Gene-wide association study between the methylenetetrahydrofolate reductase gene (MTHFR) and schizophrenia in the Japanese population, with an updated meta-analysis on currently available data

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Abstract

Methylenetetrahydrofolate reductase (MTHFR) is a critical molecule for single-carbon transfer reactions. Recent evidence suggests that polymorphisms of MTHFR are related to neural tube deficits and the pathogenesis of schizophrenia. While several studies have demonstrated associations between the gene encoding the MTHFR (MTHFR) polymorphisms and schizophrenia, these studies lack consistency. Therefore, we conducted a gene-wide association study (patients with schizophrenia = 696, control subjects = 747) and performed imputation analysis. Additionally, we performed meta-analysis on currently available data from 18 studies for two common functional polymorphisms (rs1801131 and rs1801133).

There were no significant associations with schizophrenia in the single marker analysis for the seven tagging SNPs of MTHFR. In the haplotypic analysis, a nominally significant association was observed between the haplotypes, which included four SNPs (rs1801133, rs17421511, rs17037396, and rs9651118) and the schizophrenic patients. Additionally, the imputation analysis demonstrated there were several associated markers on the MTHFR chromosomal region. However, confirmatory analyses of three tagging SNPs (rs1801133, rs17037396, and rs9651118) and the top SNP (rs17421511) for the imputation results (patients with schizophrenia = 797, control subjects = 1025) failed to replicate the haplotypic analysis and the imputation results. These findings suggest that MTHFR polymorphisms are unlikely to be related to the development of schizophrenia in the Japanese population. However, since our meta-analysis results demonstrated strong support for association of rs1801133 with schizophrenia, further replication studies based on a gene-wide approach need to be considered.

Introduction

Schizophrenia is a chronic and disabling mental disorder with a lifetime prevalence of approximately 1% in the global population (Freedman, 2003). Accumulating evidence suggests that both genetic and environmental factors contribute to the etiology of schizophrenia (Burmeister et al., 2008). Although schizophrenia has a high heritability with rates estimated at 80% (Sullivan et al., 2003), there has been no consistent replication found for the schizophrenia candidate genes (Harrison and Weinberger, 2005). Recent genome-wide association (GWA) studies have demonstrated new promising susceptibility genes for schizophrenia (O'Donovan et al., 2008), as well as for other common diseases (Rioux et al., 2007, The Wellcome Trust Case Control Consortium, 2007, Zeggini et al., 2007). Therefore, use of this methodology can be advantageous when trying to detect potential genetic factors responsible for the development of these disorders. In addition, by focusing on the specific molecular pathway related to the pathophysiology of schizophrenia, this may also be useful when trying to identify susceptibility genes that have a mild contribution to the development of the disease (Kirov et al., 2005).

Dysfunction of homocysteine metabolism has been linked to neurodevelopmental disorders, including neural tube defects (NTDs) (Blom et al., 2006, van der Put et al., 1995), schizophrenia (Allen et al., 2008, Muntjewerff et al., 2006), and depression (Lewis et al., 2006), in addition to other diseases and syndromes (Hobbs et al., 2000, Kluijtmans et al., 1996, Qian et al., 2007). Recent studies have also suggested that elevated plasma homocysteine levels are observed in major psychiatric disorders such as schizophrenia and bipolar disorder (Levine et al., 2005). Plasma homocysteine levels affect the intracellular methylation process of DNA, lipids, proteins, and neurotransmitters (Scott and Weir, 1998). Both elevated homocysteine levels along with physiological levels of its oxidized derivatives, such as homocysteic acid and homocysteine sulfinic acid, have been shown to be toxic for neurons and vascular endothelial cells (Zou and Banerjee, 2005). While levels of homocysteine are affected by various genes involved in the homocysteine metabolic pathway and by environmental factors such as folate or vitamin B12 intake (Refsum et al., 2004), methylenetetrahydrofolate reductase (MTHFR) also plays a major role in this pathway. MTHFR converts 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which serves as a carbon donor for the methylation of homocysteine, leading to the generation of S-adenosylmethionine (SAM) (Andreoli and Maffei, 1975). SAM is a major source of methyl groups in the brain (Godfrey et al., 1990) and is involved in catechol-O-methyltransferase (COMT) reactions such as the catabolism of serotonin and other catecholamines (Anguelova et al., 2003, Chen et al., 2004). Freeman et al. (1975) reported there is direct evidence linking decreased MTHFR activity to schizophrenia (Freeman et al., 1975). These findings have led to multiple genetic analyses examining the link between the MTHFR gene (gene symbol: MTHFR, GenBank accession number: NM_005957) and schizophrenia.

MTHFR is composed of twelve exons (Fig. 1) and is localized on chromosome 1p36.3 (Goyette et al., 1994). It has been suggested that this may be a susceptibility locus for schizophrenia, bipolar disorder (Kempisty et al., 2007) and major depressive disorder (McGuffin et al., 2005). Two common functional polymorphisms of MTHFR, C677T (rs1801133) and A1298C (rs1801131), are known to cause a decrease of enzyme activity and affect nucleic synthesis and DNA methylation (van der Put et al., 1998). Several studies have confirmed the possible involvement of these SNPs in psychiatric conditions such as schizophrenia (Regland, 2005) and affective disorders (Arinami et al., 1997). Subjects with homozygosity for the 677 T allele have a mild increase in their plasma homocysteine levels, and these subjects have a higher frequency of neural tube deficits and premature cardiovascular disease as compared to other similar genotype carriers (Bakker and Brandjes, 1997, Matsushita et al., 1997). The impact of this polymorphism varies according to environmental factors, such as folate, vitamin B2 or vitamin B12 (Hustad et al., 2000, Refsum et al., 2004, van der Put et al., 1995). Although some studies have reported that carriers of the 677 T allele in MTHFR are associated with an increased risk of schizophrenia (Arinami et al., 1997, Muntjewerff et al., 2005, Sazci et al., 2003), others have shown contradictive results (Kunugi et al., 1998, Vilella et al., 2005, Yu et al., 2004). The association of the MTHFR C677T variant with schizophrenia may be linked to the excitatory amino acids hypothesis or to decreased plasma concentrations of SAM that have been reported in psychiatric disorders (Andreoli and Maffei, 1975). Another functional polymorphism, A1298C, also has been shown to decrease MTHFR activity, although van der Put et al. (1998) have reported finding no significant effect of this variant on the plasma homocysteine levels.

A recent meta-analysis demonstrated an association between elevated homocysteine levels or carriers of the 677 T allele and an increased risk of developing schizophrenia (Allen et al., 2008, Muntjewerff et al., 2006). It has been suggested that potential associations between genetic variation in folate metabolism and psychiatric disorders could be plausible biological explanations for these disorders (Coppen and Bolander-Gouaille, 2005).

Taken together, MTHFR may be related to the development of schizophrenia. Although a number of studies have demonstrated associations between specific polymorphisms of MTHFR and schizophrenia, there have been no gene-based analysis studies. Therefore, it is still difficult to interpret these types of studies due to the inconsistent results that have been derived from some of the confounding factors, such as population stratifications (ethnic or gender differences) and number of samples. In the present study, we conducted an association study between MTHFR and schizophrenia in the Japanese population that was based on the gene-wide approach. In addition, we also performed a meta-analysis on the updated data currently available.

Section snippets

Subjects

The samples for this association study consisted of 696 patients with schizophrenia and 747 control subjects. The confirmation sample set for four SNPs (rs1801133, rs17421511, rs17037396, and rs9651118), which were positively associated with schizophrenia in the haplotypic analysis and the imputation analysis, consisted of 797 patients with schizophrenia and 1025 control subjects. Detailed demographical data are presented in Supplementary Table 1.

All subjects were unrelated to each other and

Results

Regarding quality control, the genotype calls of the duplicated samples showed complete concordance (data not shown), and all genotype frequencies of the tagging SNPs were consistent with the HWE. There were no significant differences between the schizophrenic patients and the control subjects in both allele and genotype distributions without imputed (untyped) SNP (rs17421511) (Table 1). In the haplotypic analysis, a nominally significant association was observed between the haplotypes

Discussion

Even though we applied the gene-based approach in the present study, we could not confirm any significant associations of the MTHFR polymorphisms with schizophrenia. In the association analysis, we examined the SNPs covering the entire gene, including all of the tagging SNPs that had at least ~ 10% MAF listed on the HapMap database. For all of the genoõtyped SNPs, there were no associations noted between the patients with schizophrenia and the controls in any of the allele frequencies after

Role of the funding source

Funding for this study was provided by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Ministry of Health of Japan, Labor and Welfare, Grant-in-Aid for Scientific Research B (No. 22390223) and C (No. 18591309) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Mext Academic Frontier, the Japan Health Sciences Foundation (Research on Health Sciences focusing on Drug Innovation) and the Core Research for

Contributors

Authors Akira Yoshimi, Nagahide Takahashi, and Toshiya Inada designed the study and wrote the protocol. Authors Akira Yoshimi and Yukiko Kawamura conducted SNPs genotyping and statistical analyses. Authors Norio Ozaki, Yukihiro Noda, and Kiyofumi Yamada managed the literature searches and analyses. Author Akira Yoshimi wrote the first draft of the manuscript and Branko Aleksic revised. All authors contributed to and have approved the final manuscript.

Conflict of interest

The authors have no conflicts to declare.

Acknowledgements

We sincerely thank the patients and healthy volunteers for participation in our study, and Ryoko Ishihara for her technical assistance. This work was supported in part by research grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the Ministry of Health of Japan, Labor and Welfare, Grant-in-Aid for Scientific Research B (No. 22390223) and C (No. 18591309) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Mext Academic

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      For the other MTHFR variant (A1298C), five studies supported the role of the C allele or CC genotype in SZ in mixed (Shi et al., 2008b; Zintzaras, 2006), Asian (Kim et al., 2011), or Caucasian cohorts (Allen et al., 2008; Gilbody et al., 2006; Shi et al., 2008b) (Supplementary Table 1). The role of the C allele in mixed ethnic cohorts was conflicted by null effects reported by three studies (Supplementary Table 3), one of which was in a larger cohort (Peerbooms et al., 2011) than the confirmed study (Shi et al., 2008b); the other two conflicting studies did not report total sample size (Kim et al., 2011; Yoshimi et al., 2010), so the reasons for this discrepancy is not definitive. The role of C allele was also conflicted by null effects reported by three studies in Caucasian cohorts (Supplementary Table 3); in this case, the conflicting null study used a smaller cohort (Zintzaras, 2006) than the confirmed study (Shi et al., 2008b), and the other two studies again failed to report total sample size (Kim et al., 2011; Yoshimi et al., 2010) so the reason for the discrepancy is unclear.

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      Fifth, the generalizability of the present meta-analysis is limited to the ethnic groups investigated, i.e. White and Asian. In line with the current results, the meta-analysis of Yoshimi et al. (2010) supported an association between MTHFR C677T with schizophrenia (Yoshimi et al., 2010), similar to earlier meta-analyses (Yoshimi: random effects OR = 1.17, 95% CI: 1.07–1.29) (Allen et al., 2008; Gilbody et al., 2007; Jonsson et al., 2008; Lewis et al., 2005; Muntjewerff et al., 2006; Shi et al., 2008; Zintzaras, 2006). Regarding MTHFR A1298C, Zintzaras (2006) concluded in his meta-analysis on 2.565 subjects that this SNP was associated with the diagnosis of schizophrenia, however not in all genetic models examined (fixed effects ORCvA = 1.16, 95% CI: 1.03–1.31; ORAC/CCvAA = 1.19, 95% CI: 1.02–1.40; OR CCvAA = 1.37, 95% CI 1.03–1.82; random effects OR CCvAC/AA = 1.33, 95% CI: 0.94–1.88) (Zintzaras, 2006).

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