Association of Common Methylenetetrahydrofolate Reductase Polymorphisms with Congenital Cardiac Defect and Hypothyroidism in Down Syndrome Children

INTRODUCTION

Down syndrome (DS) is one of the most common aneuploidies, with the incidence of 1 in 700–1000 live births.^[1-3] Its main cause is trisomy of chromosome number 21, and nondisjunction is the major pathophysiology in 95% of DS individuals. The exact molecular mechanism leading to an increased risk of nondisjunction in elderly mothers or young mothers is not fully clear. Abnormal folate metabolism has been implicated in DS since it was first documented in 1999.^[2,4] The folate pathway is involved in several cellular events, including methylation, deoxyribonucleic acid (DNA) synthesis, and other vital processes in cell metabolism. In the folate pathway depicted in Figure 1, the rate-limiting enzyme is methylenetetrahydrofolate reductase (MTHFR) which removes a methyl group from 5-10-methylene-tetra-hydro-folate (5, 10-MTHF) and converts it to 5-methylenetetra-hydro-folate (5-MTHF). The removed methyl group is crucial for the conversion of homocysteine to methionine. Following this conversion, methionine is transformed into S-adenosyl methionine (SAM), an essential compound involved in the methylation of nucleic acids and the process of cellular differentiation. Till now, several single-nucleotide polymorphisms have been studied in the MTHFR gene; however, C677T (rs1801133) and A1298C (rs1801131) are functionally known to be most relevant. For example, the MTHFR C677T polymorphism leads to a substitution of alanine with valine (p.Ala222Val) within the enzyme’s catalytic domain. This alteration reduces the enzyme’s activity, with individuals who are homozygous for this variant experiencing a reduction in enzymatic function by 50–60% at a temperature of 37°C and by approximately 65% at 46°C due to heightened sensitivity to heat.^[5] Similarly, the MTHFR A1298C (p.Glu429Ala) affects the regulatory domain, reducing enzymatic activity. Reduced MTHFR activity due to these two variations leads to hyperhomocysteinemia, an established risk factor for cardiovascular diseases. In addition, elevated homocysteine levels are observed in hypothyroid patients due to impaired activity of methionine synthase, an enzyme dependent on thyroid hormones. Similarly, children with congenital cardiac defects (CCDs) often exhibit elevated homocysteine levels, likely due to oxidative stress and hypoxia associated with this condition.^[6] Moreover, multiple comorbidities such as CCD, thyroid disorders, gastrointestinal tract problems such as coeliac disease, and respiratory infections, including otitis media, are frequently observed in DS from childhood. A meta-analysis involving over 800 hypothyroidism patients also reported the overrepresentation of MTHFR C677T polymorphism.^[7] Furthermore, associations of C 677T and A1298C variants and CCD in DS are reported.^[8] Maternal MTHFR polymorphisms are also known to be linked with an increased likelihood of having a child with DS.^[9] Despite these findings, the relationship of these two functionally relevant MTHFR polymorphisms, along with levels of homocysteine as well as cysteine in DS children with comorbidities such as hypothyroidism and/or CCD, remains poorly understood. This study aims to investigate any possible association of MTHFR C677T and A1298C polymorphisms with homocysteine and cysteine levels, as well as their correlation (if any) with various comorbidities in DS children. Evaluating these variants/factors may unravel the molecular mechanisms underlying these comorbidities in DS children and assist potential future therapeutic interventions.

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Figure 1:

Overview of folate pathway and MTHFR activity of one carbon metabolism in DS patients. MTHFR is a crucial enzyme in the folate system, that regulates methionine and homocysteine synthesis. When compared to controls, DS patients have higher frequencies of polymorphisms MTHFR C677T and MTHFR A1298C. These variants lower MTHFR activity, which in turn causes homocysteine accumulation in DS patients which further gets converted to cysteine by enzyme CBS which is present on chromosome 21 and its activity is high in DS patients that leads to higher levels of cysteine in the plasma of DS patients. Elevated levels of homocysteine and cysteine further raise the risk of several comorbidities in DS patients. SHMT: Serine hydroxy methyl transferase, MTHFR: Methylene tetrahydrofolate, reductase, SAM: S –Adenosyl –Methionine, SAH: S –AdenosylHomocysteine, CBS: Cystathione beta synthase, MTHFR: Methylenetetrahydrofolate reductase, DNA: Deoxyribonucleic acid.

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MATERIAL AND METHODS

RESULTS

Homocysteine and cysteine levels in trisomy 21 patients versus healthy controls

No statistically significant difference was observed in the homocysteine levels between individuals with DS and healthy euploid controls. However, individuals with trisomy 21 having both conditions, CCD and hypothyroidism, exhibited significantly higher homocysteine levels (P < 0.05) compared to euploid controls and DS individuals without both these conditions. Details are encrypted in Table 4 and Supplementary Figure 2a and b. Similarly, cysteine levels were significantly higher in DS individuals compared to euploid controls (P < 0.05). Moreover, all DS subgroups (those with CCD, hypothyroidism, or both) had elevated cysteine levels compared to DS individuals without both these conditions (P < 0.05) [Table 4, Supplementary Figure 3a and b].

Table 4: Comparison of plasma homocysteine and cysteine levels among DS subgroups and controls.

Clinical subgroup	Significance homocysteine	Median homocysteine	95% CI		Significance cysteine	Median cysteine	95% CI
			Lower bound	Upper bound			Lower bound	Upper bound
Reference category is controls
DS without both CCD and hypothyroidism	0.990	10.01	-9.4	6.68	0.952	257	-41.78	71.98
DS with CCD	0.869	12.87	-4.15	8.7	0.000	368	34.34	125.22
DS with Hypothyroidism	0.843	13.35	-4.69	10.37	0.287	281	-14.96	91.08
DS with both CCD, and Hypothyroidism	0.031	15.4	0.45	15.22	0.000	343	28.52	132.52
Reference category is DS without CCD and hypothyroidism
DS with CCD	0.737	12.87	-4.5	11.90	0.019	368	6.8	122.52
DS with Hypothyroidism	0.710	13.35	-4.8	13.32	0.865	281	-40	86.92
DS with both CCD, and Hypothyroidism	0.040	15.4	0.26	18.19	0.038	343	2.3	128.54
Reference category is DS with CCD
DS with Hypothyroidism	1.000	13.35	-7.13	8.22	0.217	281	-95.76	12.31
DS with both CCD, and Hypothyroidism	0.263	15.4	-1.99	13.07	1.000	343	52.29	53.77
Reference category is DS with hypothyroidism
DS with both CCD, and Hypothyroidism	0.493	15.4	-3.4	13.47	0.296	343	-17.19	102.12

Median homocysteine and cysteine concentrations and 95% confidence intervals (CI) are shown for DS individuals stratified by the presence of CCD, hypothyroidism, or both, compared to healthy controls. The Kruskal–Wallis test was used to evaluate overall differences across groups, followed by Scheffé post hoc test for pairwise comparisons using different reference categories. Statistical significance was defined as P < 0.05. The 95% CI indicates the range within which the true median difference is likely to fall. A statistically significant increase in homocysteine levels was observed in DS individuals with both CCD and hypothyroidism compared to controls (P = 0.031) and to DS individuals without either comorbidity (P = 0.040). Other subgroup comparisons did not show statistically significant differences. Similarly, significantly elevated cysteine levels (P < 0.05) were observed in DS individuals with CHD and those with both CHD and hypothyroidism compared to controls and without both CCD and hypothyroidism. No significant differences were found in other subgroup comparisons. Note: Reference range of plasma homocysteine (5–-10 ng/mL) and cysteine (250- –340 ng/mL). CCD: Congenital cardiac defect, DS: Down syndrome

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Figure 2:

ROC curve analysis of cysteine, homocysteine, MTHFR C677T, and MTHFR A1298C in detecting different comorbidities in DS. (a) ROC analysis in DS individuals without both CCD and hypothyroidism. (b) ROC analysis of CCD in DS individuals. (c) ROC analysis of hypothyroidism in DS individuals. (d) ROC analysis of both CCD and hypothyroidism in DS individuals. ROC: Receiver operating curve, MTHFR: Methylene tetrahydrofolate, reductase, DS: Down syndrome, CI: Confidence interval, CCD: Congenital cardiac defects.

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DISCUSSION

In this study, we found that there is a significantly higher frequency of MTHFR 677T (0.146) and 1298C (0.49) variants in DS children compared to controls and the normal North Indian population 0.107 and 0.20.^[12,14] We also observed that over-representation of these MTHFR variants (677T and 1298C), along with higher levels of homocysteine and cysteine in DS children, is associated with hypothyroidism and CCD, and higher levels of homocysteine are also positively associated with the presence of 677T variant.

The enzyme MTHFR is crucial for the conversion of 5, 10-MTHF to 5-MTHF and serves as a key methyl donor in the biochemical process of converting homocysteine to methionine. Disruptions in this enzymatic activity can lead to the accumulation of homocysteine, potentially interfering with DNA and ribonucleic acid (RNA) synthesis. MTHFR C677T and A1298C polymorphisms often result in reduced MTHFR activity, leading to a deficiency in SAM, a vital methyl donor for DNA methylation. Since both DNA and histone methylation are key regulators of gene expression, disruptions in these processes can impact chromatin structure, gene expression, DNA replication, and repair mechanisms. A study found that the presence of MTHFR 1298C and 677T decreases the activity of the enzyme. Heterozygotes (677CT) decrease the activity of the enzyme by 30% while homozygotes (677TT) decrease the enzyme activity by 60%. 1298AC lies in the C-terminal regulatory site, decreasing the activity of the enzyme by 10% only. Due to a decrease in the activity of the MTHFR enzyme, the levels of homocysteine increase in plasma. Several studies also found that these polymorphisms significantly increase the levels of homocysteine. ^[15-17] In a similar vein, we observed the most significant elevation in homocysteine levels in DS patients having 677CT genotype compared to those with 677CC genotype. However, no significant difference in the levels of homocysteine was observed in DS patients with 1298AA, AC, CC genotypes.

Various studies and meta-analyses found that “MTHFR C677T and A 1298C variants are associated with an increased risk of DS offspring.”^[6,18,19] However, very scant research has been performed on the prevalence of these common polymorphisms in DS patients. As only a few studies have been found in the literature on the prevalence of 677T and 1298C in DS patients and they found a higher prevalence of both these variants in DS patients as compared to controls. ^[20-23]

A study found significantly higher (P = 0.009) transmission of 677T allele to DS infants from parents.^[24] Various meta-analyses found that MTHFR C677T polymorphism is significantly associated with stroke. As in previous studies, it has been found that C677T increases the risk of hemorrhagic and ischemic stroke by 1.61 and 1.30 times.^[25] Previous studies described that the frequency of this polymorphism is significantly higher in the Asian pediatric population with stroke and is also a risk factor for stroke in children.^[26] Similarly, a study conducted by a member of our team revealed that the MTHFR C677T polymorphism is significantly more common in stroke patients, with a frequency of 18.8% in stroke cases, compared to 11% in the general North Indian population.^[12,27] Hence, in the current study on comparing the allelic frequency of 677T in DS children and the general North Indian population, we found that DS children had 3.736-fold higher occurrence of MTHFR C677T polymorphism. In addition to stroke, studies have identified the significant association of C677T with recurrent pregnancy loss in Asian and East Asian populations, Alzheimer’s disease, and thrombosis.^[28-31] Given that DS is associated with an elevated risk of stroke, Alzheimer’s disease, and pulmonary hypertension.^[32] It is plausible that the MTHFR 677T polymorphism may serve as a modifier for some of these comorbidities in DS patients. Our study also supports this hypothesis, as we observed a significant overrepresentation of the MTHFR C677T genotype in DS patients, particularly in those with hypothyroidism. Furthermore, the findings of this study are consistent with a meta-analysis that revealed a strong association between the C677T polymorphism and hypothyroidism.^[33] This study is the first on the North Indian population to demonstrate the frequency of 677T in individuals of DS having hypothyroidism as compared to controls and DS patients without comorbidities. This novel observation underscores the potential role of the C677T in modulating thyroid function in DS patients.

With regard to the A1298C polymorphism, various studies have found an association between homozygous 1298CC and CCD and atrioventricular septal defect in DS patients.^[33-37] This study also identified a significant association of the homozygous 1298CC genotype with both CCD and hypothyroidism in DS children. In contrast, no significant association was observed between the heterozygous 1298AC genotype with any of the comorbidities in DS, and this suggests that homozygous 1298CC is significantly associated with both CCD and hypothyroidism in individuals with DS.

As consistent with literature findings, this study also observed a significant association of higher levels of homocysteine with DS patients with MTHFR 677T variant and with both CCD and hypothyroidism.^[38-40] As CCD frequently involves shunting of systemic blood to pulmonary circulation, leading to hypoxic conditions that further promote the hyperhomocysteinemia by decreasing the activity of methionine synthase. Moreover, thyroid hormones (T3 and T4) are required for the normal functioning of methionine synthase, and deficiency of these hormones in hypothyroid patients may reduce the enzymatic conversion of homocysteine to methionine and lead to hyperhomocysteinemia, which is recognized as an independent risk factor for various vascular diseases. As it impairs the inner lining of endothelium and leads to reduced synthesis of nitric oxide, a potent vasodilator.^[38]

DS or trisomy 21 is the most common chromosomal aneuploidy associated with various co-morbidities such as “intellectual disability, otitis media, hypothyroidism, coeliac disease, Alzheimer’s disease, autism, CCD, psychosis, epilepsy, Acute megakaryocytic leukemia (AMKL).” To the best of our knowledge, this is the first study to investigate the relationship between MTHFR polymorphisms and the plasma levels of homocysteine and cysteine levels in children with DS from the North Indian population, with a particular focus on their potential association with common comorbidities. Understanding these molecular interactions is essential for elucidating the underlying mechanisms of DS and may help identify biomarkers or genetic risk factors that predispose individuals to associated health conditions.

CONCLUSION

The folate pathway is altered in CCD and hypothyroidism, and these two comorbidities occur very commonly in DS patients. MTHFR plays a key role in the folate pathway. But till now, very scant studies are performed on MTHFR polymorphisms in DS. Hence, in this, we checked the frequency of two most common MTHFR polymorphisms, levels of cysteine and homocysteine in trisomy 21 patients with different comorbidities, and it was discovered that differential representation of all these parameters constitutively acts as predictive biomarkers of hypothyroidism and both CCD and hypothyroidism in DS patients and can improve prognostication and counseling in families with trisomy 21 children.