Heterozygote advantage of the MTHFR gene in patients with neural-tube defect and their relatives
Authors: LR Weitkamp, DC Tackels, AGW Hunter, LB Holmes, CE Schwartz
Journal: The Lancet (1998)

The C677T mutation in the 5,10-methylene tetrahydrofolate reductase gene (MTHFR) yields a common thermolabile variant (T) with 30–50% activity of the allelic form (C). In an analysis of published association studies, patients with a neural-tube defect (NTD) were almost twice as likely to have the T/T genotype as were controls (odds ratio 1·9, 95% CI 1·3–2·8).1van der Put NMJ Eskes TKAB Blom HJ Is the common 677T mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects?.Q J Med. 1997; 90: 111-115Crossref Scopus (219) Google Scholar Initial studies of association between NTD and T/T were criticised on the grounds that controls were not matched properly to patients.2Papapetrou C Lynch SA Burn J Edwards YH Methylenetetrahydrofolate reductase and neural tube defects.Lancet. 1996; 348: 58Summary Full Text Full Text PDF PubMed Scopus (81) Google Scholar Within-family gene distribution analyses avoid the risk of population stratification inherent in association studies since observations are compared with mendelian expectations. In the only such study reported for NTD and MTHFR, C/T heterozygous parents transmitted equal numbers of C and T alleles (12:12) to offspring with NTD, consistent with the hypothesis of no association between the T allele and NTD.2Papapetrou C Lynch SA Burn J Edwards YH Methylenetetrahydrofolate reductase and neural tube defects.Lancet. 1996; 348: 58Summary Full Text Full Text PDF PubMed Scopus (81) Google Scholar From 1988 to 1990, we enrolled 168 families with a NTD pregnancy from catchment areas centred in Boston and in Ottawa (81 anencephaly, 1 encephalcoele, 86 spina bifida). The ethnic origins of the 672 grandparents of probands were 620 European, 47 Native American, three African American, and two Asian. Patients with spina bifida (83), mothers (167), fathers (168), and unaffected siblings (182) were typed for the MTHFR locus3Ou CY Stevenson RE Brown VK et al.5,10 methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects.Am J Med Genet. 1996; 63: 610-614Crossref PubMed Scopus (205) Google Scholar blind to diagnoses, sex, and pedigree relationships. Parentage was confirmed by typing other genetic markers.The table shows the number of offspring of each genotype according to sex and diagnosis. The allele transmission ratio from C/T heterozygous parents was 39C:48T to offspring with NTD χ12=0–93, p=0·33) and 94C:100T to unaffected offspring (χ12=0–19, p=0·66), both close to the 1:1 Mendelian expectation. However, among all 206 offspring of these parents the number of offspring of each genotype (C/C, C/T, or T/T) differed from mendelian expectations (χ22= 18·05, p=0·00012). The difference resulted from an excess of heterozygotes (136 observed, 105·5 expected) and a deficiency of C/C homozygotes (57 observed, 74·0 expected) and T/T homozygotes (18 observed, 29·5 expected). The results were significant both for the 65 spina bifida offspring (χ22=9–66, p=0·0080) and for the 146 unaffected offspring (χ22=9–28, p=0·0097). Taking into account sex, increased heterozygosity was observed in females with NTD, but not in males with NTD. On the other hand, among unaffected offspring, an excess of heterozygotes and a deficiency of homozygotes was observed for males and not for females. Tests for Hardy-Weinberg equilibrium (a population rather than family-based analysis) yielded similar levels of significance for the genotype frequency distortions in the offspring (see table).TableMTHFR mating types of 167 couples ascertained for a child with NTD, genotypes of 3 of their surviving affected offspring, and 181 unaffected offspringMating typeNumber of matings†One father (genotype C/T) is not shown in the table because his spouse was not typed.Number of offspring with genotype*Expected values in parenthesis were based on Mendelian probabilities for gene transmission from heterozygous parents (or from all parents in the totals). Expected values in brackets were calculated assuming Hardy-Weinberg equilibrium for each group (spina bifida male, spina bifida female, unaffected male, unaffected female). Chi-square analysis the totals for each group compared with expectation based on Mendelian gene transmission form heterozygous parents or Hardy-Weinberg equilibrium, respectively, were: spina bifida male (X2201, p=0·60;X22=0·30, p=0·58); spina bifida female (X22=10·07, p=0·0065;X22=6·41, p=0·011); unaffected male (X22=9·58, p=0·0083;X22 =8·79, p=0·0030);unaffected female (X22=2·14, p=0·34;X22=0·58, p=0·45).PatMatSpina bifidaUnaffectedMaleFemaleMaleFemaleC/CC/TT/TC/CC/TT/TC/CC/TT/TC/CC/TT/TC/CT/T30021T/TC/C41123C/CC/T211 (2·0)3 (2·0)1 (3·0)5 (3·0)4 (5·5)7 (5·5)5 (5·0)5 (5·0)C/TC/C484 (4·5)5 (4·5)2 (8·5)15 (8·5)15 (16·0)17 (16·0)11 (14·017 (14·0)C/TC/T442 (2·0)6 (4·0)0 (2·0)5 (3·5)9 (7·0)0 (3·5)3 (5·75)18 (11·5)2 (5·75)4 (6·25)16 (12·5)5 (6·25)C/CC/C277911014C/TT/T100 (1·5)3 (1·5)1 (1·0)1 (1·0)4 (2·0)0 (2·0)3 (5·5)4 (3·5)T/TC/T81 (0·5)0 (0·5)0 (0·5)1 (0·5)3 (2·0)1 (2·0)1 (1·0)1 (1·0)T/TT/T200a11Totals16714 (15·5) [14·7]16 (13·5) [14·6]3 (4·0) [3·7]17 (24·0) [21·1]31 (21·0) [22·8]2 (5·0) [6·1]33 (38·25) [39·3]53 (41·0) [40·4]4 (10·75) [10·3]34 (39·25) [35·7]46 (40·) [42·6]11 [12·7]* Expected values in parenthesis were based on Mendelian probabilities for gene transmission from heterozygous parents (or from all parents in the totals). Expected values in brackets were calculated assuming Hardy-Weinberg equilibrium for each group (spina bifida male, spina bifida female, unaffected male, unaffected female). Chi-square analysis the totals for each group compared with expectation based on Mendelian gene transmission form heterozygous parents or Hardy-Weinberg equilibrium, respectively, were: spina bifida male (X2201, p=0·60;X22=0·30, p=0·58); spina bifida female (X22=10·07, p=0·0065;X22=6·41, p=0·011); unaffected male (X22=9·58, p=0·0083;X22 =8·79, p=0·0030);unaffected female (X22=2·14, p=0·34;X22=0·58, p=0·45).† One father (genotype C/T) is not shown in the table because his spouse was not typed. Open table in a new tab To determine whether the sex difference in heterozygosity in unaffected offspring occurred in other first-degree relatives, we compared the distribution of genotypes in the fathers and mothers of the NTD probands with Hardy-Weinberg expectation. Among mothers there were 79 C/C, 73 C/T, and 15 T/T, compared with 79·88 C/C, 71·24 C/T, and 15·88 T/T expected (x^O-10, p=0·75). Thus, mothers and 91 unaffected sisters of NTD subjects combined showed no difference in genotype ratios from Hardy-Weinberg expectation (χ12=0–43, p=0·51). There were 51 C/C, 103 C/T, and 14 T/T fathers compared with 62·54 C/C, 79·92 C/T, and 25·54 T/T expected (χ12= 14·01, p=0·00018). The excess of heterozygotes among 168 fathers (29%) and 90 brothers (29%) was similar and, when the data for fathers and brothers were combined, highly significant (χ12=22–30, p=0·000002).Male NTD fetuses are thought to have a higher abortion rate than females.4Elwood JM Little J Elwood JH Epidemiology and control of neural tube defects. Oxford University Press, Oxford1992Google Scholar Virtually all of the 50% excess of female over male NTD subjects can be accounted for by an excess of C/T genotypes (31 of 50 females versus 16 of 33 males). Thus, C/T females with NTD may be less likely to abort than C/T males with NTD. Our data also suggest an interactive effect between maternal and fetal MTHFR genes in survival (eg, the large excess of C/T in NTD females from C/C mothers). If MTHFR genotype affects fetal survival, a possible explanation for the discrepancy between the results of our study and the association studies could be that the latter may have included more severely affected patients or abortuses.NTD, the most common malformation among abortuses, has a multifactorial aetiology with polygenetic susceptibility and environmental risk factors, including folate.4Elwood JM Little J Elwood JH Epidemiology and control of neural tube defects. Oxford University Press, Oxford1992Google Scholar On average, half of the NTD susceptibility alleles present in individuals with NTD are present in a parent or sibling. The highly significant excess of C/T heterozygosity in male firstdegree relatives of patients with NTD demonstrates C/T heterozygote advantage in the context of their genetic background. These results raise the issue of how selection acts on functionally different MTHFR alleles to produce, in families selected for a member with NTD, an excess of heterozygotes among affected females and unaffected males.We thank the families who participated in this study; Peter Rowley, Carole Ober, and Roger Stevenson for helpful criticism; Sally Guttormsen, Linda Kemp, Susan Tan Torres, Gollu Ramaurthy, Y-Z Du, Patricia Weill, and Luciana Yu for technical and clinical assistance; and Mary True for help in preparing the manuscript. This research was supported in part by US Public Health Service grant HD-24560, the South Carolina Department of Disabilities and Special Needs grant SG97–88, and US Public Health Service (CDC) grant U85/CCU408774. The C677T mutation in the 5,10-methylene tetrahydrofolate reductase gene (MTHFR) yields a common thermolabile variant (T) with 30–50% activity of the allelic form (C). In an analysis of published association studies, patients with a neural-tube defect (NTD) were almost twice as likely to have the T/T genotype as were controls (odds ratio 1·9, 95% CI 1·3–2·8).1van der Put NMJ Eskes TKAB Blom HJ Is the common 677T mutation in the methylenetetrahydrofolate reductase gene a risk factor for neural tube defects?.Q J Med. 1997; 90: 111-115Crossref Scopus (219) Google Scholar Initial studies of association between NTD and T/T were criticised on the grounds that controls were not matched properly to patients.2Papapetrou C Lynch SA Burn J Edwards YH Methylenetetrahydrofolate reductase and neural tube defects.Lancet. 1996; 348: 58Summary Full Text Full Text PDF PubMed Scopus (81) Google Scholar Within-family gene distribution analyses avoid the risk of population stratification inherent in association studies since observations are compared with mendelian expectations. In the only such study reported for NTD and MTHFR, C/T heterozygous parents transmitted equal numbers of C and T alleles (12:12) to offspring with NTD, consistent with the hypothesis of no association between the T allele and NTD.2Papapetrou C Lynch SA Burn J Edwards YH Methylenetetrahydrofolate reductase and neural tube defects.Lancet. 1996; 348: 58Summary Full Text Full Text PDF PubMed Scopus (81) Google Scholar From 1988 to 1990, we enrolled 168 families with a NTD pregnancy from catchment areas centred in Boston and in Ottawa (81 anencephaly, 1 encephalcoele, 86 spina bifida). The ethnic origins of the 672 grandparents of probands were 620 European, 47 Native American, three African American, and two Asian. Patients with spina bifida (83), mothers (167), fathers (168), and unaffected siblings (182) were typed for the MTHFR locus3Ou CY Stevenson RE Brown VK et al.5,10 methylenetetrahydrofolate reductase genetic polymorphism as a risk factor for neural tube defects.Am J Med Genet. 1996; 63: 610-614Crossref PubMed Scopus (205) Google Scholar blind to diagnoses, sex, and pedigree relationships. Parentage was confirmed by typing other genetic markers. The table shows the number of offspring of each genotype according to sex and diagnosis. The allele transmission ratio from C/T heterozygous parents was 39C:48T to offspring with NTD χ12=0–93, p=0·33) and 94C:100T to unaffected offspring (χ12=0–19, p=0·66), both close to the 1:1 Mendelian expectation. However, among all 206 offspring of these parents the number of offspring of each genotype (C/C, C/T, or T/T) differed from mendelian expectations (χ22= 18·05, p=0·00012). The difference resulted from an excess of heterozygotes (136 observed, 105·5 expected) and a deficiency of C/C homozygotes (57 observed, 74·0 expected) and T/T homozygotes (18 observed, 29·5 expected). The results were significant both for the 65 spina bifida offspring (χ22=9–66, p=0·0080) and for the 146 unaffected offspring (χ22=9–28, p=0·0097). Taking into account sex, increased heterozygosity was observed in females with NTD, but not in males with NTD. On the other hand, among unaffected offspring, an excess of heterozygotes and a deficiency of homozygotes was observed for males and not for females. Tests for Hardy-Weinberg equilibrium (a population rather than family-based analysis) yielded similar levels of significance for the genotype frequency distortions in the offspring (see table). To determine whether the sex difference in heterozygosity in unaffected offspring occurred in other first-degree relatives, we compared the distribution of genotypes in the fathers and mothers of the NTD probands with Hardy-Weinberg expectation. Among mothers there were 79 C/C, 73 C/T, and 15 T/T, compared with 79·88 C/C, 71·24 C/T, and 15·88 T/T expected (x^O-10, p=0·75). Thus, mothers and 91 unaffected sisters of NTD subjects combined showed no difference in genotype ratios from Hardy-Weinberg expectation (χ12=0–43, p=0·51). There were 51 C/C, 103 C/T, and 14 T/T fathers compared with 62·54 C/C, 79·92 C/T, and 25·54 T/T expected (χ12= 14·01, p=0·00018). The excess of heterozygotes among 168 fathers (29%) and 90 brothers (29%) was similar and, when the data for fathers and brothers were combined, highly significant (χ12=22–30, p=0·000002). Male NTD fetuses are thought to have a higher abortion rate than females.4Elwood JM Little J Elwood JH Epidemiology and control of neural tube defects. Oxford University Press, Oxford1992Google Scholar Virtually all of the 50% excess of female over male NTD subjects can be accounted for by an excess of C/T genotypes (31 of 50 females versus 16 of 33 males). Thus, C/T females with NTD may be less likely to abort than C/T males with NTD. Our data also suggest an interactive effect between maternal and fetal MTHFR genes in survival (eg, the large excess of C/T in NTD females from C/C mothers). If MTHFR genotype affects fetal survival, a possible explanation for the discrepancy between the results of our study and the association studies could be that the latter may have included more severely affected patients or abortuses. NTD, the most common malformation among abortuses, has a multifactorial aetiology with polygenetic susceptibility and environmental risk factors, including folate.4Elwood JM Little J Elwood JH Epidemiology and control of neural tube defects. Oxford University Press, Oxford1992Google Scholar On average, half of the NTD susceptibility alleles present in individuals with NTD are present in a parent or sibling. The highly significant excess of C/T heterozygosity in male firstdegree relatives of patients with NTD demonstrates C/T heterozygote advantage in the context of their genetic background. These results raise the issue of how selection acts on functionally different MTHFR alleles to produce, in families selected for a member with NTD, an excess of heterozygotes among affected females and unaffected males. We thank the families who participated in this study; Peter Rowley, Carole Ober, and Roger Stevenson for helpful criticism; Sally Guttormsen, Linda Kemp, Susan Tan Torres, Gollu Ramaurthy, Y-Z Du, Patricia Weill, and Luciana Yu for technical and clinical assistance; and Mary True for help in preparing the manuscript. This research was supported in part by US Public Health Service grant HD-24560, the South Carolina Department of Disabilities and Special Needs grant SG97–88, and US Public Health Service (CDC) grant U85/CCU408774.