MBC/200176; Total nos of Pages: 5;

Original article 1MBC 200176

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Hyperhomocysteinemia and the compound heterozygousstate for methylene tetrahydrofolate reductase areindependent risk factors for deep vein thrombosis amongSouth IndiansS.M. Naushad, Md Nurul Jain Jamal, R. Angalena, C. Krishna Prasadand A. Radha Rama Devi

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Objective To investigate the role of methylene

tetrahydrofolate reductase (MTHFR) (677 C!T and 1298

A!C), factor V (1691 G!A), factor II (20210 G!A) genetic

polymorphisms and hyperhomocysteinemia in the aetiology

of deep vein thrombosis (DVT) in 163 cases and 163

controls.

Methods Polymerase chain reaction-restriction fragment

length polymorphism was used for genotyping, reverse-

phase high-performance liquid chromatography for plasma

homocysteine, and Student’s t-test and Fisher exact tests

were used for statistical analysis.

Results Elevated mean plasma homocysteine levels were

observed in DVT cases irrespective of gender differences.

Homocysteine elevation above the 95th percentile of the

control group associated with 9.4-fold and 7.6-fold

increased risk for DVT in men and women, respectively.

Genotyping showed the MTHFR 677CT/1298AC genotype

(i.e. compound heterozygosity) is associated with 3.5-fold

risk for thrombosis. The factor V Leiden mutation frequency

was higher in DVT cases, but not statistically significant;

however, genetic predisposition to this mutation was

0957-5235 � 2006 Lippincott Williams & Wilkins

associated with early age of DVT onset. Factor II mutation

was absent in cases and controls. Co-segregation of two or

more risk factors was associated with 11.7-fold increased

risk for thrombosis.

Conclusions This study projects that

hyperhomocysteinemia and compound heterozygous state

for MTHFR are independent risk factors for DVT among

South Indians. Blood Coagul Fibrinolysis 17:00–00 � 2006

Lippincott Williams & Wilkins.

Blood Coagulation and Fibrinolysis 2006, 17:00–00

Keywords: deep vein thrombosis, factor V, factor II, homocysteine,methylene tetrahydrofolate reductase

Center for DNA Fingerprinting and Diagnostics, Hyderabad, India

Correspondence and requests for reprints to Dr A. Radha Rama Devi, Head,Diagnostic Division, Center for DNA Fingerprinting and Diagnostics (CDFD),7–18 Nacharam, Hyderabad 500076, Andhra Pradesh, IndiaTel: +91 40 27171462; e-mail: radha@cdfd.org.in

Sponsorship: The Department of Biotechnology, Government of India funded thisstudy.

Received ?? ?? Revised ?? ??Accepted ?? ??

IntroductionGenetic and environmental factors can alter the procoa-

gulant and anticoagulant mechanisms and cause venous

thrombosis. Venous thrombosis has variable clinical

manifestations depending on the site and extent of

thrombus formation and the underlying cause. Deep

vein thrombosis (DVT) is one of the most common

causes of morbidity and mortality in the western world

[1,2], Pulmonary embolism is the most serious compli-

cation of the disease. Different genetic polymorphisms

both in haemostatic and non-haemostatic genes have

been reported to be risk factors [3,4] and include protein

C [5,6], protein S [7], factor V Leiden, prothrombin,

antithrombin III [8], methylene tetrahydrofolate reduc-

tase (MTHFR) and dysfibrinogenemia.

Factor V Leiden (G1691A) mutation confers activated

protein C resistance [9,10], altered anticoagulant function

and loss of efficiency to serve as a cofactor in activated

factor VIII degradation [11,12]. Hence its inheritance

increases the risk for occurrence and recurrence of venous

thrombosis [13].

The G20210A mutation in the 30-untranslated region of

prothrombin is identified as a risk factor for DVT, causing

elevated prothrombin levels, and this appears to be respon-

sible for the increased risk for thrombosis. The precise

mechanism by which prothrombin levels are altered is yet

to be determined. Increased efficiency of polyadenylation

of mRNA transcripts, however, has been suggested [14].

Among the range of nonhaemostatic factors that can

interact with haemostatic determinants, plasma homo-

cysteine is of prime importance. In recent years hyper-

homocysteinemia has attracted much interest as a

potential risk factor for both venous and arterial throm-

botic disease [15,16]. By stimulating the procoagulant

mechanism or by inhibiting anticoagulant mechanism,

homocysteine exerts its effect on thrombus formation.

One of the two enzymatic pathways of remethylation of

MBC/200176; Total nos of Pages: 5;

2 Blood Coagulation and Fibrinolysis 2006, Vol ?? No??

Table 1 Genotype frequencies in deep vein thrombosis cases andcontrols

Group WW WM MM MOdds ratio (95%

confidence interval) P value

MTHFR 677 CC CT TT T 1.53 (1.46–1.60) 0.19Cases 133 29 1 31/326Controls 142 21 0 21/326

MTHFR 1298 AA AC CC C 0.86 (0.84–0.88) 0.41Cases 57 105 1 107/326Controls 63 82 18 118/326

Factor V GG GA AA A 1.52 (1.43–1.64) 0.42Cases 149 13 1 15/326Controls 153 10 0 10/326

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homocysteine to methionine involves MTHFR. An

MTHFR deficiency blocks this remethylation process,

resulting in hyperhomocysteinemia. Two common poly-

morphisms in MTHFR (i.e. C677T and A1298C) were

found to cause mild to moderate hyperhomocysteinemia.

The former mutation has greater clinical impact than the

latter. These two mutations occur in catalytic and regu-

latory domains, respectively. Inconsistent results have

been observed about the possible association of C677T

mutation in the aetiology of DVT [17,18].

There are fewer studies on the genetic risk factor evalu-

ation for DVT from India [19–22,24] and no comparable

studies from South India. The main aim of this study is to

determine the role of MTHFR, factor V, factor II genetic

polymorphisms and hyperhomocysteinemia in the aetiol-

ogy of DVT among South Indians.

Materials and methodsParticipantsAll of the 163 DVT patients (105 men and 58 women)

referred to our centre between October 1999 and

February 2004 agreed to participate in this study. The

diagnosis of DVT was based on impedance plethysmo-

graphy, Doppler ultrasonography, compression ultraso-

nography or contrast venography. Informed consent was

taken from each patient. The study protocol was

approved by bioethical committee of Center for DNA

Fingerprinting and Diagnostics. The control group com-

prised of 105 men and 58 women. Age, sex, and race or

ethnic group matched controls were selected.

MethodsFasting ethylenediamine tetraacetic acid anticoagulated

blood samples were collected on ice, and plasma was

separated immediately and frozen until the time of

analysis. Genomic DNA was isolated from the buffycoat

using the phenol–chloroform extraction method.

Total plasma homocysteine analysisSulfur-containing amino acids were derivatized using

ammonium 7-fluorobenzo-2-oxa-1,3-diazole-4-sulphonate

after treatment with tributyl N-phosphine. Reverse-phase

high-performance liquid chromatography with isocratic

separation was performed on an ODS 3v column using

0.1 mol/l KH2PO4:acetonitrile (96:4) as the mobile phase.

The standard calibration curve for concentrations ranging

from 5 to 200mmol/l was found to be linear (r¼ 0.999). This

method was validated against an enzyme-linked immun-

sorbent assay-based kit using Bland and Altman analysis.

Study of genetic polymorphisms:Factor V (G1691A), factor II (G20210A), MTHFR

(C677T) and MTHFR (A1298C) genetic polymor-

phisms were detected using polymerase chain reaction-

restriction fragment length polymorphism approaches

using MnlI, HindIII, HinfI and MboII restriction enzymes

[11,14,25].

Statistical analysisAs homocysteine levels were skewed, log-transformation

of the values was carried out and Grubb’s test was

performed to detect significant outliers. After correcting

for skewness and outliers, the 5th, 50th and 95th per-

centiles were calculated using an Excel datasheet and the

values were converted back to normal. Student’s t-test

was performed both on log-transformed data and cor-

rected data. Mean and SD values were obtained from

these statistics along with the P values. Graphpad soft-

ware was used for this analysis. For categorical variables,

Fisher’s exact test was performed using Vassarstats soft-

ware and the genotype frequencies, odds ratio and Pvalues were obtained. For all variables, a P value less than

0.05 was taken as statistically significant.

ResultsThe mean age of onset for DVT in men was 37.4�13.7 years and in women was 32.4� 13.8 (P< 0.05).

The frequencies of factor V, MTHFR C677T, MTHFR

A1298C mutations and hyperhomocysteinemia in cases

were 8.6%, 18.4%, 65% and 17.8% and the frequencies in

controls were 6.1%, 12.9%, 61.3% and 2.6%, respectively.

A total of 0.6% cases were homozygous for each mutation

and none of the controls were homozygous mutant for

the first two mutations. Eleven per cent of controls

were homozygous for the MTHFR A1298C mutation.

Factor II mutation was absent in both cases and controls

(Table 1).

MTHFR compound heterozygosity was observed in

11.7% of cases and 3.7% of controls; 1.2% of cases were

heterozygous for factor V, MTHFR C677T and MTHFR

A1298C, and none of the controls showed this combi-

nation. Co-segregation of MTHFR 677 C!T with factor

V/homocysteine or factor V with homocysteine was

associated with an 11.7-fold increased risk for thrombosis

(Table 2).

The total plasma homocysteine data was segregated

according to gender, and cut-off values were obtained

MBC/200176; Total nos of Pages: 5;

Hyperhomocysteinemia, MTHFR and DVT Naushad et al. 3

Table 2 Haplotype frequencies in deep vein thrombosis cases and controls

MTHFR 677/1298/factor V Cases Controls Odds ratio (95% confidence interval) P value

C-A-G 39 43 0.88 (0.86–0.90) 0.70C-C-G 82 89 0.84 (0.82–0.86) 0.51C-A-A 7 5 1.42 (1.37–1.48) 0.77C-C-A 5 5 1.00 (0.99–1.00) 1.00T-A-G 11 15 0.71 (0.69–0.74) 0.54T-C-G 17 6 3.05 (2.79–3.35) 0.03�T-A-A 0 0 ND NDT-C-A 2 0 ND 0.50T-C-G and T-C-A 19 6 3.46 (3.14–3.83) <0.01Two or more risk factors 11 1 11.7 (9.46–14.75) <0.005

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using the 95th percentile of controls, which in men and

women were 19.2 and 13.1mmol/l, respectively. Mean

plasma homocysteine was found to be higher in DVT

cases (16.5 vs 11.0mmol/l for men and 10.9 vs 8.4mmol/l

for women, with P< 0.0001 and P< 0.0006) (Table 3).

An increase in plasma homocysteine above the 95th

percentile of the control group was found to be associated

with 9.4-fold and 7.6-fold risk in men and women,

respectively, for venous thrombosis (Table 4).

Out of the 17 male DVT cases with hyperhomocystei-

nemia, four were compound heterozygous for MTHFR

(677CT/1298AC), one was heterozygous for MTHFR

C677T (677CT/1298AA), three were mutant for factor

V and the remaining nine cases did not have any

mutation. Out of the 12 female DVT cases with hyper-

homocysteinemia, one was homozygous for MTHFR

C677T. Out of the five controls with hyperhomocystei-

nemia, one was compound heterozygous for MTHFR

and none of the others had any mutation.

Excluding all the genetic risk factors, hyperhomocystei-

nemia alone was found to have a 5.6-fold risk for DVT

(12.3% vs 2.5%, P< 0.001).

Genetic predisposition to factor V mutation was associ-

ated with early onset for DVT (36.0� 13.5 vs 22.9�8.6 years, P< 0.0005), whereas genetic predisposition to

MTHFR 677 C!T, although associated with early age of

onset, was not statistically significant (36.1� 14.2 vs

32.5� 10.4 years, P> 0.05).

Table 3 Plasma homocysteine distribution in cases and controls

Group

Homocysteine (mmol/l)

P valueMean (SD) Median 5th–95th percentile

MenCase 16.5 (7.4) 15.0 8.9–35.0 <0.0001Controls 11.0 (4.9) 11.2 3.5–19.2

WomenCases 10.9 (4.3) 11.0 5.1–16.7 <0.0006Controls 8.4 (3.1) 8.5 3.1–13.1

Log-transformed values were used to correct skewness and statistics wereapplied to them. P<0.05 was considered statistically significant.

In DVT women, pregnancy-induced thrombophilic

status was observed in 28 cases, and oral contraceptive

(Mala-D) usage was observed in six cases.

DiscussionGenetic and environmental factors are implicated in the

pathophysiology of venous thrombosis. These are respon-

sible for bringing out a hypercoaguable state in the blood

or vessel wall injury and, when associated with venous

stasis, venous thrombosis results. Wide ethnic variations

are reported in the distribution of haemostatic and non-

haemostatic genetic polymorphisms. The study aims at

finding the relative risk factors in venous thrombosis

among a South Indian population regarding the role of

factor V, factor II, MTHFR polymorphisms and hyper-

homocysteinemia.

Venous thrombosis was more prevalent among males to

the extent of 66%. Women, however, had an early age of

onset manifesting during the reproductive age. The

reasons for such an early age of onset could be preg-

nancy-induced thrombophilic status or use of oral contra-

ceptives [26,27]. The Mala-D oral contraceptive contains

ethinyl oestradiol, whose consumption beyond 50mg/day

increases the risk for DVT [28]. Venous stasis during

pregnancy along with other risk factors may also contrib-

ute to increased DVT risk.

Among the genetic polymorphisms, factor II mutation is

reported to be absent among Indians. This is true even in

our study where there is no mutation detected in both

cases and controls, and thus factor II mutation is not a risk

factor among South Indians for venous thrombosis. The

factor V Leiden mutation also was reported to be less

Table 4 Elevated homocysteine (above the 95th percentile ofcontrols) and the risk for deep vein thrombosis

Pl. homocysteine(mmol/l) Cases Controls

Odds ratio(95% confidence

interval) P value

Men 17 2 9.35 (7.82–11.30) <0.001Women 12 3 7.63 (6.50–9.04) <0.001

Odds ratios and P values were calculated using Fisher’s exact test.

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MBC/200176; Total nos of Pages: 5;

4 Blood Coagulation and Fibrinolysis 2006, Vol ?? No??

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frequent among the Indian population. Certain endoga-

mous populations like Parsis have high prevalence up to

10%, indicating a wide ethnic variation among different

groups in India [19–24]. The relative risk associated with

factor V mutation in the current study is 1.5, which is not

statistically significant (P¼ 0.42).

The other important genetic variant in venous thrombo-

sis is the enzyme involved in methionine metabolism,

MTHFR, which is important in the remethylation of

homocysteine to methionine as it supplies 5-methyl

tetrahydrofolate as the co-substrate. The C677T

mutation in MTHFR gene is known to induce thermol-

ability, thereby lowering its activity. The A1298C

mutation does not induce thermolability, however; it

reduces the enzyme activity and can act in an additive

manner in combination with C677T [29]. The resultant

block in the conversion of homocysteine to methionine

causes homocysteine accumulation. Homocysteine is

now widely accepted to be involved in the integrity of

the vascular endothelium causing its damage. A meta-

analysis comprising of 3439 DVT cases and 5063 controls

from different parts of the world showed that MTHFR

mutants with a 5mmol/l increase in homocysteine exert

1.6-fold risk, which is reduced to 25% when there is a

3mmol/l decrease in homocysteine [18]. Mutation fre-

quencies vary from region to region and ethnicity. A study

from western India reported 14.9% C677T MTHFR

mutants among young patients of venous thrombosis

[20]. The current study from South India showed an

18.4% mutation frequency among cases and 12.9% among

controls. No statistical significance was observed in DVT

when there is a single mutation, either C677T or A1298C.

Compound heterozygosity, however, increased the risk

by 3.5-fold for DVT irrespective of the gender. The

homocysteine concentration among compound heterozy-

gous individuals was high among men and surprisingly

normal among women.

The mean homocysteine levels in DVT cases were

significantly higher than their controls (age-matched

and sex-matched). Furthermore, homocysteine levels

in men were significantly higher than women in geno-

type-matched cases and controls. The gender differences

observed could be explained on the basis of reproductive

hormones and stoichiometric formation of homocysteine

in connection with the creatine/creatinine synthesis that

is proportional to muscle mass [30–33]. Hyperhomocys-

teinemia was defined as the concentration of homocys-

teine above the 95th percentile of the control group,

which is 19.2 and 13.1mmol/l for men and women,

respectively, in the current study. In men a 9.4-fold

increased risk was observed in participants with homo-

cysteine above 19.2mmol/l, whereas in women the risk is

7.6-fold with homocysteine greater than 13.1mmol/l,

suggesting strongly that hyperhomocysteinemia is a risk

factor for DVT in both men and women.

The pathophysiology of homocysteine-related vascular

thrombosis is that high concentrations of homocysteine

activate factor V [34], reduce protein C activation, inac-

tivate the cofactor activity of thrombomodulin [35], sup-

press thrombomodulin and anticoagulant heparan sulfate

expression [36] and block tissue plasminogen activator

binding to human endothelial cells [37]. It therefore

affects both the vascular endothelium as well as the

haemostatic factors leading to thrombosis. Hyperhomo-

cysteinemia has emerged as an independent risk factor

after excluding other genetic risk factors. There is, how-

ever, a need to evaluate whether elevated plasma homo-

cysteine, itself a risk factor, or genetic or nutritional

factors influencing homocysteine have any direct impact

on thrombosis. Cattaneo et al. have shown positive associ-

ation of low vitamin B6 with the risk of DVT indepen-

dent of fasting and post-methionine-load homocysteine

levels [38]. Quere et al. have shown that methionine

levels below the median value were independently

associated with risk for DVT [39]. Oger et al. have shown

elevated plasma homocysteine, low serum folate and low

vitamin B12 as independent risk factors associated with

1.48-fold, 3.14-fold and 1.42-fold increased risk for DVT,

respectively [40]. Several studies have reported a high

prevalence of B vitamin (folate, vitamins B6 and B12)

deficiencies in Indians, which could be partly due to a

vegetarian diet [41–43]. There is a need to evaluate

whether the plasma homocysteine elevation observed

in this study is nutritional or genetic.

Genetic predisposition to MTHFR or factor V, or

both, was found associated with early onset of DVT. Co-

existence of two or more risk factors pronounced the risk

for DVT and may require prolonged anticoagulation [44].

To conclude, hyperhomocysteinemia due to nutritional

or genetic causes and compound heterozygous status for

MTHFR are independent risk factors for DVT among

South Indians. Co-segregation of two or more risk factors

inflates the risk for DVT.

AcknowledgementsThe authors thank all the patients who participated in

this study. They thank Mr Ram Prakash Singh and Mr G.

Srinivas for technical support.

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Please provide history date. Confirm author names are correctly stated Provide fax number Should reference [23] also be cited here? If not, references must be renumbered so first citations are numerical Provide name, city, state and country of supplier Provide name of kit and name, city, state and country of supplier Provide name, city, state and country of supplier Provide name, city, state and country of supplier Provide names and initials of first 6 authors et al.

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Provide names and initials of first 6 authors et al. Provide names and initials of first 6 authors et al. Provide names and initials of first 6 authors et al. Provide volume number and full page span Provide full page span or state if letter/abstract etc Provide names and initials of first 6 authors et al. Provide full page span or state if letter/abstract etc Provide full page span or state if letter/abstract etc Provide full page span or state if letter/abstract etc Provide names and initials of first 6 authors et al. Provide full page span or state if letter/abstract etc Provide names and initials of first 6 authors et al. Provide full page span or state if letter/abstract etc Provide full page span or state if letter/abstract etc

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Provide full page span or state if letter/abstract etc Provide names and initials of first 6 authors et al. Provide names and initials of first 6 authors et al. Provide names and initials of first 6 authors et al. Provide names and initials of first 6 authors et al. Provide names and initials of first 6 authors et al. Provide full page span or state if letter/abstract etc Replace Pl. in full - plasma?