Association of the C677T methylenetetrahydrofolate reductase mutation with congenital heart diseases

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1 Acta Obstet Gynecol Scand 2005: 84: Copyright # Acta Obstet Gynecol Scand 2005 Printed in UK. All rights reserved Acta Obstetricia et Gynecologica Scandinavica ORIGINAL ARTICLE Association of the C677T methylenetetrahydrofolate reductase mutation with congenital heart diseases CHIEN-NAN LEE 1,YI-NING SU 2,WEN-FANG CHENG 1,MING-TAI LIN 3,JOU-KOU WANG 3,MEI-HWAN WU 3 AND FON-JOU HSIEH 1,2 From the Departments of 1 Obstetrics and Gynecology, University Hospital, Taipei, Taiwan 2 Medical Genetics, and 3 Pediatrics, National Taiwan Acta Obstet Gynecol Scand 2005; 84: # Acta Obstet Gynecol Scand Background. To investigate whether the cytosine-to-thymine mutation at base 677 of the gene for methylenetetrahydrofolate reductase (MTHFR) is associated with congenital heart diseases (CHD), using high throughput heteroduplex analysis based upon the powerful technique of denaturing high-performance liquid chromatography. Methods. We investigated the MTHFR genotype of a cytosine-to-thymine mutation at base 677 for 213 patients of CHDs as confirmed by cardiac catheterization and also for 195 healthy controls. Results. The overall genotype frequencies of the MTHFR C677T polymorphism were not significantly different between the CHD patients and the healthy control (P ¼ 0.345). Furthermore, taking various subgroups of CHD patients into consideration, we noted a significantly increased proportion of homozygous TT genotypes for patients suffering from valvular pulmonary stenosis (PS) or pulmonary atresia with an intact ventricular septum (PA þ IVS) (p ¼ ). For patients revealing heterotaxy syndrome, a conotruncal anomaly including tetralogy of Fallot, an interruption of the aortic arch, persistent truncus arteriosus, and aortopulmonary window, no statistically significant difference existed. Conclusions. The discrepancy in the distribution of MTHFR genotypes amongst various subtypes of CHD reflects some heterogeneity in the developmental mechanism of CHD. The increased percentage of homozygous TT genotypes might contribute to the pathogenesis of valvular PS and PA þ IVS. Key words: congenital heart disease; denaturing high-performance liquid chromatography; methylenetetrahydrofolate reductase; mutation analysis Submitted 20 January, 2004 Accepted 22 April, 2004 Abbreviations: AP window: atriopulmonary window; ASD: atrial septal defect; CHD: congenital heart disease; CoA: coarctation of the aorta; DHPLC: denaturing high-performance liquid chromatography; DILV: double inlet of the left ventricle; DORV: double outlet of the right ventricle; ECD: endocardial cushion defect; IAA: interrupted aortic arch; LAI: left atrial isomerism; MTHFR: methylenetetrahydrofolate reductase; PA þivs: pulmonary atresia with an intact ventricular septum; PDA: patent ductus arteriosus; PS: pulmonary stenosis; RAI: right atrial isomerism; TGA: transposition of the great artery; TOF: tetralogy of Fallot; VSD: ventricular septal defect. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the biologically irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the methyl donor for methionine synthesis from homocysteine. The most common mutation of MTHFR is a cytosine-to-thymine substitution at base 677 that causes a substitution of valine for alanine in the functional MTHFR enzyme. Subjects exhibiting the common C677T mutation in the MTHFR gene reveal a reduced MTHFR enzyme activity for approximately 50%

2 MTHFR and congenital heart disease 1135 of the normal population with increased plasma homocysteine concentrations (1). The association between the MTHFR C677T mutation amongst infants and the congenital neural tube defects has been well identified previously (2). Methylenetetrahydrofolate reductase polymorphisms play an important role in clinic diseases. Neural tube defects and pregnancy complications appear to be linked to impaired MTHFR function. Methylenetetrahydrofolate reductase gene mutation may result in hyperhomocysteinemia which is frequently found in women with a history of recurrent early pregnancy loss (3), placental abruption (4), or preeclampsia (5), and adequate folate supplement can reduce the risk of neural tube defects. A higher incidence of homozygosity for the 677T allele has been found in patients with premature coronary artery disease (6), cerebral infarction, and venous thrombosis (7). However, the associations between MTHFR polymorphisms and vascular, arteriosclerotic, or thrombotic disease are still equivocal (8). Because MTHFR diverts folates to different metabolic pathways, changes in MTHFR activity and folate supply have direct consequences for many of the factors that have been implicated in tumorgenesis including nucleotide balance, RNA and DNA synthesis, DNA repair, and DNA methylation. A higher incidence of the homozygous 677T genotype has been reported in patients with bilateral breast cancer and those with both breast cancer and ovarian carcinoma, as opposed to those with unilateral breast cancer (9), and in patients with cervical intraepithelial neoplasia or endometrial cancer (10). There are interests between MTHFR polymorphisms and neurologic or psychiatric disease. Hyperhomocysteinemia has been reported in patients with auratic migraine, severe depression (11), and schizophrenia (12). Disturbances of folate and cobalamin metabolism may be involved in cognitive dysfunction (13). Congenital heart disease (CHD), as a cluster of common multifactorial heterogenous anomalies, occurs for approximately 1% of live-born children, but for those aborted spontaneously or stillborn, the figure is much higher (14,15). The association between periconceptional multivitamin use and a reduced risk of fetal conotruncal cardiac defects has been reported in a number of retrospective case control studies (16,17). Furthermore, an inverse relationship between daily maternal folic acid intake and the offspring s incidence of cardiac outflow tract defects has also been mentioned in the literature (18). Recently, the cytosine-to-thymine mutation at base 677 of the gene for MTHFR (C677T MTHFR) has been suggested to represent a potential risk factor for CHD for a limited number of cases (19,20). The status of the C677T genotype can be determined by a classical polymerase chain reaction (PCR)-based procedure (1,21), although the classical approaches to such assay are encumbered by various problems including the labor-intensive and time-consuming nature of the assay. In order to detect such single nucleotide polymorphisms (SNP) in a more efficient way, a powerful high throughput technique suitable for massive SNP screening in a population base is clearly warranted. Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection (22 26). The advantages of DHPLC over traditional techniques based on gel electrophoresis for the identification of base-pair substitutions and small insertions/ deletions include higher sensitivity and better reproducibility, higher throughput, and a higher level of automation. We therefore conducted this study on a large scale in order to investigate the association between a cytosine-to-thymine mutation at base 677 of the gene for MTHFR (C677T MTHFR) and CHD based on the use of a DHPLC technique. Materials and methods The present study was carried out in accordance with the ethical standards laid down in a relevant version of the 1964 Declaration of Helsinki and approved by the medical ethics committee at the National Taiwan University Hospital (Taipei, Taiwan). From July 1, 2002 to June 30, 2003, totally 213 consecutive children with suspected CHD referred from other hospitals were performed cardiac catheterization by Dr MH Wu and JK Wang at our hospital, were enrolled in this study. Underlying subtypes of CHD are summarized in Table I. Cord blood from 195 healthy fetuses devoid of any evidence of heart disease confirmed by auscultation and ultrasound examination by the pediatric cardiologist or other major systemic diseases from the same geographic area were also investigated, they being used as controls. We also traced back these families for three generations. The patients were unrelated and no unrecognized consanguinity presented. All DNA samples were extracted from peripheral whole blood or cord blood by using a Puregene DNA Isolation Kit (Gentra Systems, Inc., Minneapolis, Minnesota, USA) according to the manufacturer s instructions. The MTHFR genotypes were analyzed by PCR subsequent to the extraction of genomic DNA. If a C677T MTHFR mutation is present, it will lead to a substitution of valine for alanine in the gene-selected protein product. The primers for analysis of the A! V change are: TGAAGGAGAAGGTGTCTGCGGGA-3 0 (exonic) and 5 0 -AGGACGGTGCGGTGAGAGTG-3 0 (intronic), these primers ultimately generating a fragment of 198 bp. Polymerase chain reaction for the fragments was conducted according to a modified protocol as originally

3 1136 F.-N. Hsieh et al. Table I. The results of genotype frequency estimation of the methylenetetrahydrofolate reductase C677T polymorphism amongst patients with congenital heart disease and also for healthy controls MTHFR genotypes OR (95% CI) CT þ TT TT versus Disease entity CC CT TT CT versus CC TT versus CC versus CC CCþ CT Simple pulmonary ( )* 21.9 ( ) 7.0 ( ) z 10.0 ( ) valvular problem Valvular PS PA þ IVS Heterotaxy syndrome ( )* 2.5 ( ) 2.4 ( )* 1.6 ( ) RAI Situs inversus LAI Conotruncal anomaly ( ) 0.8 ( ) 1.0 ( ) 0.8 ( ) TOF Truncus AP window IAA Vascular ring ASD ( ) 0.4 ( ) 1.3 ( ) 0.3 ( ) PDA ( ) 0.8 ( ) 1.8 ( ) 0.6 ( ) VSD ( )* 0.4 ( ) 0.4 ( ) z 0.5 ( ) DORV ( ) 1.8 ( ) TGA ( ) 1.4 ( ) ECD DILV ( ) 0.9 ( ) CoA ( ) 2.8 ( ) Hypoplastic left ( ) heart Syndrome Others ( ) 2.2 ( ) 2.1 ( ) 1.6 ( ) Sum of CHD ( ) 1.1 ( ) 1.3 ( ) 1.0 ( ) Control *p < 0.1. p < z p < AP window, atriopulmonary window; ASD, atrial septal defect; CHD, congenital heart disease; CoA, coarctation of the aorta; PS, pulmonary stenosis; DILV, double inlet of the left ventricle; DORV, double outlet of the right ventricle; ECD, endocardial cushion defect; IAA, interrupted aortic arch; LAI, left atrial isomerism; PA þ IVS, pulmonary atresia with an intact ventricular septum; PDA, patent ductus arteriosus; RAI, right atrial isomerism; TGA, transposition of the great artery; TOF, tetralogy of Fallot; VSD, ventricular septal defect. Isolated VSD represents the cases suffering only from VSD without any other cardiac malformations. The subgroup of all CHD individuals featuring VSD represents the cases with isolated VSD and combined with other cardiac malformations. Others include congenital corrected TGA, total anomalous pulmonary venous return, arteriovenous malformation, cor triatriatum and aortic stenosis. described by Frosst et al. (1). The PCR reaction was conducted in a total volume of 25 ml containing 50 ng of genomic DNA, 0.3 ml of 10 pmol of each primer, 1 ml of 2.5 mm dntps, 0.5 units of AmpliTaq Gold TM enzyme (PE Applied Biosystems, Foster City, California, USA), and 2.5 ml of GeneAmp 10 buffer II (10 mm Tris-HCl ph ¼ 8.3, 50 mm KCl), and 2 ml of25mm MgCl 2 provided by the manufacturer. Amplification was performed in a mutliblock system thermocycler (ThermoHybaid, Ashford, UK). Polymerase chain reaction amplification was performed with an initial denaturation step at 95 C for a period of 10 min, followed by 35 cycles consisting of denaturation at 94 C for 30 s, annealing at 56 C for 1 min, and extension at 72 C for 30 s, and then an extension step of 72 C for 10 min. For heteroduplex analysis by DHPLC, an additional five-minute 95 C denaturing step followed by gradual reannealing from 95 Cto65 C over a period of 30 min for crude PCR products prior to analysis was needed. Denaturing high-performance liquid chromatography analysis was performed on a Wave 1 DNA Fragment Analysis System (Transgenomic Inc., San Jose, California, USA) as previously reported (27). In brief, crude PCR samples were injected into a temperature-equilibrated DNASep 1 cartridge (Transgenomic Inc.) following heteroduplex formation and eluted at a flow rate of 0.9 ml/min using a linear gradient of acetonitrile in 0.1 M triethylammonium acetate (TEAA), ph ¼ 7.0. Denaturing high-performance liquid chromatography grade acetonitrile ( , JT Baker, Phillipsburg, New Jersey, USA) and TEAA (Transgenomic Inc., Crewe, UK) were used to constitute the mobile phase. The mobile phase consisted of 0.1 M TEAA with 0.05% acetonitrile (eluent A) and 25% acetonitrile in 0.1 M TEAA (eluent B). The start- and end-points of the gradient were, respectively, 59% and 66% of eluent B running in 3.5 min, and the temperature for optimal heteroduplex analysis was set to 63 C. The temperature for optimal heteroduplex analysis and the selected gradient performed by mixing eluents A and B were all determined by using WAVEMAKER TM software (Transgenomic Inc.), and data were stored on the WAVE System computer. Generally, the analysis of an individual sample required a period of 8 10 min. This included regeneration and equilibration of the column subsequent to the completion of each individual analysis. Heterozygous control probes were used to confirm system performance in general, and, in particular, the accuracy of the selected oven temperature. The genotype

4 MTHFR and congenital heart disease 1137 of an alteration detected by DHPLC was predicted by comparison with profiles of CC genotypes. For the purposes of differentiating between CC, CT, and TT genotypes, two consecutive injections for each subject were required. For the first injection, only crude PCR product for each subject was needed such that the CT genotype (heterozygous) could be effectively identified from the CC and TT genotypes (homozygous). For the second injection, to further differentiate homozygous CC and TT genotypes, the DHPLC analysis was preceded by a mixing step with the reference being the known CC genotype DNA. Polymerase chain reaction products were purified by a solid-phase extraction procedure and bidirectionally sequenced with the Applied Biosystems Taq DyeDeoxy terminator cycle sequencing kit (Applied Biosystems, Foster City, California, USA) according to the manufacturer s instructions. Sequencing reactions were separated on an Applied Biosystems 3100 sequencer. On an explorative basis, genotype frequencies of patients and controls were compared by 2 analysis or Fisher s exact test. The significance level was set at p ¼ In addition, odds ratios (ORs) and 95% confidence intervals (CIs) were calculated. All statistical analyses were performed using the statistical package for social sciences (SPSS) for Windows, Release 9.0 (SPSS, Inc., Chicago, Illinois, USA). The population samples were tested for Hardy Weinberg equilibrium using 2 test versus the expected distribution pattern (p 2 þ 2pq þ q 2 ). We should use the statistical method that takes multiple testing with Bonferroni correction into account. Epi info 6 (CDC, USA) was used for the calculation of statistical powers. Results A total of 213 patients suffering CHD, and 195 healthy controls were tested for MTHFR C677T polymorphisms by using heteroduplex analysis based upon a DHPLC system. The results of DHPLC analysis were further confirmed by subsequent sequence analysis, revealing 100% validity and accuracy being demonstrated. The unmix and mix patterns of DHPLC and sequence analysis results for the CC, CT, and TT genotypes are shown in Fig. 1. By means of DHPLC analysis, heterozygous revealed characterized elution profiles which could be distinguished from the homozygous markedly. For the patients featuring structural CHD, the overall genotype frequencies of the MTHFR C677T polymorphism were as follows: CC, n ¼ 110 (51.6%); CT, n ¼ 89 (41.8%); TT, n ¼ 14 (6.6%). For the control group, the homozygous MTHFR genotype (CC) was present for 114 individuals (59%), the CT genotype for 68 (34%), and the TT genotype for 13 (7%). It was approved that the genotypes of the control group in our study are in Hardy Weinberg equilibrium. By constructing a 3 2 contigency table, we were able to compare the data and revealed that no statistically significant difference existed between the CHD patients and control population (p ¼ 0.345). Taking various subgroups of CHD patients into consideration, the genotype frequencies for the MTHFR C677T polymorphism were examined, they being listed in Table I. For patients featuring valvular pulmonary stenosis (PS) or pulmonary atresia with an intact ventricular septum (PA þ IVS), we noted a significantly increased percentage of homozygous TT genotypes (p ¼ ). The resulting OR statistic for this subgroup carrying the homozygous TT genotype compared to the controls was 21.9 (CI, ; p ¼ ). Also, the OR statistic for patients carrying the heterogenous CT or the homozygous TT genotype compared to the controls was 7.0 (CI, ; p ¼ ). The OR statistic for patients carrying the homozygous TT genotype compared to the heterogenous CT or the controls was 10.0 (CI, ; p ¼ ). We should use the statistical method that takes multiple testing into account. We also recalculated the figures in Table I. When Bonferroni correction was applied, the p values were corrected by multiplying the numbers of stratification. As a result, only two comparisons in simple pulmonary valvular problem were still significant: TT versus CC (p ¼ ) and TT versus CC þ CT (p ¼ ). For patients afflicted with heterotaxy syndrome, the proportion of TT and CT genotypes appeared to be a little higher, but not significantly so, as compared with the control group. For patients revealing a conotruncal anomaly, including tetralogy of Fallot (TOF), interruption of aortic arch, persistent truncus arteriosus, and aortopulmonary window, no statistically significant difference existed as compared with the control group. The OR for isolated ventricular septal defect (VSD) patients carrying CT or TT genotypes compared to the controls was 0.4 ( ), although VSD is associated with many kinds of CHD such as TOF, double outlet of the right ventricle, and endocardial cushion defect. If we took these disease entities into consideration, no statistically significant difference was noted. The results of subgroup analyses are summarized in Table I. Epi info 6 (CDC, USA) was used for the calculation of statistical powers. Table II summarizes the estimated overall powers on various percentages of exposure among control group and significant level. A total of 213 cases and 195 controls were available in our study result in controls per cases. As the OR worth detecting was assumed as 2.0, the fixed sample size yielded more than 80% statistical power if the significant level was set to 5% or 1%, and the exposure was TT þ CT. The TT and CT genotypes

5 1138 F.-N. Hsieh et al. (a) homoduplex (b) heteroduplex (c) unmix mix (d) unmix mix (e) unmix mix Fig. 1. Thedenaturinghigh-performance liquid chromatography and sequence analysis for the methylenetetrahydrofolate reductase C677T polymorphism. (a) The chromatography for an amplicon featuring a homoduplex. (b) The chromatography for an amplicon featuring a heteroduplex. (c) The unmix/mix chromatography and sequence analysis for the CC genotype. (d) The unmix/mix chromatography and sequence analysis for the CT genotype. (e) The unmix/mix chromatography and sequence analysis for the TT genotype. are 41.5% of exposure among control group. When TT genotype only was used as the exposure (6.7% of exposure among controls), the fixed sample size yielded statistical power of 51.3% and 27.9%, while the significant level was set to 5% and 1%. Discussion Methylenetetrahydrofolate reductase is critical for the folate-dependent remethylation of homocysteine to methionine. Methylenetetrahydrofolate reductase and folate metabolism are suggested to be epigenetic factors for DNA synthesis and tissue growth. A strong nutrient gene interaction is suggested in this situation by the observation that a low concentration of maternal blood folate (28) and no periconceptional vitamin supplementation (29) both may enhance the risk associated with the T allele. It would appear that the combination of the TT genotype and a low concentration of folate increases the concentration of homocysteine and impairs methionine formation in the embryo, and it is thought that both factors are involved in the genesis of incomplete neural tube closure. A recent study by Stern et al. (2000) (30) revealed that the TT genotype is also associated with lower DNA methylation for peripheral leukocytes compared with the CC genotype. This observation is particularly important, because it demonstrates that altered folate distribution amongst subjects featuring the TT genotype is associated with secondary metabolic effects in addition to hyperhomocysteinemia. Each of these methylation-dependent processes and substances is critical for normal tissue growth and maturation. If any of the molecules or cellular products related to DNA methylation are deficient or dysfunctional in the developing embryo, slowed and interrupted tissue growth and development could result. The link between slowed growth and birth defects makes sense, because a variety of congenital defects are associated with slowed growth leading to incorrect timing of normal developmental events. This etiological mechanism seems most likely to explain the link between the C677T MTHFR mutation and a variety of birth defects, because all the congenital anomalies reported to be associated with folate deficiency could potentially arise by this mechanism. It is interesting that the C677T MTHFR mutation has also been associated with premature cardiovascular disease and hereditary thrombophilia amongst adults (31,32). Several pieces of data now suggest that multifactorial cardiac defects may also be associated Table II. Estimated statistical powers on various percentages of exposure among controls and significant level (odds ratio was assumed as 2.0) Percentage of exposure among controls TT only ¼ 6.7% TT þ CT ¼ 41.5% a ¼ % 93.3% a ¼ % 81.2%

6 MTHFR and congenital heart disease 1139 with this particular mutation, for example, a prospective randomized study (33) has shown not only that there were no neural tube defects amongst the offspring of supplemented mothers but also that there were significantly fewer obstructive urinary tract defects and cardiac anomalies (primarily VSD) amongst these individuals. Retrospective case control studies have reported an association between periconceptional multivitamin use and a reduced risk for fetal conotruncal cardiac defects (16,17), and the Baltimore Washington Infant study found an inverse relationship between daily maternal folic acid intake and the offspring s incidence of cardiac outflow tract defects (18). The present study of children afflicted with structural CHD was conducted on the basis of the hypothesis that the CT or TT genotype in the MTHFR gene elicits an impact upon the embryonic development of commonly observed congenital malformations such as neural-tube defects or oral clefts. During embryogenesis, the development of the conotruncal structure, including the RV outflow tract and the separation of the two great vessels, emerge around 5 weeks subsequent to fertilization. By contrast, the semilunar valves develop after about 6 weeks of gestational age. Both Wenstrom et al. (20) and Junker et al. (19) have reported, in 2001, that the C677T MTHFR mutation is linked to multifactorial cardiac defects but both studies feature limited case numbers. To investigate this hypothesis on a larger scale, we study the DNA samples from patients who suffer from isolated congenital cardiac defects and compared such samples with those of the normal population by use of this rather newly established and powerful analytical technique of DHPLC. The most common techniques used for the identification of base-pair substitutions in DNA involve use of the classical PCR-based procedures (1,21). Such methods tend to be labor-intensive and time-consuming, however. Oefner and coworkers (25) have recently reported a technique for the identification of single-base substitutions and small deletions/insertions, in which heteroduplex pieces of DNA are separated from homoduplex strands by ion-pairing reversed phase liquid chromatography via a special high-performance liquid chromatography (HPLC) column. In effect, partial heat denaturation decreases the retention time of mismatched DNA molecules compared with their intact double-stranded counterparts. As a consequence of this shift in elution time, multiple peaks indicate internal sequence variations in a PCR sample. Single peak may be present for normal sequences. Although an initially sizeable capital investment in the HPLC device is required in order to undertake such an assay, we have demonstrated that the combination of lowrunning costs, a high level of assay sensitivity, and the tremendous reduction in the effort necessary for sequencing renders the DHPLC technique a most suitable method for high throughput mutation analysis. Our study did not show a significantly different distribution of MTHFR genotypes between normal and CHD groups, however, and patients suffering valvular PS or PA þ IVS were remarkably well associated with the expression of an increased proportion of homozygous TT genotypes. For patients affected with heterotaxy syndrome, a conotruncal anomaly including TOF, interruption of aortic arch, persistent truncus arteriosus, and the presence of an aortopulmonary window, no statistically significant difference was noted. In summary, our data have revealed a strong relationship between the MTHFR gene and lesions of the pulmonary valve rather than with conotruncal lesions. The discrepancy in the distribution of MTHFR genotypes amongst various subtypes of CHD individuals could reflect the heterogeneity in the developmental mechanism of CHD. Acknowledgments This work was supported by grants from the National Science Council (NSC B ), and we thank the statistical assistant of Chin-Lan Jen, College of Public Health, National Taiwan University. References 1. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard C, Matthew RG. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. 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Low folate levels and thermolabile methylenetetrahydrofolate reductase as primary determinant of mild hyperhomocystinemia in normal and thromboembolic subjects. Arterioscler Thromb Vasc Biol 1999; 19: Czeizel AE. Reduction of urinary tract and cardiovascular defects by periconceptional multivitamin supplementation. Am J Med Genet 1996; 62: Address for correspondence: Fon-Jou Hsieh Department of Obstetrics and Gynecology National Taiwan University Hospital No.7, Chung-Shan South Road Taipei Taiwan fjhsieh@ha.mc.ntu.edu.tw