFR homozygotes (15-16%) and the mutant tlMTHFR allele (36-39%) were well conserved between the three groups and were similar to the 12% homozygous rate and 36 to 38% mutant allele frequencies reported in other North American groups [10,13] (P > .3). For the tl-MTHFR mutation, there were no significant differences in either the mutant allele frequencies, the homozygote frequencies, or the distribution of genotypes between the thrombosis and three control study groups in Table 2 (P > .09). In particular, the thrombosis and volunteer blood donor groups, with closely matched age and sex distributions, had similar MTHFR mutation and allele frequencies (P >. 16). To investigate a possible cooperative interaction between the tl-MTHFR and factor V R506Q mutations, we analyzed the prevalence of the factor V R506Q mutation in each of the MTHFR genotype groups. In the thrombosis subjects with a high overall prevalence of the factor V R506Q mutation (19%), there was no significant difference in the prevalence of this mutation between the three MTHFR genotype groups (P > .2) (Table 2). There was also no significant difference in the prevalence of the factor V R506Q mutation among the venous thrombosis subjects either with or without a homozygous tl-MTHFR mutation (P > .14). In the blood donor control group with a low overall prevalence of the factor V R506Q mutation (7.6%), the frequency of this mutation was again no different both between the three MTHFR genotype groups (P > .5) and between those either with or without homozygous tl-MTHFR (P > .4) (Table 2). The data in Tables 1 and 2 were also used to calculate odds ratios for venous thrombosis for those with either or both of these common genetic mutations. As expected, between the venous thrombosis and control blood donor groups, the odds ratio of

•

Ocal et al.

65

thrombosis for those with the factor V R506Q mutation was 3.3 (95% confidence interval, 1.9-5.8; P < .0001). In comparison, the odds ratio for those with both a factor V mutation and a homozygous MTHFR mutation was not significantly different from 1 (P > .6). Although the factor V mutation, by itself, was a significant thrombotic risk factor, the odds ratio for those with either a factor V mutation or a homozygous MTHFR mutation was not significantly different from 1 (P > .1). Although homozygosity for the tl-MTHFR mutation may then predispose to hyperhomocysteinemia [11,12], a known risk factor for venous thrombosis [4,5], this common genotype does not appear to be a significant genetic risk factor for venous thrombosis, either alone or in combination with the factor V R506Q mutation.

Discussion In a group of 331 subjects with venous thrombosis, the frequency of a thermolabile MTHFR mutation linked to hyperhomocysteinemia has been shown to be no different than in several control groups. Although we have confirmed the tlMTHFR mutation as an extremely common genetic alteration in the general population, we conclude that carriers of this mutation are not at significantly increased risk for venous thrombosis. The absence of a direct link between the tl-MTHFR mutation and thrombosis appears somewhat contradictory given both the hyperhomocysteinemia of MTHFR homozygotes and the association between hyperhomocysteinemia and venous thrombosis. The reported association between the tl-MTHFR mutation and hyperhomocysteinemia may, however, be weaker than initially presumed. As we have previously stated [14], because the distribution of homocysteine values within a population is significantly skewed [22], an appropriate intergroup comparison of homocysteine levels may be best performed by either non-parametric statistical tests or mathematical transformation of the raw data to approximate a normal distribution. Both of the published reports linking hyperhomocysteinemia to the tl-MTHFR mutation, however, based their conclusions on parametric statistical tests using untransformed homocysteine data [11,12]. In comparison, in our recent study of homocysteine levels and the tl-MTHFR mutation in peripheral vascular disease subjects

66

Molecular Diagnosis Vol. 2 No. 1 March 1997

[14], we found no significant variation in homocysteine levels between MTHFR genotype groups (P = .16) using log-transformed homocysteine data (to approximate a normal distribution). These same data analyzed without log transformation, in contrast, gave the misleading impression of a "significant" (P = .04) increase in homocysteine levels in tl-MTHFR homozygotes. We propose that with appropriate statistical analysis, the conclusions of a "significant" link between the tl-MTHFR mutation and hyperhomocysteinemia may be either overstated or, as discussed below, dependent on other variables. An additional explanation for the observed nonassociation between tl-MTHFR and venous thrombosis may relate to the folic acid levels in those studied. The significant negative correlation between plasma homocysteine and folate levels [13,14,23-25] most likely stems from the requirement for 5-methyltetrahydrofolate as the methyl group donor for the remethylation of homocysteine to methionine. Our recent description of a significantly steeper slope for the homocysteine-folate correlation in tl-MTHFR homozygotes as compared with nonhomozygotes implies that the risk of hyperhomocysteinemia in these homozygotes may be dependent on their folate status [14]. In agreement with this hypothesis, Jacques et al. have shown a significant difference in homocysteine levels between tl-MTHFR genotype groups in those with low folate, but not those with high folate [13]. As nucleotide 677 may be within a folate binding region of MTHFR [10], the thermolabile enzyme may require significantly higher folate levels for efficient function. Should most of our venous thrombosis subjects be nutritionally replete, their folate levels may be high enough to permit adequate homocysteine metabolism (and thus no excess thrombotic risk), even in those homozygous for tl-MTHFR. Although unlikely, our inability to find a significant association between venous thrombosis and the tl-MTHFR mutation may be the result of the clinical heterogeneity in our thrombosis group. These subjects were selected, not on strict clinical criteria, but instead on having had a laboratory evaluation for familial or recurrent thrombophilia that included a factor V R506Q genotype determination. Additional clinical information that may have allowed us to exclude confounding variables could not, how-

ever, be obtained without compromising the patient anonymity required for performing genetic tests on archival specimens. Despite these limitations, objective clinical and laboratory parameters both suggested that the majority of those in our thrombosis cohort had bona fide venous thrombotic histories. For example, the frequency of the factor V R506Q mutation was as high in our thrombosis group (-20%) as in several other well-characterized venous thrombosis groups (18-26%) [3,16,17]. In addition, of the brief clinical descriptors that did accompany many of these blood specimens, 74% documented a venous thrombotic event that had necessitated the hypercoagulable evaluation. Despite the solid evidence linking hyperhomocysteinemia to arterial vascular disease [15], we and others have recently shown that the tl-MTHFR mutation may not be a significant genetic risk factor for either peripheral arterial vascular disease [14] or cardiovascular disease [26]. Other reports, however, have shown that in more selected groups of vascular disease patients, the prevalence of tlMTHFR homozygotes is significantly higher than in control subjects [12,27]. These discrepant findings may relate to the different vascular disease selection criteria used by each investigator group. The vascular disease subjects of both Defranchis et al. [27] and Kluitjmans et al. [12] may have been enriched for those with homocysteine-associated disease by including only subjects with premature vascular disease and by excluding those with other known prothrombotic risk factors. In contrast, we designed our unselected group of thrombosis subjects to approximate the more realworld example of a common clinical scenario: the typical hypercoagulable evaluation. The true relationship between the tl-MTHFR mutation, homocysteine levels, and vascular disease risk is then likely to be quite complex and dependent on a number of additional nutritional, biochemical, and/or genetic factors. Although, as expected, we have confirmed the factor V R506Q mutation as a significant genetic risk factor for venous thrombosis, the tI-MTHFR mutation was not a significant thrombotic risk factor, either by itself or in combination with the factor V mutation. These latter conclusions must, however, be tempered by the minimal statistical power afforded by our relatively small number of subjects with both mutations. The inability of the tl-MTHFR

MTHFR Mutation in Venous Thrombosis

mutation to synergize with the factor V mutation to produce an additional or cooperative thrombotic risk supports the conclusions of den Heijer et al. [4], who found no increased venous thrombotic risk in those with both hyperhomocysteinemia and factor V R506Q as compared with those with one or neither of these defects. In contrast, patients carrying factor V R506Q together with either a protein C deficiency [28,29], a protein S deficiency [30,31], or a more severe MTHFR deficiency [32] have a higher thromboric risk than those carrying either defect alone. Although we have been unable to confirm a specific association between the tl-MTHFR mutation and either venous thrombosis (this report) or arterial vascular disease [14], the reduced MTHFR enzymatic activity created by this mutation may have pathologic consequences when combined with additional biochemical or nutritional deficits in the homocysteine metabolic pathway. In particular, as tlMTHFR homozygotes are more sensitive to the hyperhomocysteinemic effects of folate deprivation, the vascular disease risk in these individuals may be folate as well as genotype dependent [14]. In this context, a comparison of the tl-MTHFR mutation frequencies in subjects with low folate levels either with or without vascular disease might be revealing. Until such a study is completed, the clinical utilization of the tl-MTHFR mutation test should probably be limited to the evaluation of those with premature vascular disease and unexplained mild to moderate hyperhomocysteinemia. As the population prevalence of both this mutation and vascular disease is quite high, the presence of this mutation may help predict both those who may be at risk for folate-dependent hyperhomocysteinemia and those who may respond more favorably to folate therapy.

Acknowledgments Thanks to Debbie Ocal and Chris Grant for specimen procurement and genotype advice, to Adam Evans and Todd Wisner for statistical and technical assistance, and to Tom DeLoughery and Scott Goodnight for helpful advice and critical reviews of the manuscript. Thanks also to Marcus Grompe for his DNA specimens from Canadian neonates. Received Oct. 1, 1996. Accepted Nov. 19, 1996.

•

Ocal et al.

67

References 1. Dalen JE, Alpert JS: Natural history of pulmonary embolism. Prog Cardiovasc Dis 1975; 17:259-270 2. Dahlback B: Inherited thrombophilia: resistance to activated protein C as a pathogenic factor of venous thromboembolism. Blood 1995;85:607-614 3. Bertina RM, Koeleman BP, Koster T, Rosendaal FR, Dirven R J, de Ronde H, van der Velden PA, Reitsma PH: Mutation in blood coagulation factor V associated with resistance to activated protein C. Nature 1994;369:64-67 4. Den Heijer M, Koster T, Blom HJ, Bos GM, Briet E, Reitsma PH, Rosendaal FR: Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med 1996;334:759-762 5. Den Heijer M, Blom HJ, Gerrits WB, Rosendaal FR, Haak HL, Wijermans PW, Bos GM: Is hyperhomocysteinaemia a risk factor for recurrent venous thrombosis? Lancet 1995;345:882-885 6. Fermo I, Vigano D'Angelo S, Paroni R, Mazzola G, Calori G, D'Angelo A: Prevalence of moderate hyperhomocysteinemia in patients with early-onset venous and arterial occlusive disease. Ann Intern Med 1995;123:747-753 7. Falcon CR, Cattaneo M, Panzeri D, Martinelli I, Mannucci PM: High prevalence of hyperhomocyst(e)inemia in patients with juvenile venous thrombosis. Arterioscler Thromb 1994; 14:1080-1083 8. Bienvenu T, Ankri A, Chadefaux B, Montalescot G, Kamoun P: Elevated total plasma homocysteine, a risk factor for thrombosis: relation to coagulation and fibrinolytic parameters. Thromb Res 1993;70: 123-129 9. Mudd SH, Skovby F, Levy HL, Pettigrew KD, Wilcken B, Pyeritz RE, Boers GH, Bromberg IL, Cerone R, Fowler B, Grobe H, Schmidt H, Schweitzer L: The natural history of homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet 1985;37:1-31 10. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Denheijer M, Kluijtmans LAJ, Vandenheuvel LP, Rozen R: A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nature Genet 1995;10:111-113 11. Van der Put NMJ, Steegers-Theunissen RPM, Frosst P, Trijbels FJM, Eskes TKAB, van den Heuvel LP, Mariman CM, den Heyer M, Rozen R, Blom HJ: Mutated methylenetetrahydrofolate reductase as a risk factor for spina bifida. Lancet 1995;346: 1070-1071 12. Kluijtmans LAJ, Vandenheuvel LPWJ, Boers GHJ, Frosst P, Stevens EMB, Denheijer M, Trijbels FJM,

68

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

Molecular Diagnosis Vol. 2 No. 1 March 1997

Rozen R, Blom HJ: Molecular genetic analysis in mild hyperhomocysteinemia: a common mutation in the methylenetetrahydrofolate reductase gene is a genetic risk factor for cardiovascular disease. Am J Hum Genet 1996;58:35-41 Jacques PF, Bostom AG, Williams RR, Ellison RC, Eckfeldt JH, Selhub J, Rozen R: Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations. Circulation 1996;93:7-9 DeLoughery TG, Evans A, Sadeghi A, McWilliams J, Henner WD, Taylor LM, Press RD: A common mutation in methylenetetrahydrotolate reductase: correlation with homocysteine metabolism and late onset vascular disease. Circulation 1996;94: 673074-3078 Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA 1995;274:1049-1057 Ridker PM, Hennekens CH, Lindpaintner K, Stampfer MJ, Eisenberg PR: Mutation in the gene coding for coagulation factor V and the risk of myocardial infarction, stroke, and venous thrombosis in apparently healthy men. N Engl J Med 1995; 332:912-917 Ridker PM, Miletich JP, Stampfer MJ, Goldhaber SZ, Lindpaintner K, Hennekens CH, Embolism: factor V Leiden and risks of recurrent idiopathic venous thromboembolism. Circulation 1995;92: 2800-2802 Grompe M, St-Louis M, Demers SI, al-Dhalimy M, Leclerc B, Tanguay RM: A single mutation of the fumarylacetoacetate hydrolase gene in French Canadians with hereditary tyrosinemia type I. N Engl J Med 1994;331:353-357 Press RD, Liu X-Y, Beamer N, Coull B: Ischemic stroke in the elderly: role of the common factor V mutation causing resistance to activated protein C. Stroke 1996;27:4448 Walsh PS, Metzger DA, Higuchi R: Chelex 100 as a medium for simple extraction of DNA for PCRbased typing from forensic material. Biotechniques 1991 ;10:506-513 Liu X-Y, Nelson D, Grant C, Morthland V, Goodnight SH, Press RD: Molecular detection of a common mutation in coagulation factor V causing thrombosis via hereditary resistance to activated protein C. Diagn Mol Pathol 1995;4:191-197 Nygard O, Vollset SE, Refsum H, Stensvold I, Tverdal A, Nordrehaug JE, Ueland M, Kvale G:

23.

24.

25.

26.

27.

28.

29.

30.

31.

32.

Total plasma homocysteine and cardiovascular risk profile. JAMA 1995;274:1526-1533 Hopkins PN, Wu LL, Wu J, Hunt SC, James BC, Vincent GM, Williams RR: Higher plasma homocyst(e)ine and increased susceptibility to adverse effects of low folate in early familial coronary artery disease. Arterioscler Thromb Vasc Biol 1995;15: 1314-1320 Jacobsen DW, Gatautis V J, Green R, Robinson K, Savon SR, Secic M, Ji J, Otto JM, Taylor LM Jr: Rapid HPLC determination of total homocysteine and other thiols in serum and plasma: sex differences and correlation with cobalamin and folate concentrations in healthy subjects. Clin Chem 1994; 40:873-881 Selhub J, Jacques PF, Wilson PW, Rush D, Rosenberg IH: Vitamin status and intake as primary determinants of homocysteinemia in an elderly population. JAMA 1993;270:2693-2698 Wilcken DEL, Wang XL, Sim AS, McCredie RM: Distribution in healthy and coronary populations of the methylenetetrahydrofolate reductase (MTHFR) C 677 T mutation. Arterioscler Thromb Vasc Biol 1996; 16:878-882 Defranchis R, Mancini FP, D'Angelo A, Sebastio G, Fermo I, DeStefano V, Margaglione M, Mazzolo M, DiMinno G, Andria G: Elevated total plasma homocysteine and 677 C to T mutation of the 5,10-methylenetetrahydrofolate reductase gene in thrombotic vascular disease. Am J Hum Genet 1996;59:262-264 Koeleman BP, Reitsma PH, Allaart CF, Bertina RM: Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families. Blood 1994;84:1031-1035 Gandrille S, Greengard JS, Alhencgelas M, Juhanvague I, Abgrall JF, Griffin JH, Aiach M: Incidence of activated protein C resistance caused by the Arg 506 Gln mutation in factor V in 113 unrelated symptomatic protein C-deficient patients. Blood 1995;86: 219-224 Koeleman BPC, Vanrumpt D, Hamulyak K, Reitsma PH, Bertina RM: Factor V Leiden: an additional risk factor for thrombosis in protein S deficient families. Thromb Haemost 1995;74:580-583 Zoller B, Berntsdotter A, Defrutos PG, Dahlback B: Resistance to activated protein C as an additional genetic risk factor in hereditary deficiency of protein S. Blood 1995;85:3518-3523 Mandel H, Brenner B, Berant M, Rosenberg N, Lanir N, Jakobs C, Seligsohn U: Coexistence of hereditary homocystinuria and factor V Leiden: effect on thrombosis. N Engl J Med 1996;334: 763-768