- Email: [email protected]
Influence of maternal MTHFR A1298C polymorphism on the risk in offspring of schizophrenia
BR A I N R ES E A RC H 1 3 2 0 ( 2 01 0 ) 1 3 0 –13 4
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Influence of maternal MTHFR A1298C polymorphism on the risk in offspring of schizophrenia Chen Zhang, Bin Xie, Yiru Fang, Wenhong Cheng, Yasong Du, Dongxiang Wang, Shunying Yu⁎ Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, 600 Wan Ping Nan Road, Shanghai 200030, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
Several lines of evidence have suggested that two functional methylenetetrahydrofolate
Accepted 16 December 2009
reductase gene (MTHFR) polymorphisms, C677T and A1298C, may be implicated in the
Available online 4 January 2010
etiology of schizophrenia. We examined these MTHFR polymorphisms in 111 families, composed of a patient and their parents, as well as 143 mothers of patients with
Keywords:
schizophrenia and 235 age-matched mothers who had healthy children. The maternal
Methylenetetrahydrofolate
MTHFR 1298C allele was associated with a significantly increased risk of schizophrenia
reductase
(OR = 1.63, 95%CI: 1.11–2.39, P = 0.01). The haplotype analysis showed a weak association for
Association study
the 1298C-677C haplotype (OR = 1.54, 95%CI = 1.03–2.29, P = 0.04). Analysis of Transmission
Schizophrenia
Disequilibrium Test (TDT) showed no preferential transmission of 1298C and 677T alleles
Neurodevelopment
from parents to probands (P = 0.64 and P = 0.71, respectively). Our results suggest that deficient MTHFR enzyme activity in pregnant women, related to the A1298C variant, is associated with a higher risk of having offspring affected with schizophrenia. Given the low sample size in this study, the present results seem tentative and need further studies to replicate. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
There are two large epidemiological studies demonstrating an association between prenatal starvation and increased risk of schizophrenia (Susser et al., 1996; St Clair et al., 2005). Further studies indicate that the specific candidate nutrient “folate” metabolism-related defect may play an important role in the pathogenesis of schizophrenia (Brown and Susser, 2005). This is hypothesized to occur in the process of “single-carbon cycle” (Regland, 2005). Elevated homocysteine is the common feature of aberrant sing-carbon cycle (Regland, 2005). A recent metaanalysis of eight case-control studies suggested that a 5 μM increase in homocysteine is associated with a 70% higher risk for schizophrenia (Muntjewerff et al., 2006). Moreover, a
population-based birth cohort study showed that maternal elevated homocysteine concentration during the third trimester of pregnancy was associated with over two-fold increase in schizophrenia risk in the offspring (Brown et al., 2007). Methylenetetrahydrofolate reductase (MTHFR) is the crucial enzyme in single carbon cycle and its deficiency is associated with elevated homocysteine (Brown and Susser, 2005). The MTHFR gene (MTHFR) is polymorphic in human and localized on chromosome 1p36.3, where linkage to schizophrenia has been suggested (Kohn et al., 2004). MTHFR contains two common functional variants, 677C > T and 1298A > C. In their homozygous form, the minor allele of these variants reduce the MTHFR activity to less than 60% (Frosst et al., 1995) and 30% (Weisberg et al., 2001), respectively, compared with
⁎ Corresponding author. E-mail address: [email protected] (S. Yu). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.12.049
131
BR A I N R ES E A RC H 1 3 2 0 ( 2 01 0 ) 1 3 0 – 1 34
Table 1 – TDT analysis for single alleles and haplotypes in 111 trios. Alleles
Transmitted Non-transmitted
1298C 677T
39 58
35 54
χ2
P
0.22 0.14
0.64 0.71
Table 3 – The estimated haplotype frequencies in case mothers and controls. SNP1–2
A1298C-C677T
a
A-C A-T C-C C-T
52.9 55.1 32.1 7.9
58.7 53.3 30.3 5.7
0.30 0.03 0.05 0.36
0.59 0.87 0.82 0.55
the activity of the homozygous wildtype. Case-control studies of the association between the MTHFR polymorphisms and schizophrenia have given inconsistent results (Arinami et al., 1997; Joober et al., 2000; Sazci et al., 2005; Muntjewerff et al., 2005; Kempisty et al., 2006, 2007; Kunugi et al., 1998; Tan et al., 2004; Yu et al., 2004; Vilella et al., 2005). Seven meta-analyses have been performed on this subject, all arguing for an association between these polymorphisms and schizophrenia, although the association with regard to A1298C so far seems somewhat less convincing (Allen et al., 2008; Gilbody et al., 2007; Jönsson et al., 2008; Lewis et al., 2005; Muntjewerff et al., 2006; Shi et al., 2008; Zintzaras, 2006). The recent SzGene database showed that either C677T or A1298C is one of the 24 genetic variants with a nominally significant effect on schizophrenia (Allen et al., 2008), and MTHFR was identified as a candidate gene and familiar association studies are reasonably required for replication (Shi et al., 2008). In this study, we conducted a family-based study to investigate the association of MTHFR C677T and A1298C with schizophrenia. In addition, association between maternal MTHFR polymorphisms and schizophrenia in offspring was investigated.
Odds ratio (95%CI)
χ2
P
53.5 (18.7) 62.4 (13.3) 1.54 (1.03–2.29) 4.45 0.04 106.5 (37.2) 200.4 (42.6) 0.82 (0.60–1.11) 1.67 0.20 118.5 (41.4) 202.6 (43.1) 0.96 (0.71–1.30) 0.07 0.80
Haplotypes with frequency < 0.03 are ignored in analysis.
For the TDT-analysis (Table 1), the transmission/nontransmission counts of − 1298C and − 677T were 39/35 (P = 0.64) and 58/54 (P = 0.71), respectively. In the haplotoypes constructed by A1298C-C677T, the transmission/non-transmission counts of A-C, A-T, C-C and C-T were 52.9/58.7 (P = 0.59), 55.1/53.3 (P = 0.87), 32.1/30.3 (P = 0.82) and 7.9/5.7 (P = 0.55), respectively. No significant transmission distortions were found. Table 2 shows the allele and genotype frequencies for A1298C and C677T in case mothers and control mothers. The frequency of 1298C allele was significantly higher in case mothers (21.3%) than in controls (14.3%), with an OR of 1.63 (95%CI: 1.11–2.39). Genotype analysis also found one significant association corresponding to this positive allele (P = 0.03). No significant difference was found in allele and genotype distributions of C677T between mothers of patients and controls. We performed a haplotype analysis for the variants (Table 3). The haplotype C-C combined by A1298C and C677T is significant (P = 0.04), which showed an OR of 1.54 (95%CI: 1.03–2.29). In a log additive mode of inheritance, the power of A1298C and C677T for detecting an odds ratio (OR) of 1.5 was 43.4% and 56.1% in case–parent trios, respectively. In comparison of case mothers and controls, the power of our sample was 52.8% for A1298C and 76.3% for C677T.
3. 2.
Controls (%)
Haplotype a C-C A-T A-C
Haplotype
Case (%)
Discussion
Results
Genotypes of A1298C and C677T were in Hardy–Weinberg equilibrium among parents (P = 0.19 and P = 0.37), affected offspring (P = 0.80 and P = 0.51) and controls (P = 0.68 and P = 0.33). Strong pairwise linkage disequilibrium was observed between A1298C and C677T (D' = 0.78).
In the present study, we found an association between maternal A1298C polymorphism and schizophrenia in offspring. Based on these data, maternal 1298C allele may be a risk allele for the development of schizophrenia. A1298C is a common variant (1298A → C; glutamate to alanine). Homozygote for the C allele decreased the MTHFR activity by about 30%
Table 2 – Distributions of alleles and genotypes for the two SNPs in case mothers and controls. SNP ID
Sample
N
Allele frequency (%) C
SNP1 A1298C SNP2 C677T
Case Control
143 235
Case Controls
143 235
61 (21.3) 67 (14.3) T 114 (39.9) 205 (43.6)
P
OR (95%CI)
A 225 403 C 172 265
(78.7) (85.7)
0.01
1.63 (1.11–2.39)
(60.1) (56.4)
0.31
0.86 (0.64–1.16)
Genotype frequency (%) C/C
C/A
A/A
6 (4.2) 3 (1.3) T/T 22 (15.4) 41 (17.4)
49 (34.3) 61 (26.0) T/C 70 (49.0) 123 (52.3)
88 (61.5) 171 (72.8) C/C 51 (35.7) 71 (30.2)
P
0.03
0.54
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(van der Put et al., 1998; Weisberg et al., 2001) and significantly increased homocysteine concentrations (Markan et al., 2007). Maternal elevated homocysteine could induce placental vasculopathy (Nelen et al., 2000) which influences the delivery of nutrients and oxygen from mother to fetus and leads to fetal hypoxia (Dalman et al., 2001). Corresponding adverse consequences for fetal development including impaired brain growth (Mercuri et al., 2000) and disturbances of neurotransmitter systems (Vollset et al., 2000) have also been suggested. Further evidence showed that elevated third-trimester maternal homocysteine levels may increase risk of schizophrenia in offspring, induced by developmental effects on brain structure and function through subtle damage to the placental vasculature (Brown et al., 2007). Thus, the maternal 1298C allele could play an important role in the development of schizophrenia. In this study, we report a low 1298C frequency (14%) for the controls. In contrast, previous schizophrenia study of Chinese subjects reported control 1298C allele frequency of 23% (Yu et al., 2004). A recent study reported 17% for the control 1298C allele frequency among Chinese Han women (Xu et al., 2007). This difference may be caused by complicated population admixture in China (Shi et al., 2004) and insufficient control subjects in this study. With regard to the C677T polymorphism, we did not found association between maternal C677T variant and schizophrenia in line with the study conducted by Muntjewerff et al (2007). It has been shown that the C677T variant form has a greater effect on homocysteine concentrations than the A1298C polymorphism (van der Put et al., 1998). Considering the strong linkage equilibrium between A1298C and C677T polymorphisms, possible effect of C677T polymorphism could not be ruled out. Taken together, in order to shed light on the results, larger studies are warranted. It has been well established that family-based studies are an effective approach to follow up the findings from casecontrol investigation (Schaid, 1998). In the present study, TDT analyses of the C677T and A1298C polymorphisms found no significant association of these two polymorphisms with schizophrenia in Chinese subjects. Our findings are consistent with the previous family-based study (Muntjewerff et al., 2007), which suggested no preferential transmission of C677T in case-parent trios. However, regarding maternal 1298C allele may be a genetic risk factor to the development of schizophrenia, the preferential transmission of the 1298C allele could not be expected (Labuda et al., 2002). Thus, parent-case trios studies do not seem to be an appropriate method to detect the association between A1298C and schizophrenia. This study suffers from two limitations. First, it should be noted that the limited sample size involved in this study causes low statistical power. Second, the interaction between MTHFR and potential environmental confounders of the folate metabolism is not considered in this study. In summary, although our results suggest that maternal MTHFR A1298C variant is associated with the occurrence of schizophrenia in offspring, it must be viewed as tentative rather than wholly conclusive given the small sample size, which lead to a higher potential for a type I error. The present results need further replication studies in order to demonstrate this point more fully.
4.
Experimental procedures
4.1.
Subjects
The 111 trios in our study consisted of healthy parents and offspring affected with schizophrenia from Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine. Of the schizophrenic probands, 56 (50.5%) were male and 55 (49.5%) were female. The average age of all patients was 18.79 (SD = 4.96). Consensual diagnosis of each proband was made by two independent psychiatrists according to the DSM-IV criteria for schizophrenia on the basis of the Mini International Neuropsychiatric Interview (MINI) and medical records. Including 111 mothers from the trios, we totally recruited 143 mothers (mean age = 47.35, SD = 4.61) whose offspring were affected with schizophrenia. We also enrolled 235 age-matched mothers (mean age = 49.16, SD = 5.90) from the group of hospital staff as control subjects who had healthy children (mean age = 18.64, SD = 6.03). All subjects were Han Chinese origins and signed informed consents. The study protocol and process were assessed and approved by the ethics committee at Shanghai Mental Health Center.
4.2.
Genotyping
Genomic DNA was prepared from venous blood using Tiangen DNA isolation kit (Tiangen biotech, Beijing, China). The primers for C677T and A1298C polymorphisms were designed using Primer 5.0 program (http://www.premierbiosoft.com/ primerdesign/index.html), while A1298C was detected, using a reverse mismatch primer. The sequences were accessed from National Center for Biotechnology Information (NCBI) database. Briefly, the forward and reverse primers for C677T analysis were: 5′-AGGACTACTACCTCTTCTACCTGAA-3′ and 5′AAGAACGAAGACTTCA-AAGACACGT-3′. The primers for A1298C analysis were: 5′-GGGGTCAGAAGCATATCAGT-3′ and 5′-CTCACCTGGATGGGAAAGA-3′. Genotyping was performed for C677T and A1298C by PCR-RFLP methods. For the C677T polymorphism, each PCR product was digested with TaqI at 65 °C for 2 h, followed by 1.5% agarose gel electrophoresis and ethidium bromide staining. The C → T substitution at nucleotide 677 creates a Taql digestion site. The PCR product (390 bp) with T allele was digested to 2 fragments (121 bp and 269 bp), whereas the PCR product with wild type C allele can not be cut by Taql. For the A1298C polymorphism, each PCR product was digested with Pmll at 37 °C overnight, followed by 12% polyacrylamide gel electrophoresis and detected by ethidium bromide staining. The A → C substitution at nucleotide 1298 creates a Pmll digestion site. The polymerase chain reaction (PCR) product (110 bp) with C allele was digested into 2 fragments (26 bp and 84 bp), whereas the PCR product with wild type A allele cannot be cut by Pmll. For quality control, all genotypes were called blind to the case or control status in the genotyping process. Five percent of the samples were repeated for the genotyping assay and the corresponding results were 100% concordant.
4.3.
Statistical analysis
The Haploview software, version 4.1, was used for Hardy– Weinberg equilibrium and transmission disequilibrium analysis
BR A I N R ES E A RC H 1 3 2 0 ( 2 01 0 ) 1 3 0 – 1 34
of individual SNPs in families. In comparison of case mothers and control mothers, all the parameters, allele and genotype frequencies and haplotype analysis were conducted by SHEsis software (http://analysis.bio-x.cn) (Shi and He, 2005). All tests were two tailed and significance was set at 0.05. The statistical power of our sample size was performed on Quanto program (Version 1.2.3, available at http://hydra.usc.edu/GxE).
Acknowledgments The authors are very grateful to all participants. This work was supported by grants from National Natural Science Foundation of China (30500181 and 30971047). We thank the two anonymous reviewers for their insightful comments. And, we thank Dr. Natashia Swalve, who helped in editing this manuscript.
REFERENCES
Allen, N.C., Bagade, S., et al., 2008. Systematic meta-analyses and field synopsis of genetic association studies in schizophrenia: the SzGene database. Nat. Genet. 40, 827–834. Arinami, T., Yamada, N., et al., 1997. Methylenetetrahydrofolate reductase variant and schizophrenia/depression. Am. J. Med. Genet. 74, 526–528. Brown, A.S., Susser, E.S., 2005. Homocysteine and schizophrenia: from prenatal to adult life. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 1175–1180. Brown, A.S., Bottiglieri, T., et al., 2007. Elevated prenatal homocysteine levels as a risk factor for schizophrenia. Arch. Gen. Psychiatry 64, 31–39. Dalman, C., Thomas, H.V., et al., 2001. Signs of asphyxia at birth and risk of schizophrenia: population-based case-control study. Br. J. Psychiatry 179, 403–408. Frosst, P., Blom, H.J., et al., 1995. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat. Genet. 10, 111–113. Gilbody, S., Lewis, S., et al., 2007. Methylenetetrahydrofolate reductase (MTHFR) genetic polymorphisms and psychiatric disorders: a HuGE review. Am. J. Epidemiol. 165, 1–13. Jönsson, G.E., Larsson, K., et al., 2008. Two methylenetetrahydrofolate reductase gene (MTHFR) polymorphisms, schizophrenia and bipolar disorder: an association study. Am. J. Med. Genet. B Neuropsychiatr. Genet. 147B, 976–982. Joober, R., Benkelfat, C., et al., 2000. Association between the methylenetetrahydrofolate reductase 677C- > T missense mutation and schizophrenia. Mol. Psychiatry 5, 323–326. Kempisty, B., Mostowska, A., et al., 2006. Association of 677C > T polymorphism of methylenetetrahydrofolate reductase (MTHFR) gene with bipolar disorder and schizophrenia. Neurosci. Lett. 400, 267–271. Kempisty, B., Bober, A., et al., 2007. Distribution of 1298A > C polymorphism of methylenetetrahydrofolate reductase gene in patients with bipolar disorder and schizophrenia. Eur. Psychiatry 22, 39–43. Kohn, Y., Danilovich, E., et al., 2004. Linkage disequlibrium in the DTNBP1 (dysbindin) gene region and on chromosome 1p36 among psychotic patients from a genetic isolate in Israel: findings from identity by descent haplotype sharing analysis. Am. J. Med. Genet. B Neuropsychiatr. Genet. 128B, 65–70. Kunugi, H., Fukuda, R., et al., 1998. C677T polymorphism in methylenetetrahydrofolate reductase gene and psychoses. Mol. Psychiatry 3, 435–437.
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Labuda, D., Krajinovic, M., et al., 2002. Parental genotypes in the risk of a complex disease. Am. J. Hum. Genet. 71, 193–197. Lewis, S.J., Zammit, S., et al., 2005. A meta-analysis of the MTHFR C677T polymorphism and schizophrenia risk. Am. J. Med. Genet. B Neuropsychiatr. Genet. 135, 2–4. Markan, S., Sachdeva, M., et al., 2007. MTHFR 677 CT/MTHFR 1298 CC genotypes are associated with increased risk of hypertension in Indians. Mol. Cell Biochem. 302, 125–131. Mercuri, E., Ricci, D., et al., 2000. Head growth in infants with hypoxic-ischemic encephalopathy: correlation with neonatal magnetic resonance imaging. Pediatrics 106, 235–243. Muntjewerff, J.W., Hoogendoorn, M.L., et al., 2005. Hyperhomocysteinemia, methylenetetrahydrofolate reductase 677TT genotype, and the risk for schizophrenia: a Dutch population based case-control study. Am. J. Med. Genet. B Neuropsychiatr. Genet. 135B, 69–72. Muntjewerff, J.W., Kahn, R.S., et al., 2006. Homocysteine, methylenetetrahydrofolate reductase and risk of schizophrenia: a meta-analysis. Mol. Psychiatry 11, 143–149. Muntjewerff, J.W., Hoogendoorn, M.L., et al., 2007. No evidence for a preferential transmission of the methylenetetrahydrofolate reductase 677T allele in families with schizophrenia offspring. Am. J. Med. Genet. B Neuropsychiatr. Genet. 144B, 891–894. Nelen, W.L., Bulten, J., et al., 2000. Maternal homocysteine and chorionic vascularization in recurrent early pregnancy loss. Hum. Reprod. 15, 954–960. Regland, B., 2005. Schizophrenia and single-carbon metabolism. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 1124–1132. Sazci, A., Ergul, E., et al., 2005. Association of the C677T and A1298C polymorphisms of methylenetetrahydrofolate reductase gene with schizophrenia: association is significant in men but not in women. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 1113–1123. Schaid, D.J., 1998. Transmission disequilibrium, family controls, and great expectations. Am. J. Hum. Genet. 63, 935–941. Shi, Y.Y., He, L., 2005. SHEsis, a powerful software platform for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci. Cell Res. 15, 97–98. Shi, Y.Y., Zhao, X., et al., 2004. Genetic structure adds power to detect schizophrenia susceptibility at SLIT3 in the Chinese Han population. Genome Res. 14, 1345–1349. Shi, J.J., Gershon, E.S., et al., 2008. Genetic associations with schizophrenia: meta-analyses of 12 candidate genes. Schizophr. Res. 104, 96–107. St Clair, D., Xu, M., et al., 2005. Rates of adult schizophrenia following prenatal exposure to the Chinese famine of 1959–1961. J.A.M.A. 294, 557–562. Susser, E.S., Neugebauer, R., et al., 1996. Schizophrenia after prenatal famine. Further evidence. Arch. Gen. Psychiatry 53, 25–31. Tan, E.C., Chong, S.A., et al., 2004. Genetic analysis of the thermolabile methylenetetrahydrofolate reductase variant in schizophrenia and mood disorders. Psychiatr. Genet. 14, 227–231. van der Put, N.M., Gabreëls, F., et al., 1998. A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects? Am. J. Hum. Genet. 62, 1044–1051. Vilella, E., Virgos, C., et al., 2005. Further evidence that hyperhomocysteinemia and methylenetetrahydrofolate reductase C677T and A1289C polymorphisms are not risk factors for schizophrenia. Prog. Neuropsychopharmacol. Biol. Psychiatry 29, 1169–1174. Vollset, S.E., Refsum, H., et al., 2000. Plasma total homocysteine, pregnancy complications, and adverse pregnancy outcomes: the Hordaland Homocysteine study. Am. J. Clin. Nutr. 71, 962–968.
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Weisberg, I.S., Jacques, P.F., et al., 2001. The 1298A- > C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine. Atherosclerosis 156, 409–415. Xu, W.H., Shrubsole, M.J., et al., 2007. Dietary folate intake, MTHFR genetic polymorphisms, and the risk of endometrial cancer among Chinese women. Cancer Epidemiol Biomarkers Prev. 16, 281–287.
Yu, L., Li, T., et al., 2004. No association between polymorphisms of methylenetetrahydrofolate reductase gene and schizophrenia in both Chinese and Scottish populations. Mol. Psychiatry 9, 1063–1065. Zintzaras, E., 2006. C677T and A1298C methylenetetrahydrofolate reductase gene polymorphisms in schizophrenia, bipolar disorder and depression: a meta-analysis of genetic association studies. Psychiatr. Genet. 16, 105–115.
available at www.sciencedirect.com
www.elsevier.com/locate/brainres
Research Report
Influence of maternal MTHFR A1298C polymorphism on the risk in offspring of schizophrenia Chen Zhang, Bin Xie, Yiru Fang, Wenhong Cheng, Yasong Du, Dongxiang Wang, Shunying Yu⁎ Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine, 600 Wan Ping Nan Road, Shanghai 200030, PR China
A R T I C LE I N FO
AB S T R A C T
Article history:
Several lines of evidence have suggested that two functional methylenetetrahydrofolate
Accepted 16 December 2009
reductase gene (MTHFR) polymorphisms, C677T and A1298C, may be implicated in the
Available online 4 January 2010
etiology of schizophrenia. We examined these MTHFR polymorphisms in 111 families, composed of a patient and their parents, as well as 143 mothers of patients with
Keywords:
schizophrenia and 235 age-matched mothers who had healthy children. The maternal
Methylenetetrahydrofolate
MTHFR 1298C allele was associated with a significantly increased risk of schizophrenia
reductase
(OR = 1.63, 95%CI: 1.11–2.39, P = 0.01). The haplotype analysis showed a weak association for
Association study
the 1298C-677C haplotype (OR = 1.54, 95%CI = 1.03–2.29, P = 0.04). Analysis of Transmission
Schizophrenia
Disequilibrium Test (TDT) showed no preferential transmission of 1298C and 677T alleles
Neurodevelopment
from parents to probands (P = 0.64 and P = 0.71, respectively). Our results suggest that deficient MTHFR enzyme activity in pregnant women, related to the A1298C variant, is associated with a higher risk of having offspring affected with schizophrenia. Given the low sample size in this study, the present results seem tentative and need further studies to replicate. © 2009 Elsevier B.V. All rights reserved.
1.
Introduction
There are two large epidemiological studies demonstrating an association between prenatal starvation and increased risk of schizophrenia (Susser et al., 1996; St Clair et al., 2005). Further studies indicate that the specific candidate nutrient “folate” metabolism-related defect may play an important role in the pathogenesis of schizophrenia (Brown and Susser, 2005). This is hypothesized to occur in the process of “single-carbon cycle” (Regland, 2005). Elevated homocysteine is the common feature of aberrant sing-carbon cycle (Regland, 2005). A recent metaanalysis of eight case-control studies suggested that a 5 μM increase in homocysteine is associated with a 70% higher risk for schizophrenia (Muntjewerff et al., 2006). Moreover, a
population-based birth cohort study showed that maternal elevated homocysteine concentration during the third trimester of pregnancy was associated with over two-fold increase in schizophrenia risk in the offspring (Brown et al., 2007). Methylenetetrahydrofolate reductase (MTHFR) is the crucial enzyme in single carbon cycle and its deficiency is associated with elevated homocysteine (Brown and Susser, 2005). The MTHFR gene (MTHFR) is polymorphic in human and localized on chromosome 1p36.3, where linkage to schizophrenia has been suggested (Kohn et al., 2004). MTHFR contains two common functional variants, 677C > T and 1298A > C. In their homozygous form, the minor allele of these variants reduce the MTHFR activity to less than 60% (Frosst et al., 1995) and 30% (Weisberg et al., 2001), respectively, compared with
⁎ Corresponding author. E-mail address: [email protected] (S. Yu). 0006-8993/$ – see front matter © 2009 Elsevier B.V. All rights reserved. doi:10.1016/j.brainres.2009.12.049
131
BR A I N R ES E A RC H 1 3 2 0 ( 2 01 0 ) 1 3 0 – 1 34
Table 1 – TDT analysis for single alleles and haplotypes in 111 trios. Alleles
Transmitted Non-transmitted
1298C 677T
39 58
35 54
χ2
P
0.22 0.14
0.64 0.71
Table 3 – The estimated haplotype frequencies in case mothers and controls. SNP1–2
A1298C-C677T
a
A-C A-T C-C C-T
52.9 55.1 32.1 7.9
58.7 53.3 30.3 5.7
0.30 0.03 0.05 0.36
0.59 0.87 0.82 0.55
the activity of the homozygous wildtype. Case-control studies of the association between the MTHFR polymorphisms and schizophrenia have given inconsistent results (Arinami et al., 1997; Joober et al., 2000; Sazci et al., 2005; Muntjewerff et al., 2005; Kempisty et al., 2006, 2007; Kunugi et al., 1998; Tan et al., 2004; Yu et al., 2004; Vilella et al., 2005). Seven meta-analyses have been performed on this subject, all arguing for an association between these polymorphisms and schizophrenia, although the association with regard to A1298C so far seems somewhat less convincing (Allen et al., 2008; Gilbody et al., 2007; Jönsson et al., 2008; Lewis et al., 2005; Muntjewerff et al., 2006; Shi et al., 2008; Zintzaras, 2006). The recent SzGene database showed that either C677T or A1298C is one of the 24 genetic variants with a nominally significant effect on schizophrenia (Allen et al., 2008), and MTHFR was identified as a candidate gene and familiar association studies are reasonably required for replication (Shi et al., 2008). In this study, we conducted a family-based study to investigate the association of MTHFR C677T and A1298C with schizophrenia. In addition, association between maternal MTHFR polymorphisms and schizophrenia in offspring was investigated.
Odds ratio (95%CI)
χ2
P
53.5 (18.7) 62.4 (13.3) 1.54 (1.03–2.29) 4.45 0.04 106.5 (37.2) 200.4 (42.6) 0.82 (0.60–1.11) 1.67 0.20 118.5 (41.4) 202.6 (43.1) 0.96 (0.71–1.30) 0.07 0.80
Haplotypes with frequency < 0.03 are ignored in analysis.
For the TDT-analysis (Table 1), the transmission/nontransmission counts of − 1298C and − 677T were 39/35 (P = 0.64) and 58/54 (P = 0.71), respectively. In the haplotoypes constructed by A1298C-C677T, the transmission/non-transmission counts of A-C, A-T, C-C and C-T were 52.9/58.7 (P = 0.59), 55.1/53.3 (P = 0.87), 32.1/30.3 (P = 0.82) and 7.9/5.7 (P = 0.55), respectively. No significant transmission distortions were found. Table 2 shows the allele and genotype frequencies for A1298C and C677T in case mothers and control mothers. The frequency of 1298C allele was significantly higher in case mothers (21.3%) than in controls (14.3%), with an OR of 1.63 (95%CI: 1.11–2.39). Genotype analysis also found one significant association corresponding to this positive allele (P = 0.03). No significant difference was found in allele and genotype distributions of C677T between mothers of patients and controls. We performed a haplotype analysis for the variants (Table 3). The haplotype C-C combined by A1298C and C677T is significant (P = 0.04), which showed an OR of 1.54 (95%CI: 1.03–2.29). In a log additive mode of inheritance, the power of A1298C and C677T for detecting an odds ratio (OR) of 1.5 was 43.4% and 56.1% in case–parent trios, respectively. In comparison of case mothers and controls, the power of our sample was 52.8% for A1298C and 76.3% for C677T.
3. 2.
Controls (%)
Haplotype a C-C A-T A-C
Haplotype
Case (%)
Discussion
Results
Genotypes of A1298C and C677T were in Hardy–Weinberg equilibrium among parents (P = 0.19 and P = 0.37), affected offspring (P = 0.80 and P = 0.51) and controls (P = 0.68 and P = 0.33). Strong pairwise linkage disequilibrium was observed between A1298C and C677T (D' = 0.78).
In the present study, we found an association between maternal A1298C polymorphism and schizophrenia in offspring. Based on these data, maternal 1298C allele may be a risk allele for the development of schizophrenia. A1298C is a common variant (1298A → C; glutamate to alanine). Homozygote for the C allele decreased the MTHFR activity by about 30%
Table 2 – Distributions of alleles and genotypes for the two SNPs in case mothers and controls. SNP ID
Sample
N
Allele frequency (%) C
SNP1 A1298C SNP2 C677T
Case Control
143 235
Case Controls
143 235
61 (21.3) 67 (14.3) T 114 (39.9) 205 (43.6)
P
OR (95%CI)
A 225 403 C 172 265
(78.7) (85.7)
0.01
1.63 (1.11–2.39)
(60.1) (56.4)
0.31
0.86 (0.64–1.16)
Genotype frequency (%) C/C
C/A
A/A
6 (4.2) 3 (1.3) T/T 22 (15.4) 41 (17.4)
49 (34.3) 61 (26.0) T/C 70 (49.0) 123 (52.3)
88 (61.5) 171 (72.8) C/C 51 (35.7) 71 (30.2)
P
0.03
0.54
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(van der Put et al., 1998; Weisberg et al., 2001) and significantly increased homocysteine concentrations (Markan et al., 2007). Maternal elevated homocysteine could induce placental vasculopathy (Nelen et al., 2000) which influences the delivery of nutrients and oxygen from mother to fetus and leads to fetal hypoxia (Dalman et al., 2001). Corresponding adverse consequences for fetal development including impaired brain growth (Mercuri et al., 2000) and disturbances of neurotransmitter systems (Vollset et al., 2000) have also been suggested. Further evidence showed that elevated third-trimester maternal homocysteine levels may increase risk of schizophrenia in offspring, induced by developmental effects on brain structure and function through subtle damage to the placental vasculature (Brown et al., 2007). Thus, the maternal 1298C allele could play an important role in the development of schizophrenia. In this study, we report a low 1298C frequency (14%) for the controls. In contrast, previous schizophrenia study of Chinese subjects reported control 1298C allele frequency of 23% (Yu et al., 2004). A recent study reported 17% for the control 1298C allele frequency among Chinese Han women (Xu et al., 2007). This difference may be caused by complicated population admixture in China (Shi et al., 2004) and insufficient control subjects in this study. With regard to the C677T polymorphism, we did not found association between maternal C677T variant and schizophrenia in line with the study conducted by Muntjewerff et al (2007). It has been shown that the C677T variant form has a greater effect on homocysteine concentrations than the A1298C polymorphism (van der Put et al., 1998). Considering the strong linkage equilibrium between A1298C and C677T polymorphisms, possible effect of C677T polymorphism could not be ruled out. Taken together, in order to shed light on the results, larger studies are warranted. It has been well established that family-based studies are an effective approach to follow up the findings from casecontrol investigation (Schaid, 1998). In the present study, TDT analyses of the C677T and A1298C polymorphisms found no significant association of these two polymorphisms with schizophrenia in Chinese subjects. Our findings are consistent with the previous family-based study (Muntjewerff et al., 2007), which suggested no preferential transmission of C677T in case-parent trios. However, regarding maternal 1298C allele may be a genetic risk factor to the development of schizophrenia, the preferential transmission of the 1298C allele could not be expected (Labuda et al., 2002). Thus, parent-case trios studies do not seem to be an appropriate method to detect the association between A1298C and schizophrenia. This study suffers from two limitations. First, it should be noted that the limited sample size involved in this study causes low statistical power. Second, the interaction between MTHFR and potential environmental confounders of the folate metabolism is not considered in this study. In summary, although our results suggest that maternal MTHFR A1298C variant is associated with the occurrence of schizophrenia in offspring, it must be viewed as tentative rather than wholly conclusive given the small sample size, which lead to a higher potential for a type I error. The present results need further replication studies in order to demonstrate this point more fully.
4.
Experimental procedures
4.1.
Subjects
The 111 trios in our study consisted of healthy parents and offspring affected with schizophrenia from Shanghai Mental Health Center, Shanghai Jiao Tong University School of Medicine. Of the schizophrenic probands, 56 (50.5%) were male and 55 (49.5%) were female. The average age of all patients was 18.79 (SD = 4.96). Consensual diagnosis of each proband was made by two independent psychiatrists according to the DSM-IV criteria for schizophrenia on the basis of the Mini International Neuropsychiatric Interview (MINI) and medical records. Including 111 mothers from the trios, we totally recruited 143 mothers (mean age = 47.35, SD = 4.61) whose offspring were affected with schizophrenia. We also enrolled 235 age-matched mothers (mean age = 49.16, SD = 5.90) from the group of hospital staff as control subjects who had healthy children (mean age = 18.64, SD = 6.03). All subjects were Han Chinese origins and signed informed consents. The study protocol and process were assessed and approved by the ethics committee at Shanghai Mental Health Center.
4.2.
Genotyping
Genomic DNA was prepared from venous blood using Tiangen DNA isolation kit (Tiangen biotech, Beijing, China). The primers for C677T and A1298C polymorphisms were designed using Primer 5.0 program (http://www.premierbiosoft.com/ primerdesign/index.html), while A1298C was detected, using a reverse mismatch primer. The sequences were accessed from National Center for Biotechnology Information (NCBI) database. Briefly, the forward and reverse primers for C677T analysis were: 5′-AGGACTACTACCTCTTCTACCTGAA-3′ and 5′AAGAACGAAGACTTCA-AAGACACGT-3′. The primers for A1298C analysis were: 5′-GGGGTCAGAAGCATATCAGT-3′ and 5′-CTCACCTGGATGGGAAAGA-3′. Genotyping was performed for C677T and A1298C by PCR-RFLP methods. For the C677T polymorphism, each PCR product was digested with TaqI at 65 °C for 2 h, followed by 1.5% agarose gel electrophoresis and ethidium bromide staining. The C → T substitution at nucleotide 677 creates a Taql digestion site. The PCR product (390 bp) with T allele was digested to 2 fragments (121 bp and 269 bp), whereas the PCR product with wild type C allele can not be cut by Taql. For the A1298C polymorphism, each PCR product was digested with Pmll at 37 °C overnight, followed by 12% polyacrylamide gel electrophoresis and detected by ethidium bromide staining. The A → C substitution at nucleotide 1298 creates a Pmll digestion site. The polymerase chain reaction (PCR) product (110 bp) with C allele was digested into 2 fragments (26 bp and 84 bp), whereas the PCR product with wild type A allele cannot be cut by Pmll. For quality control, all genotypes were called blind to the case or control status in the genotyping process. Five percent of the samples were repeated for the genotyping assay and the corresponding results were 100% concordant.
4.3.
Statistical analysis
The Haploview software, version 4.1, was used for Hardy– Weinberg equilibrium and transmission disequilibrium analysis
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of individual SNPs in families. In comparison of case mothers and control mothers, all the parameters, allele and genotype frequencies and haplotype analysis were conducted by SHEsis software (http://analysis.bio-x.cn) (Shi and He, 2005). All tests were two tailed and significance was set at 0.05. The statistical power of our sample size was performed on Quanto program (Version 1.2.3, available at http://hydra.usc.edu/GxE).
Acknowledgments The authors are very grateful to all participants. This work was supported by grants from National Natural Science Foundation of China (30500181 and 30971047). We thank the two anonymous reviewers for their insightful comments. And, we thank Dr. Natashia Swalve, who helped in editing this manuscript.
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