ARTICLE International Journal of Neuropsychopharmacology (2006), 9, 705–712. Copyright f 2005 CINP doi:10.1017/S1461145705006267 An investigation of the dihydropyrimidinase- like 2 (DPYSL2) gene in schizophrenia: genetic association study and expression analysis Xinzhi Zhao1,2*, Ruqi Tang1,2*, Zeping Xiao3*, Yongyong Shi1,2, Guoying Feng3, Niufan Gu3, Jianguo Shi4, Yangling Xing4, Lijuan Yan5, Hong Sang5, Shaomin Zhu6, Huijun Liu6, Wuyan Chen1,2, Jixia Liu1,2, Wei Tang1,2, Jing Zhang1,2 and Lin He2,7 1 Bio-X Center, Shanghai Jiao Tong University, Shanghai, China 2 Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China 3 Shanghai Institute of Mental Health, Shanghai, China 4 Xi’an Institute of Mental Health, Xi’an, China Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 5 Changchun Kaixuan Hospital, Changchun, China 6 JiLin Institute of Mental Health, JiLin, China 7 NHGG, Shanghai Jiao Tong University, Shanghai, China Abstract Several linkage studies support a susceptibility locus for schizophrenia on chromosome 8p21-22. In this study, we investigated a gene mapping to 8p21, dihydropyrimidinase-like 2 (DPYSL2). DPYSL2 plays an important role in axonal formation and dysfunction of DPYSL2 may result in neurodevelopmental abnormalities. In previous studies, the expression of the gene has been shown to display alteration in the brain of schizophrenia patients compared with those of healthy controls. Recently, Nakata and colleagues found polymorphisms in the 3k-end of DPYSL2 to be associated with schizophrenia, especially the paranoid type, in a Japanese population. In this study, we genotyped four SNPs in DPYSL2 in 2552 Chinese Han specimens. Case-control and TDT analyses were performed to detect association of DPYSL2 with schizophrenia. However, no allele, genotype or haplotype association was found. We investigated the expression of DPYSL2 in 29 schizophrenia patients and 54 healthy controls using quantitative real-time PCR and no difference was found between the two groups. In a comparative allele-specific expression test, we used two SNPs as markers. Only a small proportion of heterozygotes revealed a significant difference (>20%) in allele representation. The results indicated the mRNA level did not contribute mainly in the altered expression of the gene in schizophrenia patients. Although our results provided no evidence for DPYSL2 itself as a susceptibility gene for schizophrenia, recent findings have indicated that DPYSL2 may interact with other candidate genes for schizophrenia and be worthy of further studies. Received 12 June 2005; Reviewed 17 July 2005; Revised 19 August 2005; Accepted 23 August 2005; First published online 2 December 2005 Key words: Allele-specific expression, DPYSL2, genetic association, gene expression, schizophrenia. Introduction As a result of a large number of genome-wide linkage studies, many plausible candidate regions for Schizophrenia (MIM 181500) is one of the most severe schizophrenia have been found although only small mental disorders, with a worldwide incidence of 1% parts of them have been well replicated. However, a (Lewis and Lieberman, 2000). Data from twin, family, series of independent studies indicate that the 8p21-22 and adoption studies provide strong evidence that region is a locus associated with genetic susceptibility schizophrenia is predominantly a genetic disorder to schizophrenia (Blouin et al., 1998; Gurling et al., with high heritability (Tsuang, 2000). 2001; Kendler et al., 2000; Stefansson et al., 2002). Several genes located in the 8p21-22 region may Address for correspondence : Dr Lin He, Institute for Nutritional be strong candidates for susceptibility to schizo- Sciences, Shanghai Institutes for Biological Sciences, 294 Taiyuan phrenia. In the present study, we have focused on Road, Chinese Academy of Sciences, Shanghai 200031, China. dihydropyrimidinase-like 2 (DPYSL2, also named as Tel. : 00 86 21 62822491 Fax : 00 86 21 62822491 E-mail : [email protected] DRP2 or CRMP2), which is localized on 8p21 and is * These authors contributed equally to this work. highly expressed in the developing nervous system. 706 X. Zhao et al. In neurons, DPYSL2 binds to tubulin heterodimers Materials and methods to enhance microtubule assembly, which helps in Subjects the elongation of axons (Fukata et al., 2002). The hypofunction of DPYSL2 may inhibit axonal formation For the case-control investigation, 741 unrelated and may result in neurodevelopmental abnormalities patients and 752 controls were recruited. The patient (Inagaki et al., 2001), which may be a factor in the group consisted of 403 males and 338 females with pathogenesis of schizophrenia (Weinberger, 1995). a mean age of 42.74 yr (S.D.=12.58 yr), of whom 549 Two-dimensional gel electrophoresis and sequenc- originated from Shanghai and 192 from Jilin. The ing of proteins have indicated that DPYSL2 displays controls consisted of 359 males and 393 females with disease-specific alterations in the human brain. a mean age of 34.28 yr (S.D.=9.59 yr), of whom 560 Johnston-Wilson et al. (2000) reported that the DPYSL2 were from Shanghai and 192 from Jilin. Among these protein level was significantly lower in the frontal specimens, there were 675 patients with information cortex in schizophrenia, bipolar and major depression on subtypes, including 279 cases of paranoid type, patients; while Edgar et al. (2000) found increased seven cases of catatonic type, 29 cases of disorganized Downloaded from https://academic.oup.com/ijnp/article/9/6/705/666184 by guest on 01 October 2021 levels of DPYSL2 in the hippocampus in schizo- type, 76 cases of residual type and 284 cases of phrenia patients. undifferentiated type. Genotyping data from the Recently, Nakata and colleagues (2003) investigated paranoid-type schizophrenia patients (134 males five polymorphisms within DPYSL2 and performed and 145 females, mean age 45.62 yr, S.D.=13.60) were a case-control analysis in a Japanese population. additionally compared with those from the controls They found that a polymorphism, rs17666, in the (Table 1). Another subgroup of population-based 3k untranslated region of the gene was significantly specimens including 29 patients (15 males and 14 associated with schizophrenia (p=0.0097), especially females, mean age 39.68 yr, S.D.=12.66 yr) and 54 the paranoid-type (p=0.0083). However, more inde- controls (27 males and 27 females, mean age 39.66 yr, pendent replications are needed to confirm this S.D.=11.99 yr) from Shanghai were used to investigate finding. Another allele-specific gene expression study gene expression and allele-specific expression. For indicated that some cis-acting variations may alter the transmission disequilibrium test (TDT) study, 353 the expression of DPYSL2 but may be in low unrelated schizophrenia probands (187 males and 165 heterozygosity: Bray et al. (2003) used the SNP females, mean age 24.88 yr, S.D.=6.82 yr) and their rs708621 as the marker, and found that one of the 20 biological parents were recruited. All subjects were heterozygotes revealed a significant difference (39%) Han Chinese in origin. Subjects with schizophrenia in allele representation. Since polymorphisms in the were strictly diagnosed by two independent psy- untranslated region may alter the gene expression chiatrists according to the DSM-III-R criteria. A (Conne et al., 2000), an allele-specific gene expression standard informed consent in the protocol, which was study may be able to detect the potential function of reviewed and approved by the Shanghai Ethical the SNP rs17666. Committee of Human Genetic Resources, was given In the present study, we analysed the association by the participants, after the nature of study had been between four SNPs (rs327234, rs2289592, rs708621 and fully explained. rs17666) in DPYSL2 and schizophrenia in a Chinese Han population, using a total of 2552 specimens. We Genotyping choose SNPs in the study according to the following criteria: the SNP rs17666, which showed association The genomic structure of DPYSL2 and the location with schizophrenia in the Japanese population, used of the SNPs genotyped in the present study are in the study of Nakata et al. (2003); rs708621 used in shown in Figure 1. All the four SNPs were selected the study of Bray et al. (2003); rs327234 and rs2289592 from the dbSNP (http://www.ncbi.nlm.nih.gov/ with proper localization and heterozygosity. We also SNP). Genomic DNA was prepared from venous investigated the gene expression in the lymphocytes blood using the phenol chloroform extraction method. 1 of 83 samples. After synthesizing cDNA, real-time The four SNPs were genotyped using TaqMan tech- polymerase chain reaction (PCR) was performed to nology on an ABI 7900 system (Applied Biosystems, investigate whether the expression of DPYSL2 was Foster City, CA, USA). All probes and primers were altered in schizophrenia patients compared with designed by the Assay-by-DesignTM or Assay-on- controls. Template-directed primer extension was DemandTM service of Applied Biosystems. The stan- used to detect allele-specific expression of rs17666 dard PCR reactions of 5 ml were carried out using 1 and rs708621. TaqMan Universal PCR Master Mix reagent kits An investigation of the DPYSL2 gene in schizophrenia 707 Table 1. Genotype and allele frequencies of SNPs and association analysis of each SNP in population-based samples Genotypeb Alleleb Marker na T/T T/C C/CHWE (p) p value T C p value OR rs327234 Control