Brief report 221

Genetic association study between methyl-CpG-binding domain genes and schizophrenia among Chinese family trios Bing Xiea,d, Yaqin Yua,b, Xiangfei Menga,b, Qiong Yua,b, Jieping Shia,b, Hong Sangc and Changgui Koua,b This study investigates the genetic association between methyl-CpG-binding domain (MBD) gene polymorphisms and schizophrenia. A total of 200 family trios consisting of fathers, mothers, and affected offspring with schizophrenia were recruited as our participants. Four tag SNPs on MBD1 (rs125555, rs140689, rs140687, and rs140686), three tag SNPs on MBD2 (rs3876254, rs7614, and rs1145317), and three tag SNPs on MBD3 (rs7252741, rs4807934, and rs4807122) genes were tested using the PCR-based ligase detection reaction (PCR-LDR). The transmission disequilibrium test showed that rs1145317 on the MBD2 gene was significantly overtransmitted from parents to schizophrenic offspring (P = 0.026). The haplotype-based haplotype relative risk test revealed that the haplotype rs7614–rs1145317 (A–G) was associated with schizophrenia (P = 0.029). Our finding suggests that the

Introduction Schizophrenia is a severe psychiatric disease affecting about 1% of the world’s population (Freedman, 2003), with high heritability (80–85%) and complex transmission (Tandon et al., 2008). Genetic studies have identified several candidate risk genes or genomic regions for schizophrenia (Allen et al., 2008; Purcell et al., 2009; Shi et al., 2009; Stefansson et al., 2009; Kiyomi et al., 2013), and epidemiological studies have revealed several environmental risk factors (Morgan and Fisher, 2007; Van et al., 2010; Brown, 2011). However, the etiology of schizophrenia still remains largely unknown. Nevertheless, the interaction of many genes with each other and with environmental risk factors may lead to the onset of schizophrenia (Van et al., 2008; Vilain et al., 2013). Epigenetic mechanisms such as DNA methylation and histone modification can explain the interaction between genetic and environmental factors at a molecular level, and accumulating evidence suggests that such epigenetic alterations are involved in the pathogenesis of schizophrenia (Akbarian, 2010; Gavin and Sharma, 2010; Dean, 2013; Dempster et al., 2013). The epigenetic genes, such as histone deacetylase (HDAC) genes and methyl-CpGbinding domain (MBD) genes, as well as DNA methyltransferase genes may be associated with the occurrence of schizophrenia. We previously reported on the Supplemental digital content is available for this article. Direct URL citations appear in the printed text and are provided in the HTML and PDF versions of this article on the journal's website (www.psychgenetics.com). 0955-8829 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins

MBD2 gene may be a susceptibility gene for schizophrenia. Psychiatr Genet 24:221–224 © 2014 Wolters Kluwer Health | Lippincott Williams & Wilkins. Psychiatric Genetics 2014, 24:221–224 Keywords: haplotype, MBD genes, schizophrenia, tag single nucleotide polymorphisms, transmission disequilibrium test a

Research Center for Genomic Medicine, bDepartment of Epidemiology and Biostatistics, School of Public Health, Jilin University, cChangchun Mental Health Hospital, Changchun and dCentral Laboratory, The First Hospital of Hebei Medical University, Shijiazhuang, China Correspondence to Changgui Kou, PhD, Research Center for Genomic Medicine, School of Public Health, Jilin University, 1163 Xinmin Street, Changchun 130021, Jilin, China Tel/fax: + 86 0431 85619437; e-mail: [email protected] Received 15 August 2013 Revised 18 March 2014 Accepted 20 April 2014

association of HDAC-2 and HDAC-3 genes with schizophrenia, but no statistically significant difference was found (Han et al., 2013). Besides HDAC genes, MBD genes also play a central role in epigenetic mechanisms, and provide the molecular link between DNA methylation and histone modification (Hendrich and Bird, 1998; Klose and Bird, 2006). Although, MBD genes could be expressed in many different tissues, the absent and/or mutated phenotype is more likely present in the nervous system (Chahrour et al., 2008). A study on the expression of MBD genes indicated that MBD1, MBD2, and MBD3 are broadly expressed in the murine brain (Hendrich and Tweedie, 2003). In addition, some patients with an 18q deletion (such as 18q21 deletion), where MBD1 and MBD2 genes are localized, have been diagnosed with neurological disease (Mahr et al., 1996). All signs indicate that common variations in MBD genes confer risk for schizophrenia. In this study, we select relevant epigenetic genes, MBD1, MBD2, and MBD3 genes, as the target genes and analyze their association with schizophrenia among Chinese family trios using the transmission disequilibrium test (TDT) and haplotype-based haplotype relative risk (HHRR) test.

Methods Family trios

In this study, a total of 200 Chinese family trios (600 participants) of Han descent, comprising fathers, DOI: 10.1097/YPG.0000000000000042

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222 Psychiatric Genetics 2014, Vol 24 No 5

mothers, and affected offspring with schizophrenia, were recruited for genetic analysis at Jilin University (Changchun, China). The participants were from the Northeastern area of China. The patients (126 male and 74 female), aged 26.6 ± 6.3 years, were admitted to a psychiatric hospital between 2008 and 2011. Consensual diagnosis of each patient was made by two independent psychiatrists according to the International Statistical Classification of Diseases and Related Health Problems – tenth revision criteria for schizophrenia and excluded the diagnosis of any other psychiatric disease. Healthy individuals from the trios were assessed through brief psychiatric assessment by research psychiatrists. Our study was carried out between January 2011 and January 2012 and authorized by the Ethics Committee of Jilin University; written informed consent was obtained from the next of kin of the participants. Genetic analysis

The reference details of MBD1, MBD2, and MBD3 genes in the Chinese Han population are available from the Hapmap website (http://www.hapmap.org). Tag single nucleotide polymorphisms (tag SNPs) were selected on the basis of their map position using HaploView 4.1 (Broad Institute of MIT and Harvard, USA) (Barrett et al., 2005). All tag SNPs selected were from phase II genotyping data of the HapMap project, which limited the inclusion criteria to the Chinese Han population: MAF greater than 0.10 and r2 greater than 0.8. Four tag SNPs on MBD1 (rs125555, rs140689, rs140687, and rs140686), three tag SNPs on MBD2 (rs3876254, rs7614, and rs1145317), and three tag SNPs on MBD3 (rs7252741, rs4807934, and rs4807122) genes were selected to represent the genetic information of all tag SNPs that qualified the inclusion criteria. Genomic DNA was extracted from saliva samples using a genomic DNA extraction kit (Promega, Wisconsin, USA). SNPs were successfully genotyped using the PCR-based ligase detection reaction (Xie et al., 2011). The probe and primer sequences for the PCR-based ligase detection reaction in MBD1, MBD2, and MBD3 genes have been shown in supplementary material I and II (Supplemental digital content 1 and 2, http://links.lww.com/PG/A107, http://links.lww.com/PG/A108). PCR amplification was performed in a 20 μl reaction volume containing 1 μl 50 ng genomic DNA, 0.6 μl 3.0 mmol/l MgCl2, 2 μl 2 mmol/l each dNTP, 0.2 μl 1 U HotStar Taq DNA polymerase (Qiagen, Hilden, Germany), 2 μl 1 × buffer, 4 μl 1 × Q solution (Qiagen, Hilden, Germany), 0.4 μl mixed primer (supplementary material I, Supplemental digital content 1, http://links. lww.com/PG/A107), and 9.8 μl double distilled H2O. The conditions required for PCR amplification were as follows: 95°C for 15 min; 35 cycles of 94°C for 30 s, 59°C for 1.5 min, and 72°C for 1 min; and final elongation at 72°C

for 7 min. The LDR reaction involves a 10 μl volume containing 1.0 μl 1 × buffer, 1 μl 12.5 pmol/μl of each of the LDR mixed probes (supplementary material II, Supplemental digital content 2, http://links.lww.com/PG/A108), 0.05 μl 2 U/μl Taq DNA Ligase (New England Biolabs, Ipswich, Massachusetts, USA), 1 μl 100 ng/μl PCR product, and 6.95 μl double distilled H2O. The cycling condition of LDR reaction were as follows: 30 cycles at 95°C for 2 min, 94°C for 30 s, and 50°C for 2 min. Statistical analyses

Statistical analyses included family-based association analyses carried out using the PBAT statistical package incorporated into SNP & Variation Suite 7 (http://www. goldenhelix.com/SNP_Variation/PBAT/index.html; Lange et al., 2004). For nuclear families, the PBAT package was used to perform the sample power calculation, to assess Hardy–Weinberg equilibrium for genotype distributions, for TDT analysis, for the HHRR test, and for the Bonferroni test with 1000 permutations. TDT was applied to test allelic association for a single locus, and the HHRR test was applied to test for multiple locus association. Bonferroni’s test gives a corrected significance level for these 10 SNPs. Linkage disequilibrium (LD) was estimated on the Haploview 4.1 software. D′ values were used as the LD measure to represent the strength of LD between pairs of SNPs. Statistical power was computed on the online software Genetic Power Calculator (http://pngu.mgh.harvard.edu/ ~ purcell/gpc/; Purcell et al., 2003). Statistical significance was defined as P less than 0.05.

Results The distribution of allelic and genotypic frequencies of MBD1 SNPs (rs125555, rs140689, rs140687, and rs140686), MBD2 SNPs (rs3876254, rs7614, and rs1145317), and MBD3 SNPs (rs7252741, rs4807934, and rs4807122) are shown in supplementary material III (Supplemental digital content 3, http://links.lww.com/PG/A109). None of the 10 SNPs deviated from HWE in either the patient group or the parent group (P > 0.05, supplementary material IV, Supplemental digital content 4, http://links.lww.com/PG/A110). Strong pairwise LD was observed in all tested SNPs (all |D′| > 0.712, supplementary material V, Supplemental digital content 5, http://links.lww.com/PG/A111). TDT analysis of individual SNPs found that there was a significant association between rs1145317 and schizophrenia (χ2 = 4.928, P = 0.026). Of 364 parents, 195 were heterozygous at rs1145317, and they transmitted 82A alleles and 113G alleles to their affected offspring (Table 1). In addition, HHRR analysis showed that the rs7614–rs1145317 haplotype (A–G) was significantly overtransmitted (global P = 0.029, d.f. = 3; Table 2). In the sample size power calculation using the PBAT package, we found that the present sample size had greater than 83.5% power for

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MBD gene SNPs and schizophrenia Xie et al. 223

The transmission disequilibrium test analysis of allelic association between single nucleotide polymorphisms and schizophrenia

Table 1

Allele frequencya

TDT

Gene

Tag SNP

Type

Patients

Parents

T/NT

χ2

P

MBD1

rs125555 rs140689 rs140687 rs140686 rs3876254 rs7614 rs1145317 rs7252741 rs4807934 rs4807122

C/G A/T G/T A/G A/G A/G A/G C/T A/G C/T

0.137 0.141 0.412 0.366 0.444 0.478 0.401 0.261 0.450 0.144

0.123 0.153 0.444 0.376 0.471 0.494 0.444 0.289 0.481 0.141

38/48 45/54 94/74 93/86 83/102 97/85 82/113 66/86 103/80 46/44

1.163 0.818 2.381 0.274 1.951 0.791 4.928 2.632 2.891 0.044

0.281 0.366 0.123 0.601 0.162 0.374 0.026c 0.105 0.089 0.833

MBD2

MBD3

b

MBD, methyl-CpG-binding; NT, nontransmitted; SNP, transmission disequilibrium test; T, transmitted; TDT, transmission disequilibrium test. a The frequency of minor allele. b The base with minor allele frequency is in boldface. c Associations remain significant after Bonferroni correction with 1000 permutations.

Table 2

The haplotype analysis of multimarkers

Gene

Haplotypes

χ2

d.f.

P

MBD1

rs125555–rs140689 rs140689–rs140687 rs140687–rs140686 rs125555–rs140689–rs140687 rs140689–rs140687–rs140686 rs125555–rs140689–rs140687–rs140686 rs3876254–rs7614 rs7614–rs1145317 rs3876254–rs7614–rs1145317 rs7252741–rs4807934 rs4807934–rs4807122 rs7252741–rs4807934–rs4807122

2.121 4.076 2.917 5.284 5.088 12.190 2.830 9.042 11.300 5.114 3.357 10.950

3 3 3 6 7 13 3 3 7 3 3 7

0.548 0.253 0.405 0.508 0.649 0.513 0.419 0.029a 0.126 0.164 0.340 0.141

MBD2

MBD3

a Associations remain significant permutations.

after Bonferroni correction with 1000

alleles. The statistical analysis showed that the power of each site was greater than 0.861 and suitable for genetic analysis in these selected samples (supplementary material V, Supplemental digital content 5, http://links. lww.com/PG/A111). The sample size and genetic power calculations were based on a disease prevalence of 1% and a type I error of 0.05.

Discussion Previous genetic studies on schizophrenia have mainly focused on candidate genes from functional pathways, but few genes involved in epigenetic profiles have been reported. This study aims to reveal the role of the MBD genes (epigenetic gene) in schizophrenia. We genotyped 10 tag SNPs on the MBD1, MBD2, and MBD3 genes and analyzed individual and joint genetic associations between the MBD 1–3 genes and schizophrenia in Han Chinese family trios. Our results revealed the following: first, that a significant association between rs1145317 at the MBD2 gene and schizophrenia exists, and second that the haplotype rs7614–rs1145317 was

significantly overtransmitted from parents to schizophrenic offspring. Bonferroni’s correction was used to adjust the significance level of multiple comparisons, and after correction the adjusted P-value for the association between rs1145317 and schizophrenia was statistically significant (P = 0.026), indicating that there was an association between rs1145317 and schizophrenia. Therefore, our finding suggests that the MBD2 gene may be a susceptibility gene for schizophrenia. However, the present study failed to support the genetic association of the MBD1 and MBD3 loci with schizophrenia. It is well known that a complicated disease like schizophrenia depends on the interaction of multiple factors and no particular genes are uniquely responsible for the disease. Surely, environmental factors also play a role in the occurrence of the disease (Tsuang, 2000; Mittal et al., 2008). Many studies have suggested an association between MBD1 and MBD3 genes and other complex disorders. For example, Li et al. (2005) found that the R269C mutation in the MBD1 gene may be associated with autism. In addition to clinical data, accumulating evidence from animal models has linked the severe effects of MBD genes with regulation of brain function. For example, in the adult rat brain, MBD1 and MBD3 are strongly expressed in neurons of the hippocampus and cortex (Fan and Hutnick, 2005). MBD1 null mice have multiple behavioral abnormalities, such as learning deficits, reduced social interactions, heightened anxiety, and greater susceptibility to depression (Zhao et al., 2003). MBD3 null mice are not viable and embryonic lethal, with no normal-appearing MBD3 null embryos recovered after implantation (Hendrich et al., 2001). Recent evidence indicates that DNA methylation may serve as a contributing mechanism in memory formation and storage. These emerging findings suggest a role for MBD genes in learning and long-term memory maintenance, which certainly can be associated with schizophrenia (Day and Sweatt, 2010). These results suggest an important role for MBD genes in nervous system development and function, and abnormalities in these genes may lead to some mental disorders. Our results provide significant evidence supporting the association of MBD2 gene polymorphisms with schizophrenia in Han Chinese. These findings reinforce the idea that some epigenetic gene variants are associated with schizophrenia. It is well known that the incidence of complicated diseases like schizophrenia depends on the interaction of multiple factors. Usually, no single gene is uniquely responsible for schizophrenia and environmental factors also play a role in its occurrence. Limited sample size, etiological heterogeneity, and clinical heterogeneity may result in inconsistent results, showing different associations between the MBD genes and schizophrenia; therefore, the conclusion of this study may be viewed in

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224 Psychiatric Genetics 2014, Vol 24 No 5

this context. Further genetic studies using a larger sample size and environmental interaction studies will undoubtedly add further valuable insight into the implications of the relationship between MBD gene polymorphisms and schizophrenia.

Acknowledgements The authors thank all the families who participated in this study and the psychiatrists and mental health workers who helped identify the studied participants. This work was supported by the National Natural Science Foundation of China (Grant No. 30972536). Study concept and design: Yaqin Yu and Hong Sang. Acquisition of data: Xiangfei Meng and Qiong Yu. Analysis and interpretation of data: Changgui Kou. Drafting of the manuscript: Bing Xie. Critical revision of the manuscript for important intellectual content: Yaqin Yu and Changgui Kou. Statistical analysis: Bing Xie and Changgui Kou. Administrative, technical, and material support: Yaqin Yu, Jieping Shi, and Hong Sang. Study supervision: Yaqin Yu and Changgui Kou. Conflicts of interest

There are no conflicts of interest.

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