Polymorphisms of Methylene Tetrahydrofolate Reductase (MTHFRC677T and A1298C) and Probable Depression in Postpartum Period in South Indian Women


 Background: Two common variants of Methylene tetrahydrofolate reductase (MTHFR) are C677T and A1298C, which have been associated with general depression. Main objective of the study was to explore how these functional polymorphisms of MTHFR gene associate with the risk to develop postpartum depression. Method: 434 women were recruited in a study period of 3 years (January 2014 to December 2017) in a tertiary care centre. EPDS was administered at 6 weeks postpartum, with a cut-off score of ≥10 to define probable postpartum depression. Plasma was used for the estimation of circulating vitamin B12 metabolites and whole blood for extraction of DNA. Real time PCR using Taqman probes was used for genotyping the proposed SNP’s. Results: T allele of MTHFR C677T was associated with the risk of probable PPD (p = 0.024, OR = 1.67; 95% CI: 1.06-2.62) and presence of suicidal ideations in these women (p<0.001, OR = 2.90; 95% CI: 1.61-5.24). Haplotype 677T-1298A was associated with both the risk of probable PPD (p = 0.029, OR = 1.66; 95% CI: 1.05-2.64) and suicidal ideations (p <0.001, OR = 2.933; 95% CI: 1.60-5.35), whereas 677C-1298A was protective against suicidal ideations (p = 0.011; OR = 0.558; 95% CI: 0.35-0.88). Women with risk allele T were also associated with higher levels of circulating holotranscobalamin levels, and lower levels of vitamin B12, whereas risk allele C of A1298C had lower levels of holotranscobalamin. Limitations: Cross-sectional studyConclusion: Polymorphic variants of MTHFR gene might indicate susceptibility to develop probable depression in postpartum period. SignificanceMTHFR C677T has been widely reported in the literature to be associated with general depression. The association of this variant in susceptibility to develop postpartum depression is not widely studied. We observed T allele of MTHFR C677T to be associated with the risk of to develop depression and suicidal ideation in postpartum period. Higher levels of MMA and holotranscobalamin, and lower levels of vitamin B12 were associated with T allele of MTHFR C677T. Polymorphic variants of MTHFR gene might indicate susceptibility to develop depression in postpartum period.


Introduction
). Both these vitamins play a crucial rule in the remethylation of cellular hcy to methionine, which further leads to the synthesis of S-adenosyl methionine (SAM). SAM is responsible for non-genomic methylations essential for neurotransmitter synthesis and genomic methylation, which regulated reuptake of neurotransmitters. Degradation of hcy also protects the cells from its excess, which can disrupt oxidative state of the cell (Fox and Stover, 2008).
Certain enzymes and proteins play a crucial role in maintaining the optimum cellular concentration and functions of folate and vitamin B12. Reduced activity of methylene tetrahydrofolate reductase (MTHFR) enzyme, a major regulatory enzyme of folate-B12-methionine cycle is associated with depression in general population (Lok et al, 2013;Peerbooms et al, 2011). C677T and A1298C are the most common polymorphic forms of MTHFR gene loci. Both these single nucleotide polymorphism (SNPs) are exonic in location, causing production of a thermolabile enzyme that degrades faster; hence, they can in uence intracellular concentration of metabolites like hcy, along with decreased regeneration of methionine (Frosst et al., 1995;Weisberg et al.,1998). We hypothesize that the polymorphic variants of MTHFR (C677T and A1298C) are associated with the susceptibility to develop postpartum depression in South Indian women. Therefore, in the present study, we aimed to explore the association of MTHFR C677T and A1298C genotypes on probable postpartum depression. We also analyzed the effect of these polymorphisms on the circulating folate and vitamin B12 metabolites. We calculated the sample size for genetic polymorphism using 0.12 as the minor allele frequency of MTHFR gene in Indian population (Sukla and Raman, 2012), and the disease prevalence of 26.3% as reported by Savarimuthu et al., (Savarimuthu et al, 2010) in the software Genetic Power Calculator (Purcell et al, 2003). Sample size was estimated at 5% level of signi cance with 80% power. Estimated sample size considered was 217 subjects in each group (total 434 study subjects) to ful ll one of the study objectives of genotype-phenotype interaction. Written informed re-consent was obtained prior to including women in the present study.

Material And
We de ned probable PPD as Edinburg Postpartum Depression Scale (EPDS), score of ≥ 10, which has shown good validity in measuring depressive symptoms during pregnancy and postnatal depression in this population (Benjamin et al., 2005;Cox et al., 1987). Further group strati cation was done in cases based on presence or absence of suicidal ideation, which was assessed on EPDS item 10: "The thought of harming myself has occurred to me" (yes, quite often; sometimes; hardly ever; never). We excluded women with previous history of depression. Socio-demographic, obstetric and psychiatric history associated with PPD was collected. Women included in the study had their antenatal follow up and delivery in JIPMER, where folate supplements are provided by the hospital as a part of patient care. At this point, we collected 5 ml of venous blood in EDTA vial for plasma metabolites and genotyping.

Genotyping
We checked the quality of extracted DNA using NanoDrop™ 2000 (Thermo Fisher Scienti c Inc., USA) and a minimum 260:280 ratio of > 1.8 acceptable for SNP studies. Samples with a ration of less than 1.8 were re-extracted by commercially available extraction kits (QIAmp DNA Blood mini kit, Qiagen, Germany).
Genotyping for Single Nucleotide Polymorphisms (SNP) MTHFR C677T (assay ID C-1202883-20) and A1298C (assay id C-850486-20) was performed using a Taqman SNP genotyping assay in CFX 96 (BioRad Laboratories Inc., USA), using an allelic discrimination mode. In all, 50 ng DNA was ampli ed in a 10 µL reaction mixture containing 5 µL of Taqman genotyping master-mix (Applied Biosystems), 0.25 µL (5 pmol) of each primer & probe (Applied Biosystems), and 3.75 µL of nuclease free water. The ampli cation conditions consisted of denaturation at 95°C for 10 minutes followed by 40 cycles of denaturation at 95°C for 15 seconds, and then by annealing and elongation at 65°C for 60 seconds. Genotype call rate for both C677T and A1298C was 100%.

Measurement of folate and vitamin B12 metabolites in circulation
Plasma was separated by centrifugation at 2000g for 15 minutes and stored at -80C till further analysis. Determination of vitamin B12, holotranscobalmin (holotc), 5-methyl THF, SAM, Hcy, Methyl malonyl Co-A (MMA), and serotonin in plasma was performed using commercially available ELISA kits.

Statistical methods
We used chi-square test for categorical variables and student t test for continuous variables using SPSS version 20.0 (SPSS Inc., Chicago, USA). To nd the Hardy-Weinberg equilibrium (HWE), and the difference in both allele and genotype frequency, we used chi-square test using online calculator DeFinetti (http://ihg.gsf.de/cgi-bin/hw/hwa1.p1). Further, association of MTHFR genotypes with the risk for probable PPD was carried out by logistic regression analysis by creating dominant, recessive & additive models that was adjusted to signi cantly different socio-demographic, obstetric and nutritional factors between two groups. Correction for multiple testing was carried out by Bonferroni's method. The results were expressed as percentage, odds ratio (OR) and their 95% con dence intervals. Combined genotype frequencies were calculated by direct counting. Haplotype frequencies were estimated and linkage disequilibrium (LD test was performed in

Single locus analysis
Both MTHFR C677T and A1298C were in Hardy Weinberg Equilibrium (p = 0.116 and 0.812 respectively) in healthy controls. Among the two SNP's, the minor allele of MTHFR C677T signi cantly associated with the risk for probable PPD (p = 0.024, OR = 1.67; 95% CI: 1.06-2.62) ( Table 1). Homozygous risk genotype (TT) of MTHFR C677T was more common in women with probable PPD (p = 0.041), whereas we observed no such difference in case of MTHFR A1298C (p = 0.955) ( Table 1).   (Table 3). We found no difference in genotype or allele frequency of A1298C in suicidal ideations (Table 3). The genetic association analyses suggested that MTHFR C677T was signi cantly associated with suicidal ideations in women with probable PPD under dominant model (Table 4). We observed no such association with MTHFR A1298C.

Combined genotypes
In the whole cohort, we observed four common combined genotypes-CC/AC (42.3%), CC/AA (22.8%), CC/CC (18.2%) and CT/AA (8.7%). The distribution of combined genotype did not vary between women with and without probable PPD (p = 0.348). Table 5 summarizes the results of LD analyses and haplotype frequencies of both the polymorphisms of MTHFR gene. We found MTHFR C677T and A1298C to be at high linkage disequilibrium (D'=0.88; r2 = 0.066), with haplotype "677C-1298A" to be the most common in our cohort. The distribution of haplotype frequencies was also different between two groups with "677T-1298A" being more prevalent in women with probable PPD (Table 5). On strati cation of women with probable PPD by the presence or absence of suicidal ideation, haplotype "677T-1298A" conferred a high risk for suicidal ideation, whereas "677C-1298A" was found to be protective (Table 6).

Association of MTHFR gene polymorphisms with circulating vitamin B12 metabolites
A Kruskal -Wallis H test was run to determine if there were differences in the metabolite levels among MTHFR genotypes. In the whole cohort (n = 434), we observed risk allele T of MTHFR C677T to be associated with higher levels of holotc (Fig. 1). Pair wise analysis of holotc showed signi cant differences between CC versus TT genotypes (p = 0.045), and CT versus TT genotypes (p = 0.044). Lower vitamin B12 levels were observed with the T allele of MTHFR C677T, although it was not statistically signi cant (Fig. 1). On the other hand, risk allele C of MTHFR A1298C associated with lower holotc levels (Fig. 2). MTHFR A1298C genotype did not show signi cant associations with any other circulating metabolite. Our results showed that the T allele of MTHFR C677T has association with the risk and severity (in terms of suicidal ideations) of PPD. Women with the risk allele T had higher levels of holotc, and lower levels of vitamin B12. Further, haplotype 677T-1298A associated with both the risk of probable PPD and suicidal ideations, whereas 677C-1298A was found to be protective against suicidal ideations. These ndings support our hypothesis that polymorphic variants of MTHFR gene might be associated with probable depression in postpartum period.

Discussion
Similar to our study, T allele of MTHFR C677T associated with general depression in Irish (Kelly et al., 2004), Norway (Folate, 2003), We found that haplotype 677T-1298A to be associated with the risk of probable PPD. It also conferred a high risk for suicidal ideation, whereas 677C-1298A was found to be protective against severe PPD. No study to date has examined the association of these haplotypes with the risk and severity of depressive symptoms in postpartum period.
On analyzing the association of MTHFR C677T and A1298C with circulating folate and vitamin B12 metabolites, we observed that the risk allele T of MTHFR C677T associated with higher levels of holotc and lower levels of vitamin B12 (Figure 1), whereas risk allele C of MTHFR A1298C associated with lower holotc levels (Fig 2). Rise of holotc levels with the risk allele of MTHFR C677T might be interpreted as a compensatory mechanism of the body to decreased vitamin B12 levels. The effect of MTHFR A1298C on vitamin B12 and holotc levels need to be con rmed further in larger prospective studies.
Strength of this study is the inclusion of ethnically similar population (Dravidians), where all the women received folate supplementation during pregnancy.
One limitation of our study is its cross-sectional nature. As, we could not follow these women for a longer time; effect of MTHFR genotype on the progression of probable PPD could not be determined. Also, there may be other social factors contributing to depression in postpartum period other than the ones collected by us in the study, which may in uence the results.

Conclusions
In conclusion, we observed T allele of MTHFR C677T and 677T-1298A compound genotype to be associated both with the risk of developing probable depression and suicidal ideations in the postpartum period. Haplotype 677C-1298A was protective against the risk of suicidal ideations. Risk allele T MTHFR C677T was also associated with increased holotc levels and decreased vitamin B12 levels. These preliminary results might carry a translational value in recognizing women susceptible to depression in postpartum period and should be con rmed further in larger, prospective studies.