Schizophrenia is a mental illness that is characterized by three kinds of symptoms: positive symptoms, or things caused by schizophrenia, include hallucinations, paranoia and disordered thoughts; negative symptoms, or things schizophrenia interferes with, include loss of interest in activities, lack of emotion, and loss of motivation; finally, cognitive symptoms are the ways schizophrenia interferes with comprehension. These include memory loss and difficulty understanding information. The prevalence rate of this disease is ~1%. The pathophysiology of this disease is not fully known, but studies shows that several components might be involved in this condition such as- 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 or neurotransmitters (Mudd et al., 2001).
Methylenetetrahydrofolate reductase (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. In this a point mutation occurs at 677th position which results in a C to T transition, this makes a substitution of an alanine to valine residue at 222nd position in the MTHFR protein. This change makes the protein thermolabile means its enzymatic activity reduces at high temperature so the re-methylation of homocysteine is reduced which results in the elevation of plasma homocysteine and also a higher homocysteine to methionine level this ultimately results in low SAM so the methylation is also affected.
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The present study was designed to check the association of MTHFR C677T with schizophrenia. Since studies exploring MTHFR C677T polymorphism in schizophrenia show conflicting results, so we performed a meta-analysis with the previous published literature and our data is also included.
MATERIALS AND METHOD
After taking ethical clearance from the Institutional Ethics Committee, a total of 85 cases of Schizophrenia were recruited from the OPD of Department of Psychiatry of Swaroop Rani Nehru Medical hospital, affiliated to, Motilal Nehru Medical College, Allahabad for this study. The diagnosis criterion was based on DSM IV. The age group was 13-70 years and mean age was 34.82 Â± 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 Â± 14.08 years. Informed written consent was taken prior to blood sample collection. Blood samples were collected in EDTA coated vials and genomic DNA was extracted by the method of Bartlett and White (2003). 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.
The chi square (Ï‡2) 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 in between the cases and controls. Statistical analysis was performed on OpenEpi version 2.3. All statistical tests were two-sided and differences were taken as significant when p-value was less than 0.05.
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, short communication, letter to editor and case reports were excluded. Care was also taken to check those studies where authors were the same. After this, 26 articles remained out of which in 3 articles data was un-retrieval and two articles by the same author so at final 20 articles are found suitable to include in this study including our study. 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).
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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 geography means the region from where the study belonged i.e. Asia, Europe or America. 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 the MIX version 1.7.
The control population was in Hardy-Weinberg Equilibrium (p= 0.606) and it is pure from inbreeding (inbreeding coefficient F = -0.055). Power of this study was 80% (Quanto). 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) and Chi square was 3.892 (p=0.048).
The meta-analysis comprises 3939 cases of schizophrenia and 4566 controls. Out of 20 studies included in this meta-analysis, 12 studies were from Asia, 5 from Europe, 2 from America and 1 from Africa. We also performed sub-group analysis which is based on geographic distribution of population. In combined and in Asian meta-analysis low heterogeneity was observed, in European sub-group meta-analysis moderate heterogeneity was observed while in American studies no heterogeneity was found.
For combined meta-analysis I2 was 56.14% so we used fixed effect model and OR was 1.17 (95% CI: 1.10-1.25) and publication bias was also found p=0.038. In Asian studies a total of 2587 cases and 2846 controls were observed, I2 = 57.67% so fixed effect model was applied and OR was 1.18 (95% CI: 1.09-1.28) and publication bias was also found p=0.021. While the European studies comprises 1117 cases and 1498 controls and they have shown slight heterogeneity with I2 = 66.68% and random effect model was applied. The OR was 1.17 (95% CI: 0.95-1.44) and no publication bias was found p=0.700. In American studies a total of 210 cases and 197 controls were observed, no heterogeneity was found I2 = 0% and fixed effect OR was 1.18 (95% CI: 0.89-1.56).
In the present study we found that the T allele of MTHFR C677T is strongly associated with schizophrenia in our population. Furthermore, result of meta-analysis also shows little association of T allele with schizophrenia.
The frequency of T allele greatly varies in different ethnic groups. Phase 1 of 1000 genomes project reveals the overall frequency of 0.32 of minor allele. In African it is lowest only 0.11 and in American it is highest 0.49, while in European it is 0.35 and in East Asian it is 0.37 (http://asia.ensembl.org/Homo_sapiens/Variation/Population?db=core;r=1:11855878-11856878;v=rs1801133;vdb=variation;vf=1366988). In the Indian population, frequency of this SNP is found to be 0.14 (Indian Genome Consortium), which is considerably lower than that reported for CEU (0.24), CHB (0.51) and JPT (0.36) and close to YRI (0.11) population (HapMap data). More specifically, in Eastern Uttar Pradesh the frequency of this minor allele is 0.12 (Rai et al, 2012). In the present study we get considerably low frequency of T allele in the control samples.
MTHFR 677T allele shows reduction in its enzymatic activity, so it can not efficiently carried out the conversion of homocysteine to methionine (REF). There is an alternate pathway for the regulation of homocysteine by CÎ²S, but in brain this pathway is suspected (REF), so the level of homocysteine is high in the brain. Increased level of homocysteine has been linked to various disorders/diseases such as neural tube defects (van der Put et al., 1995), schizophrenia (Allen et al., 2008; Muntjewerff et al., 2006), depression (Lewis et al., 2006), Down syndrome (Hobbs et al., 2000), cardiovascular disease (Kluijtmans et al., 1996) and hypertension (Qian et al., 2007). High level of plasma homocysteine decreases the level of SAM so it affects the intracellular methylation process of DNA, lipids, proteins, and neurotransmitters. 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).
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Although our study has a power of 80% but more number of samples are required to make a clear cut association of MTHFR 677T allele and schizophrenia. In our study a major number of patients belong to cities. In India a major portion of patients who might be showing the positive symptoms of schizophrenia did not go to psychiatrists as their relatives thought that it is something supernatural phenomenon and rather then going to a psychiatrist they prefer to visit any priest.
Moreover, the brain of schizophrenic patients shows altered methylation pattern (Singh et al, 2003).
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Case N (%)
Control N (%)
OR (95% CI), p-value
1.83 (0.76-4.5), 0.165
5.58 (0.26-118.35), 0.137