Interaction between MTHFR Polymorphisms and Maternal Age Increases the Risk of Congenital Heart Defects in Down Syndrome

Congenital heart disease (CHD) comprises one-third of all congenital anomalies in newborns and is the most frequent cause of infant deaths. Long-term morbidity and mortality from CHD pose a substantial challenge to healthcare systems globally (Dolk et al., 2011). The exact etiology of CHD remains uncertain, the disease is heterogeneous, but it is believed to be multifactorial: environmental factors contribute (Gladki et al., 2015; Gorini et al., 2014; Huhta and Linask, 2013); and twin studies suggest familial aggregation of CHD (Kuo et al., 2017). Specific recurrence patterns and phenotypes also suggest that genetically-determined developmental mechanisms exert a strong influence in CHD etiology (Ellesøe et al., 2018).

Several cohort studies have established a relationship between Down syndrome (DS) and CHD (Ferencz et al., 1989; Kidd et al., 1993), with 4%-10% of all CHD cases associated with DS and 40%-60% of DS patients having CHD (de Rubens-Figueroa et al., 2003). Several lines of evidence indicate that maternal hyperhomocysteinemia is an independent risk factor for CHD (Malik et al., 2017), and recent population studies in humans have indicated that folic acid supplementation before and/or during pregnancy can decrease CHD risk (Czeizel et al., 2015; Leirgul et al., 2015; Li et al., 2013). This evidence prompted several follow-on studies examining the role of folic acid in CHD risk (Parnell and Correa, 2017; Xu et al., 2016; Feng et al., 2015).

Folates serve as cofactors for nucleotide synthesis and co-substrates of DNA methyltransferases (Stover and Field, 2011). DNA methylation plays a key role in embryonic development, and abnormally methylated genes have been detected in fetuses with both isolated and syndromic heart malformations (Serra-Juhe et al., 2015). Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is a key enzyme in folate and homocysteine (Hcy) metabolism (Bhaskar et al., 2011). The gene encoding human MTHFR is known to have two important functional polymorphisms, C677T/A222V/rs1801133 and A1298C/Glu429Ala/rs1801131, which generate a thermolabile form of MTHFR that subsequently increases total plasma Hcy and decreases serum folate levels (Goyette et al., 1994). A recent computational modeling study demonstrated that the rs1801133 polymorphism causes conformational changes in the tertiary structure of MTHFR by reducing its FAD-binding ability (Abhinand et al., 2016). However, results from studies examining the relationship between MTHFR rs1801133 and rs1801131 polymorphisms and CHD risk have been inconsistent (Zhang et al., 2018; Asim et al., 2017; Yu et al., 2017; Guo et al., 2017). We recently highlighted the high burden of DS and CHD in the Al Madinah region of Saudi Arabia (Abdulhadi et al., 2016). Here we investigated associations between MTHFR polymorphisms (rs1801133 and rs1801131) and the risk of CHD and DS co-occurrence in a Saudi population.

The Institutional Ethics Committees of Maternity and Children Hospital and Centre for Genetics and Inherited Diseases (CGID), Taibah University, Al Madinah, Kingdom of Saudi Arabia granted ethical approval. As many of the subjects in the CHD group were minors (<18 years old), written informed consent was obtained from the parents or legal guardians of the children enrolled in this study as necessary.

Ninety-nine CHD patients with (n=26) or without (n=73) DS attended the Pediatric Cardiology Clinic at the Maternity and Children Hospital, Al Madinah, Kingdom of Saudi Arabia were enrolled in this study. Participants with mosaic, translocation of chromosome 21, non-cardiac congenital anomalies were excluded from this study. The main inclusion criteria for DS was cases had full trisomy 21 confirmed by karyotype and/or Fluorescent in situ Hybridization (FISH) for chromosome 21. CHD and CHD in DS samples were collected after checking the family history, physical and clinical examination, electrocardiograms (ECG), chest x-rays, 2D-echocardiography, Karyotypes or FISH data from the patient’s case sheet. One hundred and twenty-six healthy subjects without CHD or a family history of CHD were recruited as controls. All participants were of Saudi Arabian ethnicity.

Two ml of whole peripheral blood was collected from each participant after obtaining informed consent. DNA was extracted using the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany). Two MTHFR SNPs [rs1801133: C___1202883_20 and rs1801131: C____850486_20] were genotyped using primers and probes purchased from Applied Biosystems, Foster City, CA. Reactions were carried out in a final volume of 5 μL (2.5 μL TaqMan PCR Master Mix, 0.125 μL TaqMan Genotyping Assay Mix, 1.375 μL nuclease free water, and 1 μL of 10 ng/μL DNA) in a 96-well optical microplate (Applied Biosystems). At least two no-template controls without any DNA were included on each plate. The PCR conditions were initial denaturation at 95°C for 10 min followed by 40 denaturation cycles (95°C for 15 s) and annealing/extension (60°C for 1 min). PCR amplification was performed using Applied Biosystems’ StepOnePlus Real-Time PCR system, and fluorescence was also measured using its

OR, odds ratio; CI, confidence interval; MAF, minor allele frequency; aHWp, Hardy-Weinberg p value.

Table II.- The distribution of MTHFR genotypes and other risk factors among CHD patients and their association with Down syndrome.

OR, odds ratio; CI, confidence interval.

Sequence Detection Software (SDS) v. 2.3. A genotype call rate over 99% was considered for the analysis. As a quality control measure, 10% of samples selected at random were included for duplicate genotyping.

Allele frequencies of both polymorphisms in CHD and control groups were calculated by the gene counting method. The genotype distribution for each polymorphism was evaluated for Hardy–Weinberg equilibrium. The strength of the association between MTHFR gene polymorphisms and CHD was evaluated using the χ2 test and odds ratios (OR) with 95% confidence intervals (CI). The influence of different genotypes on the relationship between DS and maternal age and their interaction was examined using the Mantel-Haenszel stratified analysis. All statistical analyses were performed in SPSS v. 14.0 (IBM Statistics, Chicago, IL).

Of 99 patients with CHD, 26 (26.3%) had DS. Among DS patients, atrioventricular septal defect (AVSD) was the most common cardiac defect followed by ventricular septal defect (VSD) and atrial septal defect (ASD). The distributions of MTHFR rs1801133 and rs1801131 genotypes and alleles in CHD and control groups are shown in Table I. The MTHFR rs1801133 (p=0.402) and rs1801131 (p=0.357) genotype distributions in the control group followed Hardy­Weinberg equilibrium (p=0.359).

Genotypes and alleles were not statistically different between controls and individuals with CHD (Table I). MTHFR rs1801133 and rs1801131 polymorphisms were not associated with CHD risk (Table I) and, in CHD individuals, MTHFR rs1801133 and rs1801131 genotypes failed to show any significant association with DS (Table II).

Univariate analysis showed that increased maternal age (≥35 years) contributed to CHD risk in DS patients (OR: 5.32, 95% CI: 1.43-19.82; p=0.013), while increased paternal age (≥40 years) had no effect on CHD in DS patients (OR: 2.45, 95% CI: 0.66-9.19; p=0.182) (Table II). Significant heterogeneity in the effect of maternal age on DS was observed among different genotypes of MTHFR rs1801133 and rs1801131 polymorphisms. The M-H combined OR for maternal age and rs1801133 (p<0.001) was 6.33 and for maternal age and rs1801131 was 7.42 (p<0.001) (Table III).

CHD, congenital heart disease; M-H, Mantel-Haenszel.*Homogeneity test p value.

The pathogenesis of CHD is complex and remains poorly understood due to its multifactorial etiology. Individuals with DS show several common cardiac malformations such as VSDs, atrioventricular canals, and the tetralogy of Fallot (Tandon and Edwards, 1973). As folate serves as a DNA methyltransferase co-substrate, reduced folate causes hypomethylation and consequently abnormal segregation of chromosomes or chromosome nondisjunction (Fenech, 2011; James et al., 1999; Blom and Smulders, 2011). One common factor in these folate abnormalities is MTHFR rs1801133 polymorphisms, with decreased enzyme activity causing impaired re-methylation of Hcy to methionine and subsequent hyperhomocysteinemia (Yigit et al., 2013).

However, the association between maternal genetic polymorphisms in folate metabolism genes and DS risk is still controversial (Yang et al., 2013; Brandalize et al., 2009, 2010). Significantly elevated Hcy levels in mothers of children with CHD suggest that maternal hyperhomocysteinemia is an independent risk factor for CHD (Malik et al., 2017; Lu et al., 2011; Verkleij-Hagoort et al., 2007). Due to the high prevalence of CHDs in neonates born with DS and DS survivors in Al Madinah, CHDs are considered as an important health problem in Saudi Arabia, a problem further exacerbated by widespread consanguinity in this region (Abdulhadi et al., 2016). Thus, maternal supplementation with folic acid is likely to be associated with a reduced risk of CHD in DS. Given this perspective and to inform management, we considered the association between MTHFR polymorphisms and CHD in DS susceptibility in a Saudi population.

We did not identify any significant association between MTHFR polymorphisms and CHD risk. Both polymorphisms had a confounding effect on the relationship between CHD in DS and maternal age. The presence of the maternal MTHFR rs1801133 “T” allele was associated with an increased risk of DS in a Brazilian population (Brandalize et al., 2009), while another study of a Croatian population reported no association between maternal MTHFR polymorphisms and CHD in DS (Bozovic et al., 2011; Elsayed et al., 2013). MTHFR rs1801131 was over-transmitted in patients with CHD in DS ASDs (Locke et al., 2010). A meta-analysis showed that MTHFR rs1801131 polymorphisms reduced the risk of CHD in patients without DS (Yu et al., 2017), while another suggested that the maternal MTHFR rs1801133 “T” allele was a risk factor for DS-affected pregnancies (Rai et al., 2014). Finally, a recent study showed that the MTHFR gene was twice as likely to be promoter methylated in mothers of children with DS with CHD than mothers of children with DS without CHD (Asim et al., 2017).

The following strengths and limitations must be taken into consideration when interpreting our results. Significant strengths include the inclusion of subjects with CHD diagnosed by echocardiography and clinical cardiac examination and DS by karyotype or FISH for chromosome 21. However, the study also has several limitations: plasma Hcy levels were not determined and correlated with DS-related CHD or MTHFR variants, and maternal MTHFR gene polymorphisms were not tested. Further, the study is relatively small and retrospective, which may impact on the validity of the results.

In conclusion, polymorphisms in MTHFR may interact with other confounding variables and contribute to the increased risk of CHD in DS.

This project was funded by King Abdulaziz City for Science and Technology (KACST), award numbers (AT-34-369).

The authors have no conflicts of interest.