Correspondence to: Ah Rang Cho, Department of Psychiatry, East-West Neo Medical Center, School of Medicine, Kyung Hee University, 149, Sangil-dong, Gangseo-gu, Seoul 134-727, Korea
Tel: +02-440-6174, E-mail: [email protected] Received May 13, 2008, Accepted May 24, 2008.
Association between Methylenetetrahydrofolate Reductase (MTHFR) Gene Polymorphisms and Korean Patients with Schizophrenia
*Department of Psychiatry, East-West Neo Medical Center,
†Department of Surgery,
‡
Department of Thoracic and Cardiovascular Surgery, School of Medicine, Kyung Hee University, Seoul, Korea
Ah Rang Cho*, Yong Ho Kim
†, Bum Shik Kim
‡High levels of homocysteine promote oxidative stress, and aberrant homocysteine metabolism is linked to neurodevelopmental disorders including schizophrenia. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of 5,10-methy- lenetetrahydrofolate to 5-methyltetrahydrofolate, a cosubstrate for homocysteine remethylation to methionine. The present study was conducted to examine the association between the MTHFR gene and schizophrenia in Korean population. Subjects of 279 patients with schizophrenia and 296 healthy controls were recruited. We genotyped two single nucleotide polymorphisms (SNPs, rs2274976 and rs1801133) using the Illumina Sentrix Array Matrix chip and direct sequencing. The rs2274976 (Arg594Gln) is located on exon 12 of the MTHFR gene region, and the rs1801133 (Ala222Val) located on exon 5. For the analysis of genetic data, SNPStats, Haploview, HapAnalyzer, SNPAnalyzer, and Helixtree programs were used. Multiple logistic regression analysis (codominant, dominant, and recessive models) was also used. Patients with schizophrenia were evaluated according to clinical manifestations using the Operational Criteria Checklist (OPCRIT). Two SNPs (rs2274976 and rs1801133) were not associated with schizophrenia. However, one SNP showed a significant association with one phenotype of psychotic symptoms. Running commanding voices, which could be clinically considered serious feature of auditory hallucinations, was different in the recessive model (p=0.018; p=0.036 after Bonferroni correction). Recently, rs59514310 replaces rs1801133 as new number (SNP database, BUILD 129). In present study, a significant association was observed between rs1801133 and running commanding voices. The result suggests that the MTHFR gene may be a susceptibility gene for schizophrenia with auditory hallucination. (Korean J Str Res 2008;16:85∼91)
Key Words: Auditory hallucination, Methylenetetrahydrofolate reductase (MTHFR), Schizophrenia, Single nucleotide poly- morphism
INTRODUCTION
Schizophrenia is a complex neurobehavioral disorder, and affects
approximately 1 percent of the general population (Brown et al., 2005). It is characterized by multifarious clinical manifestations including psychotic symptoms such as hallucinations, delusional thinking, and other negative symptoms responsible for social withdrawal and occupational dysfunction. Etiology and pathophy- siology of schizophrenia are mostly unclear. However, evidences are accumulating that both genetic and environmental factors contribute to the etiology of schizophrenia (Cannon et al., 2002).
There are substantial potential explanations for supporting
neurodevelopmental hypothesis in the etiology of schizophrenia.
Aberrant homocysteine metabolism has been linked to neuro- developmental disorders, such as neural tube defects (Blom et al., 2006), Down syndrome (Da Silva et al., 2005), Parkinson's dis- ease, cognitive impairment (Reutens and Sachdev, 2002), and schizophrenia (Muntjewerff et al., 2006). Elevated levels of homo- cysteine may be generated superoxide by a biochemical mech- anism involving nitric oxide synthase. Methylenetetrahydrofolate reductase (MTHFR) is the essential enzyme in folate-mediated single-carbon transfer reactions of homocysteine remethylation (Mudd et al., 2001). Decreased MTHFR activity seems to exert elevation of plasma homocysteine level. Homocysteine itself has been shown to be toxic for neurons (Mattson and Shea, 2003) and endothelial cells (Jacobsen, 2001). The MTHFR gene is located on chromosome 1p36.3. The C677T single nucleotide polymorphism (SNP) in the MTHFR gene is known as the most common inherited cause of elevated homocysteine (Brattstrom et al., 1998). According to recent study, elevated plasma homocy- steine concentration is an independent risk marker for schizo- phrenia (Muntjewerff et al., 2006). Several molecular genetic studies have been reported the association between MTHFR and schizophrenia. Especially C677T and A1288C SNPs were known as two common genetic mutations of the MTHFR gene related with schizophrenia. The relationship of the MTHFR 677T allele in patients with schizophrenia have been reported (Yu et al., 2004; Muntjewerff et al., 2005; Sazci et al., 2005; Vilella et al., 2005). Also, a recent meta-analysis (Lewis et al., 2005) supports the relationship between 677TT genotype and schizophrenia risk.
The present study aimed to identify the possible association between other SNPs in the MTHFR gene except C677T and A1298C and schizophrenia.
MATERIALS AND METHODS
1. Subjects
Two hundred seventy nine schizophrenia patients (182 males and 97 females) diagnosed according to DSM-IV were recruited.
The control group consisted of 296 healthy Koreans (143 males and 153 females), had visited primary medical care center for routine examination. Subjects without personal and family histories of psychiatric disorders were selected based on self-
reporting records. Written informed consents were obtained from all subjects. This research was approved by the Institutional Review Board (IRB) of Kyung Hee University Medical Center, Seoul, Korea.
2. Clinical assessments
All patients with schizophrenia were evaluated using the Operational Criteria Checklist for Psychotic Illness (OPCRIT) (McGuffin et al., 1991). We categorized clinical phenotypes by using OPCRIT that is based on a lifetime history of mania, reality distortion, depression, disorganization and negative symp- toms (Dikeos et al., 2006). Also, the association between two SNPs of the MTHFR gene and Scale for Assessment of Negative Symptoms (SANS) scores representing negative symptoms (Iancu I et al., 2005) was examined in the patients group.
3. SNP selection and genotyping
All typed SNPs of the MTHFR gene were downloaded from the HapMap database (http://www.hapmap.org/genome build 34).
Since we considered functional significance of SNPs in the possi- ble region to be changed the expression from the MTHFR gene, we selected tag SNPs using the aggressive tagging option of the Tagger program (http://www.broad.mit.edu/mpg/tagger/). Furth- ermore, we added two preconditions, nonsynonymous type and high heterozygosities, to SNPs selection criteria. Finally, two coding SNPs (rs2274976 and rs1801133) were selected. The rs2274976 (Arg594Gln) is located on exon 12 of the MTHFR gene region, and the rs1801133 (Ala222Val) located on exon 5.
However, the rs59514310 replaces the rs1801133 as new number
(SNP database, BUILD 129). DNA was isolated from a peri-
pheral blood sample using the Core One
TMBlood Genomic DNA
Isolation Kit (CoreBioSystem
TM, Seoul, Korea). SNP genotyping
was performed using direct sequencing method. For sequencing,
we made primers of each SNP. Sense (5'-CACACCCAGC-
TCTGACTCACC-3') and antisense (5'-CTCCACTCTCCTTCG-
TGTCTCT-3') primers for the rs2274976 were used (size 726
bp). For the rs1801133, sense (5'- TCTATGGCCACCAAGTG-
CAGGCCT-3') and antisense (5'-CTCTCAGGTCCAGAACTTG-
CAC-3') primers were also used (size 649 bp).
Korean Patients with Schizophrenia
SNP Genotype Control Schizophrenia
Freq % Freq %
rs2274976 (exon 12, Arg594Gln) G/G 250 84 233 84
A/G 45 15 46 16
A/A 1 1 0 0
rs1801133 (exon 5, Ala222Val) (rs59514310, BUILD 129) G/G 91 31 94 34
A/G 144 49 135 49
A/A 61 21 49 18
N/A 0 0 1 0
SNP: single nucleotide polymorphism, Freq: Frequency, N/A: Non applicable.
Table 1. Genotype frequencies of methylenetetrahydrofolate reductase (MTHFR) SNPs in Korean schizophrenia and control subjects.
SNP
Schizophrenia
Genotype With AH Without AH
Model OR 95% CI
Freq % Freq % LCL UCL p
rs1801133
G/G 16 29 78 35 Codominant 0.42 0.19 0.95 0.060
A/G 23 42 112 50 Dominant 0.76 0.40 1.45 0.400
A/A 16 29 33 15 Recessive 0.42 0.21 0.84 0.018
SNP: single nucleotide polymorphism, Freq: Frequency, AH: auditory hallucination, OR: Odds Ratio, CI: Confidence intervals, LCL: Lower Confidence Limit, UCL: Upper Confidence Limit, p: p-value.
Table 2. Association between the rs1801133 SNP and schizophrenia.
4. Statistical analysis
The present study was designed a case-control study. Geno- typic and allelic data of the two groups (patient and control groups) in each selected polymorphism were compared by Chi- square test or Fisher's exact test using the SPSS version 12.5 program (SPSS. Inc. Ltd. Chicago, Ill, USA). Hardy-Weinberg equilibrium was examined for genotype distributions. All haplo- type frequencies in this study were analyzed using HapAnalyzer and Haploview. We adjusted age and gender between the patients and controls using SNPStats (Solé et al., 2006). Also, we calculated the odd ratio (OR) with 95% confidence interval (CI) and corresponding p values to analyze association between SNPs and schizophrenia by using SNPStats. We set the level of significance at 0.05 of p-value in all statistical tests.
RESULTS
We assessed two SNPs (rs2274976 and rs1801133) of the
MTHFR gene in both control subjects and patients with schi- zophrenia. Genotype distributions of two SNPs were consistent with Hardy-Weinberg equilibrium. The rs2274976 and rs1801133 showed complete LD block. The genotype and allele distributions of rs2274976 and rs1801133 did not show any significant differences between the patient and control groups, respectively (Table 1). Haplotype frequencies of rs227496 and rs180133 also showed no significant differences.
Next, we investigated the association between two SNPs in the
MTHFR gene and clinical manifestations of schizophrenia listed at
OPCRIT. One SNP showed a significant association with one
phenotype of psychotic symptoms. Running commanding voices,
which could be clinically considered serious feature of auditory
hallucinations, revealed a significant different in the recessive
model (p=0.018, OR=0.42, 95%CI=0.21∼0.84). This result
was statistically significant even if its p-value was changed to
0.036 after the Bonferroni correction (Table 2). Although
problems including delusional thinking are common and impor-
tant positive symptoms of schizophrenia, however, we did not
find any significant associations between persecutory delusions and two SNPs. There were no significant association between two SNPs and other clinical phenotypes assessed by OPCRIT. Finally, the association between two SNPs of the MTHFR gene and SANS scores representing negative symptoms (Iancu I et al., 2005) was examined in the patients group. The result did not show any significant association.
DISCUSSION
In this study, statistically significant association was observed between rs180133 polymorphism and running commanding voices in Korean schizophrenia. Although auditory hallucinations appear in other psychiatric syndromes or in general population, internal voices remain the hallmark of psychoses (Sanjuán et al., 2006). Several genetic polymorphisms could be involved in the vulnerability to auditory hallucination in psychotic patients. San- juán et al. (2005) reported the association between short allele of serotonin transporter gene (5-HTTLPR) and the emotional response to auditory hallucination. Also, a significant association between FOXP2 polymorphism and the frequency and intensity of auditory hallucination was found (Sanjuán et al., 2006). Several studies have been observed an association between the CCK-AR gene polymorphisms and schizophrenia with auditory hallu- cinations (Zhang et al., 2000; Sanjuan et al., 2004). Auditory hallucinations are associated with increased metabolic activity in several cortical areas such as the inferior frontal/insular, anterior cingulate and temporal cortex, as well as the right thalamus and several hippocampal areas (Shergill et al., 2000).
Neurodevelopmental disruption during prenatal and perinatal periods has been proposed to play a role in the etiology of schizophrenia (Brown et al., 2005). Prenatal and perinatal risk factors include prenatal viral infection, nutritional deficiency, fetal hypoxia and perinatal insults are known to produce neurodevelop- mental insults and have been implicated with schizophrenia (Brown et al., 1996; 2002). Hypothesis that homocysteine induces neuronal death are based on the presumption that homocysteine might act as an excitotoxin of N-methyl-D-aspartate receptor and lead to apoptosis. In several animal studies, perinatal administ- ration of NMDA receptor antagonists was induced schizo- phrenia-like symptoms (di Simone et al., 2004; El Khodor et al.,
2004). On the other hand, homocysteine might cause placental vasculopathy, which may induce fetal hypoxia and other fetal developmental disruption including impaired brain growth and neurotransmitter dysfunction (Mercuri et al., 2000; Vollset et al., 2000). MTHFR is the crucial enzyme in folate metabolism.
MTHFR acts enzymatically on 5,10-methylenetetrahydrofolate to produce 5-methyltetrahydrofolate, a cofactor for methionine. A C677T mutation in the MTHFR gene had been identified and reduced the overall enzyme activity (Frosst et al., 1995). The C677T polymorphism in the MTHFR gene has been reported to associate with hyperhomocysteinemia, bipolar affective disorder and schizophrenia (Kempisty et al., 2006). The second common mutation of MTHFR is A1298C polymorphism. Association of 1298CC genotype polymorphism with schizophrenia was observed by existing studies (Kempisty et al., 2007). Especially, Sazci et al.
(2005) reported a significant association between A1298C poly- morphism and schizophrenia in male patients. Our data from two SNPs did not show any significant differences of genotype and allele frequencies according to gender difference in schizophrenia group. Another study reported that hyperhomocysteinemia was a non-specific risk factor for female bipolar affective disorder and schizophrenia (Reif et al., 2005). A1298C polymorphism was also associated with bipolar affective disorder (Reif et al., 2005). In this study, there was no significant association between two SNPs and manic or depressive symptom clusters evaluated by OPCRIT.
Besides our negative association finding between the MTHFR
gene and poor concentration of schizophrenia, there is substantial
clinical overlap between negative symptoms and cognitive impair-
ment in schizophrenia patients (Harvey et al., 2006). Roffman et
al. (2007) suggested that C677T polymorphism partially effects
on verbal fluency test (VFT) performance reflect, in part, its
effects on negative symptoms. We also investigated whether
rs2274976 and rs1801133 SNPs are associated with negative
symptoms measured by SANS of schizophrenia patients, but,
there were no significant results. In the investigation of 200
schizophrenia outpatients by Roffman et al. (2007), individuals
with homozygosity for the MTHFR 677T allele performed
significantly more poorly than C/C or C/T subjects in VFT, and
Wisconsin card-sorting test executive dysfunction. Schizophrenia is
also characterized by significant cognitive deficits including
impaired executive function (Sharma and Antonova, 2003). Consi-
Korean Patients with Schizophrenia
dering the theoretical issue that cognitive impairment might reflect heritable abnormalities of brain function in schizophrenia, further study using neuropsychological assessment tools in order to make cognitive dysfunction of schizophrenia concrete is needed.
Our data could explain the association of the MTHFR gene except well-known two genetic mutation sites, C677T and A1298C polymorphism, with schizophrenia in Korean population.
Further studies for other subtypes of schizophrenia without auditory hallucination or non-schizophrenic patients with auditory hallucination may be helpful to clarify the role of the MTHFR gene in the vulnerability to auditory hallucination.
In conclusion, the rs1801133 SNP of MTHFR exhibited a significant association with auditory hallucination, one of positive symptoms of schizophrenia.
ACKNOWLEDGMENTS
This study was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (R11-2005-014), and partially supported by the Governance Program of Kyung Hee University.
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Korean Patients with Schizophrenia
