Association Of Mthfr C677t Polymorphism And Schizophrenia

Published Date: 02 Nov 2017

1. Introduction

Schizophrenia is a complex psychiatric disorder with a lifetime risk of ~1%. The pathophysiology of this disease is not yet fully known. Three theories were proposed for the explanation of etiopathogenesis of schizophrenia. Two of them were biochemical hypotheses- dopaminergic and serotonergic mechanism (Carlsson et al., 1963; Crow, 1980; Weinberger et al., 1987). The third one is neurodevelopmental hypothesis considering interaction between genetic risk and environmental factors contributing to the development of this disease (Vilella et al., 2005). Numerous linkage analyses and candidate gene studies have been conducted for schizophrenia (Badner et al., 2002; Kirov et al., 2005; Lewis et al., 2003). By these studies a no. of genes are found to be associated to this condition, one of the important gene is Methylenetetrahydrofolate reductase (MTHFR). This gene is related to the altered DNA methylation, folate deficiency and high level of homocysteine. The level of homocysteine is regulated by the folic acid metabolism/ homocysteine pathway. Elevated homocysteine level affects the methylation of DNA, proteins, lipids and neurotransmitters (Mudd et al., 2001).

MTHFR is located at the short arm of 1st chromosome (1p36.3). This complementary DNA sequence is 2.2 kb long and has 11 exons (Goyette et al., 1998). It makes a protein of 77KD, methylenetetrahydrofolate reductase, which has 656 residues. MTHFR catalyzes the 5, 10- methylenetetrahydrofolate to 5-methylenetetrahydrofolate which gives its methyl group to homocysteine and homocysteine converted into methionine. Now this methionine converted into S-adenocylmethionine (SAM), which is the universal methyl donor for all the cellular methylation reactions. So far, more than 40 polymorphisms are reported in this gene, but the most studied polymorphism is C677T, which lies at the 4th exon. In this a point mutation occurs at 677th position which results in a Cytosine to Thymine transition, this makes a substitution of an Alanine to Valine residue at 222nd position in the MTHFR protein (Frosst et al., 1995; Rozen et al., 1997). This change makes the protein thermolabile (Frosst et al., 1995).

Several studies have shown association of MTHFR C677T polymorphism with schizophrenia (Arinami et al, 1997; Joober et al, 2000; Kempisty et al, 2006; Feng et al, 2009 and Lajin et al, 2012) though; studies showing negative results are also reported (Muntjewerff et al, 2003; Philibert et al, 2006 and Tsutsumi et al, 2011). Therefore, the present study was designed to evaluate the role of MTHFR C677T polymorphism in the susceptibility to schizophrenia. Since studies exploring role of MTHFR C677T polymorphism in schizophrenia show conflicting results, we also performed Meta analysis of this polymorphism in schizophrenia.

2. Materials and methods

2.1. Study Subjects

After taking ethical clearance from the Institutional Ethics Committee, a total of 85 cases of Schizophrenia were recruited for the present study from the OPD of Department of Psychiatry of Swaroop Rani Nehru Medical hospital, affiliated to, Motilal Nehru Medical College, Allahabad. The age group was 13-70 years and mean age was 34.82 ± 14.3 years. The same number of healthy controls was also taken for this study, the age group of control samples was 15-70 years and mean age was 34.4 ± 14.08 years. Informed written consent was taken prior to blood sample collection.

2.2. Sample collection

Blood samples were collected in EDTA coated vials and genomic DNA was extracted by the method of Bartlett and White (2003). The DNA was stored in -200C until genotyping.

2.3. Genotyping

Genotyping was performed by PCR-RFLP method as described by Frosst et al (1995). MTHFR C677T PCR product (198 bp) was digested with Hinf I restriction enzyme at 370C, and genotype was classified as CC (198 bp), CT (198 bp, 175 bp and 23 bp) and TT (175 bp and 23 bp). PCR product and digested amplicon were visualized on 2% agarose gel.

2.4. Statistical analysis

The chi square (χ2) goodness of fit test was performed on control samples for any deviation from Hardy Weinberg Equilibrium by using online program available at http://ihg.gsf.de/cgi-bin/hw/hwa2.pl. The Odds ratio (OR) and chi square test was performed to check the association of the cases and controls. Statistical analysis was performed by computer program OpenEpi version 2.3 (Dean et al., 2009). All statistical tests were two-sided and differences were taken as significant when p-value was less than 0.05.

2.5. Meta-analysis

NCBI PubMed database was searched on 13th October, 2012 with the key word "MTHFR and Schizophrenia" and a total of 80 articles were retrieved from year 1994 to 2012. Articles in languages other than English, review, non-relevant, and case reports were excluded. Care was also taken to check those studies where authors were the same. After this, 28 articles remained out of which in 3 articles data was un-retrieval and two articles by the same author, so the older study was removed (Muntjewerff et al., 2005). In this way 24 articles were found suitable to fit our inclusion criteria (Figure 1). We also added our present study so we had 25 studies. In 1 study (Yu et al., 2004) two populations was taken one from China and other from Scotland. So this study also divided into two groups according to ethnicity. From all the articles which follow the inclusion criteria the following information was extracted: author, country where the work had been done, polymorphisms studied, number of controls and cases, year and journal (Table 1).

The pooled Odds Ratio and 95% CI were used to assess the association of T allele of MTHFR C677T and schizophrenia. We had also done sub-group analysis based on ethnicity i.e. Caucasian and Asian. The OR was estimated either by using fixed effects (Mantel–Haenszel) or random effects (DerSimonian and Laird) models depending upon heterogeneity. Heterogeneity in meta-analysis refers to the variation among study outcomes. I2 statistics is a measure to check the heterogeneity in between the studies. It is the percentage of total variation across studies due to heterogeneity rather than chance. If I2 > 50% then random effect model was used (which gives wider confidence intervals) otherwise fixed effect model applied. To assess the publication bias funnel plot was made and to detect the asymmetry of funnel plot Egger test was performed and p<0.05 was considered as statistically significant publication bias. All the Meta-analysis was performed by MIX program version 1.7 (Bax et al., 2006).

3. Results

3.1. Association of MTHFR C677T polymorphism and schizophrenia

The control population was in Hardy-Weinberg Equilibrium (p= 0.606) and it is pure from inbreeding (inbreeding coefficient F = -0.055). The power of this study was 80% (Quanto) (Gauderman and Morrison, 2006). The most prevalent genotype is CC in both the cases and controls. The frequency of minor allele was 0.111 and 0.052 in cases and controls respectively. There is a significant association in between the cases and control with OR=2.246 (95% CI: 0.995-5.36), p=0.048. (Table: 3)

3.2. Meta-analysis

3.2.1. Characteristics of included studies

The meta-analysis comprises 5731 cases of schizophrenia and 7603 controls. Out of 26 studies included in this meta-analysis, 13 studies were reported for Asian population and 12 for Caucasian and 1 from African population. Since African population has only one study so we excluded it from the sub-group analysis and only Asian and Caucasian populations were included in the sub-group analysis. Asian studies comprise 2817 cases and 3097 controls while Caucasian studies have 2889 cases and 4481 controls.

3.2.2. Qualitative synthesis

Pooled summary estimate, heterogeneity test and publication bias results for the association of MTHFR C677T allele/genotype and schizophrenia risk are shown in Table: 4. Furthermore, we categorized studies on the basis of ethnicity of study subjects: Asian and Caucasian. MTHFR C677T polymorphism shows association in overall studies and in Asian population but no such association was observed in the Caucasian population.

3.2.3. Publication bias

Publication bias was found in overall studies but no publication bias was found in Caucasian sub-group while it is present in the Asian studies.

4. Discussion

In the present study T allele of MTHFR C677T is strongly associated with schizophrenia in our North Indian population. Furthermore, result of meta-analysis shows that the T allele and TT genotype are association with schizophrenia, association was also found in the dominant and recessive models but no such association was found with CT genotype (see table 4). Our meta-analysis results were similar to the previously published meta-analyses. In the allelic model we found the OR of 1.15 (p=0.001) which is similar to Allen et al., 2008 OR= 1.16, Gilbody et al., 2006 OR= 1.17, and Zintazras, 2006 OR= 1.14. In the TT vs. CC model our OR was 1.34 (p=0.003) which is also similar to Lewis et al., 2004 (OR= 1.48), Muntjewerff et al., 2006 (OR= 1.36) and Zintazaras, 2006 (OR= 1.37). In the sub-group analysis Asian studies were shown association but no association was found in the Caucasian sub-group. Publication biasness was found in the overall analysis and in the Asian sub-group but not in the Caucasian sub-group analysis. The reason for publication biasness might be the exclusion of some of the studies from Asia which were in the languages other than English.

The frequency of T allele greatly varies in different ethnic groups. According to the HapMap database (http://hapmap.ncbi.nlm.nih.gov): the highest frequency is found in Chinese [Han Chinese in Beijing, China (CHB)] i.e 0.48, 0.31 in European [Utah residents with Northern and Western European ancestry (CEU)], 0.38 in Japanese [Japanese in Tokyo, Japan (JPT)], in Indians it is 0.16 [Gujarati Indians in Houston, Texas (GIH)] and in Africans it ranges from 0.08-0.12 [African ancestry in Southwest USA (ASW), Yoruban in Ibadan, Nigeria (YRI), Luhya in Webuye, Kenya (LWK) and Maasai in Kinyawa, Kenya (MKK)]. In the Indian population, frequency of T allele is 0.14 (Indian Genome Consortium). More specifically, in Eastern Uttar Pradesh the frequency of this minor allele is 0.12 (Rai et al, 2012 a and b). In the present study we get considerably low frequency of T allele in the control samples than the previously reported frequency of this gene in Eastern Uttar Pradesh population.

The T allele of the MTHFR gene shows reduction in its enzymatic activity, so it can not efficiently carried out the conversion of homocysteine to methionine (Leclerc et al., 2003) hence, the level of homocysteine increases. Increased level of homocysteine has been linked to various disorders/diseases such as neural tube defects (van der Put et al., 1995), depression (Lewis et al., 2006), Down syndrome (Hobbs et al., 2000), cardiovascular disease (Kluijtmans et al., 1996) and hypertension (Qian et al., 2007). Homocysteine is rapidly taken up by the neurons through a specific membrane transporter, resulting in high level intracellular homocysteine (Grieve et al., 1992). Since the brain lacks two major metabolic pathways for the elimination of homocysteine: the betaine remethylation and transsulfuration so the brain may be particularly vulnerable to high level of homocysteine (Finkelstein, 1998). Elevated homocysteine level along with their oxidized derivatives, such as homocysteic acid and homocysteine sulfinic acid, has been shown to be toxic for neurons and vascular endothelial cells (Zou and Banerjee, 2005). Plasma homocysteine regulation is depends on various genes involved in the homocysteine metabolic pathway and by environmental factors such as folate or vitamin B12 intake. MTHFR plays a major role in this pathway as it converts 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which serves as a carbon donor for the methylation of homocysteine, and generates S-adenosylmethionine (SAM).

Although our study has a power of 80% but more number of samples are required to make a clear cut association of MTHFR C677T with schizophrenia. Moreover, the frequency of MTHFR C677T varies from population to population and the prevalence of the schizophrenia is constant between populations (~1%). It suggests that gene-gene and gene-environmental interactions play some role in the development of this disease so for the future studies these factors should also be considered.

5. Conclusion

In summary, MTHFR C677T polymorphism plays a role in the development of schizophrenia in north Indian population. Moreover, evidence from Meta-analysis further reinforces the association of MTHFR C677T polymorphism with schizophrenia.