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Family-based investigation of the C677T polymorphism of the methylenetetrahydrofolate reductase gene in ischaemic heart disease
Atherosclerosis 165 (2002) 293 /299 www.elsevier.com/locate/atherosclerosis
Family-based investigation of the C677T polymorphism of the methylenetetrahydrofolate reductase gene in ischaemic heart disease Mark S. Spence a, Paul G. McGlinchey a, Chris C. Patterson b, Christine Belton c, Gillian Murphy a, Dorothy McMaster c, Damian G. Fogarty c, Alun E. Evans b, Pascal P. McKeown a,c,* a
Regional Medical Cardiology Centre, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA, UK Department of Epidemiology and Public Health, The Queen’s University of Belfast, Mulhouse Building, Grosvenor Road, Belfast, Northern Ireland BT12 6BJ, UK c Department of Medicine, The Queen’s University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast, Northern Ireland BT12 6BJ, UK b
Received 26 February 2002; received in revised form 6 June 2002; accepted 26 June 2002
Abstract Background: Elevated homocysteine is associated with ischaemic heart disease (IHD). The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene results in reduced MTHFR enzyme activity and reduced methylation of homocysteine to methionine resulting in mild hyperhomocysteinaemia. Case-control association studies of the role of the C677T MTHFR polymorphism in IHD have produced conflicting results. We therefore used newly described family-based association tests to investigate the role of this polymorphism in IHD, in a well-defined population. Methods: A total of 352 individuals from 129 families (discordant sibships and parent /child trios) were recruited. Linkage disequilibrium between the polymorphism and IHD was tested for using the combined transmission disequilibrium test (TDT)/sib-TDT and pedigree disequilibrium test (PDT). Homocysteine levels were measured. Results: Both the TDT/sib-TDT and PDT analyses found a significantly reduced transmission of the T allele to affected individuals (P/0.016 and P/0.021). There was no significant difference in homocysteine levels between affected and unaffected siblings. TT homozygotes had mean homocysteine levels significantly higher than those of TC heterozygotes (P B/0.001) and CC homozygotes (P B/0.001). Conclusions: These data suggest that in contrast to the conventional hypothesis the T allele may be protective against IHD, independent of homocysteine levels. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ischaemic heart disease; Methylenetetrahydrofolate reductase; Gene polymorphism
1. Introduction Ischaemic heart disease (IHD) remains a leading cause of morbidity and mortality in the developed world. It is a complex phenotype arising from the interaction of genetic and environmental factors. A genetic component to IHD is suggested by studies that
* Corresponding author. Tel.: /44-28-9024-0503; fax: /44-289031-2907 E-mail address: [email protected] (P.P. McKeown).
have found a family history of IHD to be a strong independent risk factor for IHD, even when other risk factors are controlled for [1,2]. The Swedish twin study also clearly demonstrated that, at younger ages, death from IHD is influenced by genetic factors in both men and women [3]. To date, most researchers investigating the genetic basis of IHD have used case-control association studies. In such studies an allele is said to be associated with the trait if it occurs at a significantly higher frequency among affected individuals as compared with unrelated controls. Polymorphisms in various genes, including the C677T polymorphism in the
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methylenetetrahydrofolate reductase (MTHFR) gene have been reported to be associated with IHD. The MTHFR gene codes for the 5,10-MTHFR enzyme */ this catalyses the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which serves as a methyl donor in the reaction converting homocysteine to methionine. The implication of this polymorphism in the pathogenesis of IHD is based on its involvement in homocysteine metabolism. In 1969, McCully [4] proposed that homocysteine might be involved in the pathogenesis of atherosclerosis in the general population since patients with congenital homocystinuria (characterised by markedly elevated plasma homocysteine levels) developed premature vascular disease. Since then many studies have confirmed that elevated plasma total homocysteine (tHcy) is associated with an increased risk of IHD [5]. About 10 /13% of white populations are homozygous for a C to T substitution at base pair 677 of the MTHFR gene leading to the exchange of an alanine for a valine [6]. This polymorphism results in a thermolabile variant of MTHFR that is associated with decreased enzyme activity [7]. In the presence of suboptimal folate intake homozygous TT individuals have elevated tHcy levels [8]. Given the relationship between this C677T polymorphism of the MTHFR gene and elevated homocysteine levels it has been proposed as a candidate genetic risk factor for cardiovascular disease. However, subsequent case-control studies have shown conflicting results and a recent meta-analysis concluded that although those bearing the TT genotype had higher plasma homocysteine levels than those with the CC genotype they did not have an increased risk of cardiovascular disease [9]. This controversy has resulted in considerable debate as to whether the C677T MTHFR polymorphism is a risk factor for IHD. It is recognised that there are inherent difficulties in studying the genetics of complex diseases. A major problem with case-control studies is that bias may be introduced by unrecognised differences in the samples of cases and controls selected, resulting in erroneous conclusions. In the present study we investigated the presence of linkage disequilibrium between the C677T MTHFR polymorphism and IHD using two recently described family-based association methods in a well-defined Irish population. We used the combined transmission disequilibrium test (TDT)/sib-TDT [10] and pedigree disequilibrium test (PDT) [11]. Both methods have been designed specifically for the study of complex diseases such as IHD, as they overcome the problems of population admixture inherent in traditional case-control methods. We report the first use of these methods in the investigation of IHD. Since this polymorphism is considered to influence the risk of IHD through its effect on homocysteine we also measured homocysteine levels in all patients studied.
2. Methods
2.1. Study population Between August 1999 and October 2000 we recruited 352 individuals from 129 families. All subjects were Caucasian whose parents and grandparents were born in Ireland. Each family was required to have at least one member affected with proven premature IHD (disease onset 0/55 years for males and 0/60 years for females) and at least one surviving unaffected sibling and/or both parents surviving. The affected siblings were recruited from those referred to the cardiology centres in the Royal Victoria Hospital and Belfast City Hospital, Northern Ireland. IHD was defined as the presence of one or more of the following features: (1) a history of acute myocardial infarction (as defined by WHO criteria) [12]; (2) a history of unstable angina (typical chest pain with dynamic ECG changes or minor elevations in cardiac markers); (3) obstructive coronary artery disease angiographically ( E/70% luminal stenosis). For inclusion the unaffected siblings were required to: (1) be older than the affected sibling was at the onset of IHD; (2) have no symptoms of angina or possible myocardial infarction by WHO questionnaire assessment [13]; (3) have no history of IHD diagnosed by a doctor and (4) have a resting 12 lead electrocardiogram showing no evidence of ischaemia or previous myocardial infarction (independently coded using the ‘Minnesota code’ [14], with codes 1.1 /1.2 indicating probable myocardial infarction and codes 1.3, 4.1 /4.4,5.1 /5.3,7.1 indicating possible ischaemia). Parents were not phenotyped for IHD as this is not required for the two family-based association tests we selected. All subjects underwent physical examination including height, weight, and blood pressure measurement. They also provided demographic information and medical history (including IHD risk factors) using standardised questionnaires. The following definitions were used for the IHD risk factors: hypertension, systolic blood pressure greater than 140 mmHg and/or diastolic pressure greater than 90 mmHg or current treatment with antihypertensive drugs; smoking, current smoking or smoking up to 3 months prior to recruitment; hypercholesterolemia, total cholesterol greater than 5.0 mmol/l or current treatment with a cholesterol lowering drug; diabetes mellitus, random blood glucose level greater than 11.1 mmol/l or a previous diagnosis of diabetes mellitus. The study was approved by the Research Ethics Committee of Queen’s University Belfast and fully informed consent was obtained from all subjects.
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2.2. DNA procedures DNA was extracted from peripheral whole blood using a salting out method [15]. The MTHFR genotypes were determined by PCR amplification of the region containing the mutation followed by Hinf I digestion and agarose gel electrophoresis as previously described by Frosst et al. [6]. Genotyping was repeated in 10% of the samples randomly selected as a quality control measure. Each gel was read by 2 observers unaware of the subject’s disease status. 2.3. Homocysteine determination tHcy (free and protein bound) was assayed by high performance liquid chromatography with fluorescent detection as described by Ubbink et al. [16]. 2.4. Statistical analysis We used two recently described family-based association tests, the TDT/sib-TDT and PDT to assess the presence of linkage disequilibrium between the C677T MTHFR polymorphism and IHD. Both the combined TDT/sib-TDT and PDT determine the presence of linkage disequilibrium by testing for unequal transmission of either allele from parents to affected offspring and/or unequal sharing of either allele between discordant sibships. The combined TDT/sib-TDT [10] combines the TDT with the sib-TDT. The TDT tests for preferential transmission of a genetic marker, from a heterozygous parent to their affected offspring. The sib-TDT compares the marker frequency in affected individuals versus their unaffected siblings. Trios (both parents and affected offspring) are informative for the TDT test if there is an affected child, and at least one parent heterozygous at the marker. Disease discordant sibships are informative for the sib-TDT if there is at least one affected and one unaffected sibling with different marker genotypes. Only one trio or discordant sibship was included from each family to ensure the analyses gave valid tests of association. In families with multiple disease discordant sibships the sibship with the maximally discordant genotype was selected as described by Curtis [17]. The TDT/sib-TDT was performed using publicly available software (http://genomics.med.upenn.edu/spielman/tdt.htm) and the results were also verified using a spreadsheet. The PDT [11] is a recently described family-based association test. In contrast to the combined TDT/sibTDT which requires the selection of a single nuclear family from any extended pedigrees when testing for linkage disequilibrium, the PDT was designed to allow the use of data from related trios and discordant sibships from extended pedigrees when testing for
295
linkage disequilibrium. It is also applicable to data consisting of trios without extended pedigrees. Informative extended pedigrees contain at least one informative trio and/or discordant sibship as described for the combined TDT/sib-TDT. This test was performed on our data using publicly available software (http:// www.chg.mc.duke.edu/software/pdt.html) and verified using a spreadsheet. Prospective power calculations for family-based association studies of complex diseases are problematic, as they require a model of inheritance to be specified and the number of families that will be informative can be difficult to predict. We therefore chose to assess power retrospectively. Analysis of variance was used to compare means for quantitative variables and the x2-test was used for qualitative variables. A multiple regression analysis was performed to investigate the effects of various characteristics on homocysteine levels. The distribution of homocysteine was skewed and was logarithmically Table 1 Family structure Structure
Number of families
1 affected sib and 1 unaffected sib 61 only 1 affected sib and 2 unaffected 20 sibs 1 affected child and both parents 13 only (trio) 1 affected sib and 1 unaffected sib 11 and mother 1 affected sib and 1 unaffected sib 4 and both parents 2 affected sibs and 1 unaffected 4 sib 1 affected sib and 3 unaffected 3 sibs 2 affected sibs and 2 unaffected 3 sibs 1 affected sib and 2 unaffected 2 sibs and both parents 1 affected and 4 unaffected sibs 1 1 affected and 1 unaffected sib 1 and father 2 affected sibs and 1 unaffected 1 sib and both parents 2 affected sibs and 1 unaffected 1 sib and father 1 affected sib and 2 unaffected 1 sibs and father 2 affected sibs and 3 unaffected 1 sibs 2 affected sibs and 4 unaffected 1 sibs 3 affected sibs and 1 unaffected 1 sib Total 129
Number of individuals 122 60 39 33 16 12 12 12 10 5 3 5 4 4 5 6 4 352
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296 Table 2 Characteristics of siblings
Age when IHD diagnosed, years (mean9SD) Age at study entry, years (mean9SD) Sex, males % Non-smoker, % Hypertension, % Diabetes mellitus, % Hypercholesterolemia, % Body mass index, kg/m2 (mean9SD) Homocysteine, mmol/l (geometric mean; IR)
Affected siblings (N 142)
Unaffected siblings (N 156)
P
46.096.3 49.597.9 79.6 15.5 28.9 9.2 91.5 28.894.6 9.5; 7.3 /11.5
na 54.698.6 51.9 41.0 46.8 1.9 80.8 28.594.7 8.9; 7.1 /11
na B 0.001 B 0.001 B 0.001 0.001 0.006 0.01 0.57 0.18
na, not applicable; SD, standard deviation; IR, interquartile range.
transformed (base 10) before analysis. Family effects were included in the model and robust standard errors were used to take account of the intra-familial correlation in homocysteine levels. All statistical tests were performed at the 5% significance level (two-tailed).
3. Results Our study sample of 352 individuals was drawn from 129 families. The structure of these families is shown in Table 1. The characteristics of the affected and unaffected siblings are shown in Table 2. The affected siblings were statistically significantly more likely to be male, smokers, suffer from diabetes mellitus and have increased cholesterol compared to the unaffected siblings. In contrast the affected siblings were less likely to have hypertension. For the entire study sample the genotype frequencies were: CC (41.5%), CT (46.3%) and TT (12.2%), giving a T allele frequency of 35.4%. 3.1. Combined TDT/sib-TDT After genotyping and after selection of a single discordant sibship or trio per family 47 discordant sibships and 16 transmissions from 12 trios (a total of 130 individuals) were informative for analysis. There was a statistically significantly less frequent transmission Table 3 Combined TDT/sib-TDT analysis Transmission of T allele to affected individuals
TDT Sib-TDT Combined TDT/sib-TDT
Observed
Expected
5 27 32
8 34.5 42.5
Z 2.41, P 0.016 (two-tailed with continuity correction).
of the T allele to affected individuals (P /0.016, twotailed), as shown in Table 3. 3.2. Pedigree disequilibrium test After genotyping 59 pedigrees (202 individuals) were informative for analysis. There was a deficit of 19 T alleles among the 89 contributing discordant sibships. There was also a deficit of 5 T alleles in the 13 informative trios (including 2 from 1 family). This generates a PDT test statistic of z/2.31 (P /0.021, two-tailed). 3.3. Homocysteine For the sample of all 352 individuals the geometric mean for homocysteine was 9.5 mmol/l (95% confidence intervals; 9.1, 9.9). As shown in Table 2 there was no significant difference in homocysteine levels between affected and unaffected siblings. The homocysteine level according to genotype for the whole population is shown in Table 4. TT homozygous individuals had mean homocysteine levels statistically significantly higher than those of CT heterozygotes (P B/0.001) and CC homozygotes (P B/0.001). A multiple regression analysis as shown in Table 5 determined that TT genotype was associated with significantly increased homocysteine levels. Male sex and increasing age were also associated with increased levels although the latter result just failed to attain significance when robust standard errors were used which took account of intra-familial clustering in homocysteine levels. In contrast IHD phenotype, diabetes mellitus, smoking, hypertension, hypercholesterolemia and obesity had no observable effects.
4. Discussion Using two family-based association tests we have found significantly reduced transmission of the T allele
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Table 4 Plasma homocysteine levels by MTHFR genotype Genotype
Homocysteine, mmol/l geometric mean (95% CI)
CC (N 146)
CT (N 163)
TT (N 43)
9.0 (8.5, 9.5)
9.2 (8.8, 9.8)
12.7 (10.5, 15.3)
CI, confidence intervals. Homocysteine levels for the TT genotype are significantly higher than for the CT (P B 0.001) or CC (P B 0.001) genotypes.
to affected individuals, indicating significant linkage disequilibrium between the wild type (C) variant of the C677T polymorphism of the MTHFR gene and premature IHD. These results suggest that the T allele may be protective against IHD. Despite many studies showing that elevated homocysteine is a risk factor for IHD controversy remains as to whether the C677T polymorphism of the MTHFR gene is a risk factor for IHD. Interest in this polymorphism stems from the observation in most populations studied, including an Irish population similar to ours [18], that those with the TT genotype have higher homocysteine concentrations than those with the CC genotype. Our homocysteine results also confirm this relationship between homocysteine and MTHFR C677T genotype. This is consistent with the observation that the C677T polymorphism encodes a thermolabile variant of MTHFR that has decreased enzyme activity [7]. We undertook our study because of conflicting reports from case-control studies regarding the relationship between the C677T MTHFR polymorphism and cardiovascular disease [9]. Our results are intriguing as they raise the possibility that the T allele is protective against IHD. This is in
contrast to the conventional hypothesis that the C677T polymorphism in the gene encoding the enzyme MTHFR is likely to confer increased risk given its relationship to increased homocysteine levels. There was no statistically significant difference in homocysteine levels between the affected siblings and unaffected siblings in our study. Are our findings biologically plausible? The MTHFR enzyme operates at an important metabolic locus regulating the availability of 1carbon units of folate not only for remethylation of homocysteine, but also for synthesis of thymidine and purines. Reduced MTHFR activity will tend to cause elevated homocysteine levels but also lead to higher availability of folate for DNA synthesis, which could have beneficial effects on the cardiovascular system (e.g. during repair of endothelial damage) [19]. Demuth et al. [20] also found in asymptomatic subjects that elevated homocysteine was associated with lumen enlargement and wall thickening of the carotid artery, both of which are seen in the development of cardiovascular disease. In contrast the TT genotype was associated with a decreased internal diameter of the carotid artery, independent of plasma homocysteine. Moreover a recent study of 68 normal young men, investigating
Table 5 Multiple regression analysis estimating the effects of various characteristics on homocysteine levels* Characteristic
Coefficienta b9SE (b)
Sex Male v Female
0.05190.026
0.05
1.12 (1.00, 1.26)
Age Per decade
0.04990.033
0.07
1.12 (0.99, 1.27)
Genotype TC v CC TT v CC
0.01390.027 0.15390.058
0.69 0.009
1.03 (0.89, 1.20) 1.42 (1.09, 1.85)
Phenotype Unaffected sibs v affected sibs Parents v affected sibs
0.02890.027 0.06390.082
0.31 0.44
0.94 (0.83, 1.06) 0.86 (0.59, 1.26)
Family b
Multiplicative effect (95% CI) 10b (10b1.96 SE (b), 10b1.96 SE (b))
P
B 0.001
* Logarithm base 10 transformation applied to homocysteine levels for regression analysis. Diabetes mellitus, smoking, hypertension, hypercholesterolemia and obesity had no significant effect and were omitted from the model. a Robust standard errors take into account the intrafamily correlations in homocysteine levels. b Family was included as a random effect in the model.
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the impact of the T allele on endothelial function assessed by venous plethysmography found that the CC genotype was associated with a significant impairment in endothelial dependent vasodilatation. The authors suggested that the T allele may protect the endothelium in young normal subjects [21]. Interestingly it has also been reported that TT homozygosity is associated with a lower risk of colon cancer [22]. It is therefore conceivable that the T allele of the C677T MTHFR polymorphism protects against IHD by mechanisms independent of homocysteine. One of the major strengths of our study is its use of family-based association tests which have important advantages over the case-control method used in other studies investigating the role of the C677T polymorphism in IHD. Family-based association tests have been specifically designed for the investigation of complex diseases and avoid the effects of confounding due to population stratification. To our knowledge, this is the first report of the use of family-based association tests to investigate the role of candidate genes involved in susceptibility to IHD. Both the combined TDT/sibTDT and PDT provided consistent results from our data. Part of the rationale for the development of the PDT was to increase the utilisation of individuals from extended pedigrees compared to the combined TDT/sibTDT. In our study after genotyping, 202 individuals were informative for the PDT analysis compared to 130 for the combined TDT/sib-TDT. However, in spite of allowing utilisation of more subjects this did not result in a reduction in the P value for the PDT analysis. Our findings indicate significant linkage disequilibrium between the wild type (C) variant of the C677T polymorphism of the MTHFR gene and premature IHD. It is possible that it is not this wild type variant of the C677T polymorphism that is in linkage disequilibrium with IHD but rather an as yet unidentified region with which it is in close proximity. It is also possible that our results represent a type 1 (false positive) error and similar family-based association studies should be replicated in other independent populations. However, using the method described by Spielman and Ewens [10] for the combined TDT/sibTDT we retrospectively estimate that, a sample of 62 minimally informative families has over 90% power to detect as significant (P B/0.05; two-tailed) a deviation from 50 to 70% in the rate of transmission of an allele to affected individuals. In conclusion, we report the first application of family-based association tests, the combined TDT/sibTDT and PDT to the investigation of susceptibility genes for IHD. We investigated the C677T polymorphism of the MTHFR gene and demonstrated less frequent transmission of the T allele to individuals affected with IHD. The TT genotype was associated with increased homocysteine levels. In contrast to the conventional
hypothesis this suggests that in our study population the T allele may be protective against IHD, independent of homocysteine.
Acknowledgements This research was supported by a Royal Victoria Hospital Research Fellowship (MS), the Northern Ireland Chest, Heart and Stroke Association, the Research and Development Office, Northern Ireland and the Heart Trust Fund (Royal Victoria Hospital). We thank Judith Troughton (Department of Medicine, Queen’s University Belfast) who performed the Homocysteine measurements.
References [1] Shea S, Ottman R, Gabrieli C, Stein Z, Nichols A. Family history as an independent risk factor for coronary artery disease. J Am Coll Cardiol 1984;4:793 /801. [2] Colditz GA, Rimm EB, Giovannucci E, Stampfer MJ, Rosner B, Willett WC. A prospective study of parental history of myocardial infarction and coronary artery disease in men. Am J Cardiol 1991;67:933 /8. [3] Marenberg ME, Risch N, Berkman LF, Floderus B, de Faire U. Genetic susceptibility to death from coronary heart disease in a study of twins. N Engl J Med 1994;330:1041 /6. [4] McCully KS. Vascular pathology of homocysteinemia: implications for the pathogenesis of arteriosclerosis. Am J Pathol 1969;56:111 /28. [5] Danesh J, Lewington S. Plasma homocysteine and coronary heart disease: systematic review of published epidemiological studies. J Cardiovasc Risk 1998;5:229 /32. [6] Frosst P, Blom HJ, Milos R, et al. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111 /3. [7] Kang SS, Zhou J, Wong PWK, Kowalisyn J, Strokosch G. Intermediate homocysteinemia: a thermolabile variant of methylenetetrahydrofolate reductase. Am J Hum Genet 1988;43:414 / 21. [8] Malinow MR, Nieto FJ, Kruger WD, et al. The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylenetetrahydrofolate reductase genotypes. Arterioscler Thromb Vasc Biol 1997;17:1157 /62. ¨ hrvik J, Brudin L. Common [9] Brattstro¨m L, Wilcken DEL, .O methylenetetrahydrofolate reductase gene mutation leads to hyperhomocysteinemia but not to vascular disease: the result of a meta-analysis. Circulation 1998;98:2520 /6. [10] Spielman RS, Ewens WJ. A sibship test for linkage in the presence of association: the sib transmission/disequilibrium test. Am J Hum Genet 1998;62:450 /8. [11] Martin ER, Monks SA, Warren LL, Kaplan NL. A test for linkage and association in general pedigrees: the pedigree disequilibrium test. Am J Hum Genet 2000;67:146 /54. [12] Nomenclature and criteria for diagnosis of ischaemic heart disease. Report of the Joint International Society and Federation of Cardiology/World Health Organisation task force on standardization of clinical nomenclature. Circulation 1979; 59: 607 /9. [13] Rose GA, Blackburn H, Gillum RF, Prineas RJ. Cardiovascular Survey Methods (2nd ed.): World Health Organisation monograph series no. 56; 1982.
M.S. Spence et al. / Atherosclerosis 165 (2002) 293 /299 [14] Blackburn H, Keys A, Simonson E, Rautaharju P, Punsar S. The electrocardiogram in population studies: a classification system. Circulation 1960;21:1160 /75. [15] Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. [16] Ubbink JB, Vermaak WJH, Bissbort S. Rapid high-performance liquid chromatographic assay for total homocysteine levels in human serum. J Chromatogr 1991;565:441 /6. [17] Curtis D. Use of siblings as controls in case-control association studies. Am J Hum Gen 1997;61:319 /33. [18] Harmon DL, Woodside JV, Yarnell JWG, et al. The common ‘thermolabile’ variant of methylenetetrahydrofolate reductase is a major determinant of mild hyperhomocysteinaemia. Q J Med 1996;89:571 /7.
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[19] Hankey GJ, Eikelboom JW. Homocysteine and vascular disease. Lancet 1999;354:407 /13. [20] Demuth K, Moatti N, Hanon O, Benoit MO, Safar M, Girerd X. Opposite effects of plasma homocysteine and the methylenetetrahydrofolate reductase C677T mutation on carotid artery geometry in asymptomatic adults. Arterioscler Thromb Vasc Biol 1998;18:1838 /43. [21] Butler R, Morris AD, Struthers AD. The T allele of the C677T 5,10-methylenetetrahydrofolate reductase (MTHFR) gene polymorphism may protect endothelial function in young, normal subjects. Arterioscler Thromb Vasc Biol 2002;22:193 /4. [22] Chen J, Giovannucci EL, Hunter DJ. MTHFR polymorphism, methyl-replete diets and the risk of colorectal carcinoma and adenoma among US men and women: an example of geneenvironment interactions in colorectal tumorigenesis. J Nutr 1999;129:560S /4S.
Family-based investigation of the C677T polymorphism of the methylenetetrahydrofolate reductase gene in ischaemic heart disease Mark S. Spence a, Paul G. McGlinchey a, Chris C. Patterson b, Christine Belton c, Gillian Murphy a, Dorothy McMaster c, Damian G. Fogarty c, Alun E. Evans b, Pascal P. McKeown a,c,* a
Regional Medical Cardiology Centre, Royal Victoria Hospital, Grosvenor Road, Belfast BT12 6BA, UK Department of Epidemiology and Public Health, The Queen’s University of Belfast, Mulhouse Building, Grosvenor Road, Belfast, Northern Ireland BT12 6BJ, UK c Department of Medicine, The Queen’s University of Belfast, Institute of Clinical Science, Grosvenor Road, Belfast, Northern Ireland BT12 6BJ, UK b
Received 26 February 2002; received in revised form 6 June 2002; accepted 26 June 2002
Abstract Background: Elevated homocysteine is associated with ischaemic heart disease (IHD). The C677T polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene results in reduced MTHFR enzyme activity and reduced methylation of homocysteine to methionine resulting in mild hyperhomocysteinaemia. Case-control association studies of the role of the C677T MTHFR polymorphism in IHD have produced conflicting results. We therefore used newly described family-based association tests to investigate the role of this polymorphism in IHD, in a well-defined population. Methods: A total of 352 individuals from 129 families (discordant sibships and parent /child trios) were recruited. Linkage disequilibrium between the polymorphism and IHD was tested for using the combined transmission disequilibrium test (TDT)/sib-TDT and pedigree disequilibrium test (PDT). Homocysteine levels were measured. Results: Both the TDT/sib-TDT and PDT analyses found a significantly reduced transmission of the T allele to affected individuals (P/0.016 and P/0.021). There was no significant difference in homocysteine levels between affected and unaffected siblings. TT homozygotes had mean homocysteine levels significantly higher than those of TC heterozygotes (P B/0.001) and CC homozygotes (P B/0.001). Conclusions: These data suggest that in contrast to the conventional hypothesis the T allele may be protective against IHD, independent of homocysteine levels. # 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ischaemic heart disease; Methylenetetrahydrofolate reductase; Gene polymorphism
1. Introduction Ischaemic heart disease (IHD) remains a leading cause of morbidity and mortality in the developed world. It is a complex phenotype arising from the interaction of genetic and environmental factors. A genetic component to IHD is suggested by studies that
* Corresponding author. Tel.: /44-28-9024-0503; fax: /44-289031-2907 E-mail address: [email protected] (P.P. McKeown).
have found a family history of IHD to be a strong independent risk factor for IHD, even when other risk factors are controlled for [1,2]. The Swedish twin study also clearly demonstrated that, at younger ages, death from IHD is influenced by genetic factors in both men and women [3]. To date, most researchers investigating the genetic basis of IHD have used case-control association studies. In such studies an allele is said to be associated with the trait if it occurs at a significantly higher frequency among affected individuals as compared with unrelated controls. Polymorphisms in various genes, including the C677T polymorphism in the
0021-9150/02/$ - see front matter # 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0 0 2 1 - 9 1 5 0 ( 0 2 ) 0 0 2 3 9 - 3
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methylenetetrahydrofolate reductase (MTHFR) gene have been reported to be associated with IHD. The MTHFR gene codes for the 5,10-MTHFR enzyme */ this catalyses the irreversible conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which serves as a methyl donor in the reaction converting homocysteine to methionine. The implication of this polymorphism in the pathogenesis of IHD is based on its involvement in homocysteine metabolism. In 1969, McCully [4] proposed that homocysteine might be involved in the pathogenesis of atherosclerosis in the general population since patients with congenital homocystinuria (characterised by markedly elevated plasma homocysteine levels) developed premature vascular disease. Since then many studies have confirmed that elevated plasma total homocysteine (tHcy) is associated with an increased risk of IHD [5]. About 10 /13% of white populations are homozygous for a C to T substitution at base pair 677 of the MTHFR gene leading to the exchange of an alanine for a valine [6]. This polymorphism results in a thermolabile variant of MTHFR that is associated with decreased enzyme activity [7]. In the presence of suboptimal folate intake homozygous TT individuals have elevated tHcy levels [8]. Given the relationship between this C677T polymorphism of the MTHFR gene and elevated homocysteine levels it has been proposed as a candidate genetic risk factor for cardiovascular disease. However, subsequent case-control studies have shown conflicting results and a recent meta-analysis concluded that although those bearing the TT genotype had higher plasma homocysteine levels than those with the CC genotype they did not have an increased risk of cardiovascular disease [9]. This controversy has resulted in considerable debate as to whether the C677T MTHFR polymorphism is a risk factor for IHD. It is recognised that there are inherent difficulties in studying the genetics of complex diseases. A major problem with case-control studies is that bias may be introduced by unrecognised differences in the samples of cases and controls selected, resulting in erroneous conclusions. In the present study we investigated the presence of linkage disequilibrium between the C677T MTHFR polymorphism and IHD using two recently described family-based association methods in a well-defined Irish population. We used the combined transmission disequilibrium test (TDT)/sib-TDT [10] and pedigree disequilibrium test (PDT) [11]. Both methods have been designed specifically for the study of complex diseases such as IHD, as they overcome the problems of population admixture inherent in traditional case-control methods. We report the first use of these methods in the investigation of IHD. Since this polymorphism is considered to influence the risk of IHD through its effect on homocysteine we also measured homocysteine levels in all patients studied.
2. Methods
2.1. Study population Between August 1999 and October 2000 we recruited 352 individuals from 129 families. All subjects were Caucasian whose parents and grandparents were born in Ireland. Each family was required to have at least one member affected with proven premature IHD (disease onset 0/55 years for males and 0/60 years for females) and at least one surviving unaffected sibling and/or both parents surviving. The affected siblings were recruited from those referred to the cardiology centres in the Royal Victoria Hospital and Belfast City Hospital, Northern Ireland. IHD was defined as the presence of one or more of the following features: (1) a history of acute myocardial infarction (as defined by WHO criteria) [12]; (2) a history of unstable angina (typical chest pain with dynamic ECG changes or minor elevations in cardiac markers); (3) obstructive coronary artery disease angiographically ( E/70% luminal stenosis). For inclusion the unaffected siblings were required to: (1) be older than the affected sibling was at the onset of IHD; (2) have no symptoms of angina or possible myocardial infarction by WHO questionnaire assessment [13]; (3) have no history of IHD diagnosed by a doctor and (4) have a resting 12 lead electrocardiogram showing no evidence of ischaemia or previous myocardial infarction (independently coded using the ‘Minnesota code’ [14], with codes 1.1 /1.2 indicating probable myocardial infarction and codes 1.3, 4.1 /4.4,5.1 /5.3,7.1 indicating possible ischaemia). Parents were not phenotyped for IHD as this is not required for the two family-based association tests we selected. All subjects underwent physical examination including height, weight, and blood pressure measurement. They also provided demographic information and medical history (including IHD risk factors) using standardised questionnaires. The following definitions were used for the IHD risk factors: hypertension, systolic blood pressure greater than 140 mmHg and/or diastolic pressure greater than 90 mmHg or current treatment with antihypertensive drugs; smoking, current smoking or smoking up to 3 months prior to recruitment; hypercholesterolemia, total cholesterol greater than 5.0 mmol/l or current treatment with a cholesterol lowering drug; diabetes mellitus, random blood glucose level greater than 11.1 mmol/l or a previous diagnosis of diabetes mellitus. The study was approved by the Research Ethics Committee of Queen’s University Belfast and fully informed consent was obtained from all subjects.
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2.2. DNA procedures DNA was extracted from peripheral whole blood using a salting out method [15]. The MTHFR genotypes were determined by PCR amplification of the region containing the mutation followed by Hinf I digestion and agarose gel electrophoresis as previously described by Frosst et al. [6]. Genotyping was repeated in 10% of the samples randomly selected as a quality control measure. Each gel was read by 2 observers unaware of the subject’s disease status. 2.3. Homocysteine determination tHcy (free and protein bound) was assayed by high performance liquid chromatography with fluorescent detection as described by Ubbink et al. [16]. 2.4. Statistical analysis We used two recently described family-based association tests, the TDT/sib-TDT and PDT to assess the presence of linkage disequilibrium between the C677T MTHFR polymorphism and IHD. Both the combined TDT/sib-TDT and PDT determine the presence of linkage disequilibrium by testing for unequal transmission of either allele from parents to affected offspring and/or unequal sharing of either allele between discordant sibships. The combined TDT/sib-TDT [10] combines the TDT with the sib-TDT. The TDT tests for preferential transmission of a genetic marker, from a heterozygous parent to their affected offspring. The sib-TDT compares the marker frequency in affected individuals versus their unaffected siblings. Trios (both parents and affected offspring) are informative for the TDT test if there is an affected child, and at least one parent heterozygous at the marker. Disease discordant sibships are informative for the sib-TDT if there is at least one affected and one unaffected sibling with different marker genotypes. Only one trio or discordant sibship was included from each family to ensure the analyses gave valid tests of association. In families with multiple disease discordant sibships the sibship with the maximally discordant genotype was selected as described by Curtis [17]. The TDT/sib-TDT was performed using publicly available software (http://genomics.med.upenn.edu/spielman/tdt.htm) and the results were also verified using a spreadsheet. The PDT [11] is a recently described family-based association test. In contrast to the combined TDT/sibTDT which requires the selection of a single nuclear family from any extended pedigrees when testing for linkage disequilibrium, the PDT was designed to allow the use of data from related trios and discordant sibships from extended pedigrees when testing for
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linkage disequilibrium. It is also applicable to data consisting of trios without extended pedigrees. Informative extended pedigrees contain at least one informative trio and/or discordant sibship as described for the combined TDT/sib-TDT. This test was performed on our data using publicly available software (http:// www.chg.mc.duke.edu/software/pdt.html) and verified using a spreadsheet. Prospective power calculations for family-based association studies of complex diseases are problematic, as they require a model of inheritance to be specified and the number of families that will be informative can be difficult to predict. We therefore chose to assess power retrospectively. Analysis of variance was used to compare means for quantitative variables and the x2-test was used for qualitative variables. A multiple regression analysis was performed to investigate the effects of various characteristics on homocysteine levels. The distribution of homocysteine was skewed and was logarithmically Table 1 Family structure Structure
Number of families
1 affected sib and 1 unaffected sib 61 only 1 affected sib and 2 unaffected 20 sibs 1 affected child and both parents 13 only (trio) 1 affected sib and 1 unaffected sib 11 and mother 1 affected sib and 1 unaffected sib 4 and both parents 2 affected sibs and 1 unaffected 4 sib 1 affected sib and 3 unaffected 3 sibs 2 affected sibs and 2 unaffected 3 sibs 1 affected sib and 2 unaffected 2 sibs and both parents 1 affected and 4 unaffected sibs 1 1 affected and 1 unaffected sib 1 and father 2 affected sibs and 1 unaffected 1 sib and both parents 2 affected sibs and 1 unaffected 1 sib and father 1 affected sib and 2 unaffected 1 sibs and father 2 affected sibs and 3 unaffected 1 sibs 2 affected sibs and 4 unaffected 1 sibs 3 affected sibs and 1 unaffected 1 sib Total 129
Number of individuals 122 60 39 33 16 12 12 12 10 5 3 5 4 4 5 6 4 352
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296 Table 2 Characteristics of siblings
Age when IHD diagnosed, years (mean9SD) Age at study entry, years (mean9SD) Sex, males % Non-smoker, % Hypertension, % Diabetes mellitus, % Hypercholesterolemia, % Body mass index, kg/m2 (mean9SD) Homocysteine, mmol/l (geometric mean; IR)
Affected siblings (N 142)
Unaffected siblings (N 156)
P
46.096.3 49.597.9 79.6 15.5 28.9 9.2 91.5 28.894.6 9.5; 7.3 /11.5
na 54.698.6 51.9 41.0 46.8 1.9 80.8 28.594.7 8.9; 7.1 /11
na B 0.001 B 0.001 B 0.001 0.001 0.006 0.01 0.57 0.18
na, not applicable; SD, standard deviation; IR, interquartile range.
transformed (base 10) before analysis. Family effects were included in the model and robust standard errors were used to take account of the intra-familial correlation in homocysteine levels. All statistical tests were performed at the 5% significance level (two-tailed).
3. Results Our study sample of 352 individuals was drawn from 129 families. The structure of these families is shown in Table 1. The characteristics of the affected and unaffected siblings are shown in Table 2. The affected siblings were statistically significantly more likely to be male, smokers, suffer from diabetes mellitus and have increased cholesterol compared to the unaffected siblings. In contrast the affected siblings were less likely to have hypertension. For the entire study sample the genotype frequencies were: CC (41.5%), CT (46.3%) and TT (12.2%), giving a T allele frequency of 35.4%. 3.1. Combined TDT/sib-TDT After genotyping and after selection of a single discordant sibship or trio per family 47 discordant sibships and 16 transmissions from 12 trios (a total of 130 individuals) were informative for analysis. There was a statistically significantly less frequent transmission Table 3 Combined TDT/sib-TDT analysis Transmission of T allele to affected individuals
TDT Sib-TDT Combined TDT/sib-TDT
Observed
Expected
5 27 32
8 34.5 42.5
Z 2.41, P 0.016 (two-tailed with continuity correction).
of the T allele to affected individuals (P /0.016, twotailed), as shown in Table 3. 3.2. Pedigree disequilibrium test After genotyping 59 pedigrees (202 individuals) were informative for analysis. There was a deficit of 19 T alleles among the 89 contributing discordant sibships. There was also a deficit of 5 T alleles in the 13 informative trios (including 2 from 1 family). This generates a PDT test statistic of z/2.31 (P /0.021, two-tailed). 3.3. Homocysteine For the sample of all 352 individuals the geometric mean for homocysteine was 9.5 mmol/l (95% confidence intervals; 9.1, 9.9). As shown in Table 2 there was no significant difference in homocysteine levels between affected and unaffected siblings. The homocysteine level according to genotype for the whole population is shown in Table 4. TT homozygous individuals had mean homocysteine levels statistically significantly higher than those of CT heterozygotes (P B/0.001) and CC homozygotes (P B/0.001). A multiple regression analysis as shown in Table 5 determined that TT genotype was associated with significantly increased homocysteine levels. Male sex and increasing age were also associated with increased levels although the latter result just failed to attain significance when robust standard errors were used which took account of intra-familial clustering in homocysteine levels. In contrast IHD phenotype, diabetes mellitus, smoking, hypertension, hypercholesterolemia and obesity had no observable effects.
4. Discussion Using two family-based association tests we have found significantly reduced transmission of the T allele
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Table 4 Plasma homocysteine levels by MTHFR genotype Genotype
Homocysteine, mmol/l geometric mean (95% CI)
CC (N 146)
CT (N 163)
TT (N 43)
9.0 (8.5, 9.5)
9.2 (8.8, 9.8)
12.7 (10.5, 15.3)
CI, confidence intervals. Homocysteine levels for the TT genotype are significantly higher than for the CT (P B 0.001) or CC (P B 0.001) genotypes.
to affected individuals, indicating significant linkage disequilibrium between the wild type (C) variant of the C677T polymorphism of the MTHFR gene and premature IHD. These results suggest that the T allele may be protective against IHD. Despite many studies showing that elevated homocysteine is a risk factor for IHD controversy remains as to whether the C677T polymorphism of the MTHFR gene is a risk factor for IHD. Interest in this polymorphism stems from the observation in most populations studied, including an Irish population similar to ours [18], that those with the TT genotype have higher homocysteine concentrations than those with the CC genotype. Our homocysteine results also confirm this relationship between homocysteine and MTHFR C677T genotype. This is consistent with the observation that the C677T polymorphism encodes a thermolabile variant of MTHFR that has decreased enzyme activity [7]. We undertook our study because of conflicting reports from case-control studies regarding the relationship between the C677T MTHFR polymorphism and cardiovascular disease [9]. Our results are intriguing as they raise the possibility that the T allele is protective against IHD. This is in
contrast to the conventional hypothesis that the C677T polymorphism in the gene encoding the enzyme MTHFR is likely to confer increased risk given its relationship to increased homocysteine levels. There was no statistically significant difference in homocysteine levels between the affected siblings and unaffected siblings in our study. Are our findings biologically plausible? The MTHFR enzyme operates at an important metabolic locus regulating the availability of 1carbon units of folate not only for remethylation of homocysteine, but also for synthesis of thymidine and purines. Reduced MTHFR activity will tend to cause elevated homocysteine levels but also lead to higher availability of folate for DNA synthesis, which could have beneficial effects on the cardiovascular system (e.g. during repair of endothelial damage) [19]. Demuth et al. [20] also found in asymptomatic subjects that elevated homocysteine was associated with lumen enlargement and wall thickening of the carotid artery, both of which are seen in the development of cardiovascular disease. In contrast the TT genotype was associated with a decreased internal diameter of the carotid artery, independent of plasma homocysteine. Moreover a recent study of 68 normal young men, investigating
Table 5 Multiple regression analysis estimating the effects of various characteristics on homocysteine levels* Characteristic
Coefficienta b9SE (b)
Sex Male v Female
0.05190.026
0.05
1.12 (1.00, 1.26)
Age Per decade
0.04990.033
0.07
1.12 (0.99, 1.27)
Genotype TC v CC TT v CC
0.01390.027 0.15390.058
0.69 0.009
1.03 (0.89, 1.20) 1.42 (1.09, 1.85)
Phenotype Unaffected sibs v affected sibs Parents v affected sibs
0.02890.027 0.06390.082
0.31 0.44
0.94 (0.83, 1.06) 0.86 (0.59, 1.26)
Family b
Multiplicative effect (95% CI) 10b (10b1.96 SE (b), 10b1.96 SE (b))
P
B 0.001
* Logarithm base 10 transformation applied to homocysteine levels for regression analysis. Diabetes mellitus, smoking, hypertension, hypercholesterolemia and obesity had no significant effect and were omitted from the model. a Robust standard errors take into account the intrafamily correlations in homocysteine levels. b Family was included as a random effect in the model.
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the impact of the T allele on endothelial function assessed by venous plethysmography found that the CC genotype was associated with a significant impairment in endothelial dependent vasodilatation. The authors suggested that the T allele may protect the endothelium in young normal subjects [21]. Interestingly it has also been reported that TT homozygosity is associated with a lower risk of colon cancer [22]. It is therefore conceivable that the T allele of the C677T MTHFR polymorphism protects against IHD by mechanisms independent of homocysteine. One of the major strengths of our study is its use of family-based association tests which have important advantages over the case-control method used in other studies investigating the role of the C677T polymorphism in IHD. Family-based association tests have been specifically designed for the investigation of complex diseases and avoid the effects of confounding due to population stratification. To our knowledge, this is the first report of the use of family-based association tests to investigate the role of candidate genes involved in susceptibility to IHD. Both the combined TDT/sibTDT and PDT provided consistent results from our data. Part of the rationale for the development of the PDT was to increase the utilisation of individuals from extended pedigrees compared to the combined TDT/sibTDT. In our study after genotyping, 202 individuals were informative for the PDT analysis compared to 130 for the combined TDT/sib-TDT. However, in spite of allowing utilisation of more subjects this did not result in a reduction in the P value for the PDT analysis. Our findings indicate significant linkage disequilibrium between the wild type (C) variant of the C677T polymorphism of the MTHFR gene and premature IHD. It is possible that it is not this wild type variant of the C677T polymorphism that is in linkage disequilibrium with IHD but rather an as yet unidentified region with which it is in close proximity. It is also possible that our results represent a type 1 (false positive) error and similar family-based association studies should be replicated in other independent populations. However, using the method described by Spielman and Ewens [10] for the combined TDT/sibTDT we retrospectively estimate that, a sample of 62 minimally informative families has over 90% power to detect as significant (P B/0.05; two-tailed) a deviation from 50 to 70% in the rate of transmission of an allele to affected individuals. In conclusion, we report the first application of family-based association tests, the combined TDT/sibTDT and PDT to the investigation of susceptibility genes for IHD. We investigated the C677T polymorphism of the MTHFR gene and demonstrated less frequent transmission of the T allele to individuals affected with IHD. The TT genotype was associated with increased homocysteine levels. In contrast to the conventional
hypothesis this suggests that in our study population the T allele may be protective against IHD, independent of homocysteine.
Acknowledgements This research was supported by a Royal Victoria Hospital Research Fellowship (MS), the Northern Ireland Chest, Heart and Stroke Association, the Research and Development Office, Northern Ireland and the Heart Trust Fund (Royal Victoria Hospital). We thank Judith Troughton (Department of Medicine, Queen’s University Belfast) who performed the Homocysteine measurements.
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