RESEARCH ARTICLE Neuropsychiatric Genetics Association of RANBP1 Haplotype With Smooth Pursuit Eye Movement Abnormality Hyun Sub Cheong,1 Byung Lae Park,1 Eun Mi Kim,1 Chul Soo Park,2 Jin-Wook Sohn,2 Bong-Jo Kim,2 Jae Won Kim,3 Ki-Hoon Kim,4 Tae-Min Shin,4 Ihn-Geun Choi,5 Sang-Woo Han,6 Jaeuk Hwang,6 InSong Koh,7 Hyoung Doo Shin,1,8** and Sung-Il Woo6* 1 Department of Genetic Epidemiology, SNP Genetics, Inc., Seoul, Republic of Korea 2 Department of Psychiatry, College of Medicine, Gyeongsang National University, Jinju, Gyeongsang Nam Do, Republic of Korea 3 Division of Life Science, Research Institute of Life Science, Gyeongsang National University, Jinju, Gyeongsang Nam Do, Republic of Korea Department of Biomedical Engineering, Yonsei University, Wonju, Republic of Korea 4 5 Department of Neuropsychiatry, Hallym University, Han-Gang Sacred Heart Hospital, Seoul, Republic of Korea 6 Department of Neuropsychiatry, Soonchunhyang University Hospital, Seoul, Republic of Korea Department of Physiology, College of Medicine, Hanyang University, Seoul, Republic of Korea 7 8 Department of Life Science, Sogang University, Seoul, Republic of Korea Received 7 April 2010; Accepted 30 September 2010 Schizophrenia is a multifactorial disorder and smooth pursuit eye movement (SPEM) disturbance is proposed as one of the most consistent neurophysiological endophenotype in schizophrenia. The aim of this study was to examine the genetic association of RANBP1 polymorphisms with the risk of schizophrenia and with the risk of SPEM abnormality in schizophrenia patients in a Korean population. Two SNPs of RANBP1 were genotyped by TaqMan assay. Their genetic effect of single/ haplotype polymorphisms on the risk of schizophrenia and SPEM abnormality from 354 patients and 396 controls were performed using c2 and multiple regression analyses. Although no RANBP1 polymorphisms were associated with the risk of schizophrenia, a common haplotype, RANBP1-ht2 (rs2238798G–rs175162T), showed significant association with the risk of SPEM abnormality among schizophrenia patients after multiple correction (Pcorr ¼ 0.002–0.0003). The results of present study provide the evidence that RANBP1 on 22q11.21 locus might be causally related to the SPEM abnormality rather than the development of schizophrenia. 2010 Wiley-Liss, Inc. Key words: RANBP1; schizophrenia; single-nucleotide polymorphism; smooth pursuit eye movement INTRODUCTION Schizophrenia (MIM 181500) is regarded as a multifactorial disorder [McGrath et al., 2004] with heterogenous clinical subtypes. Smooth pursuit eye movement (SPEM) disturbance may be a neurophysiological endophenotype which is an intermediate variable linking schizophrenia phenotype more directly to the corresponding genotypes. SPEM disturbance is reportedly found in approximately 40–80% of patients of schizophrenia and <10% of healthy control 2010 Wiley-Liss, Inc. How to Cite this Article: Cheong HS, Park BL, Kim EM, Park CS, Sohn J-W, Kim B-J, Kim JW, Kim K-H, Shin T-M, Choi I-G, Han S-W, Hwang J, Koh I, Shin DH, Woo S-I. 2011. Association of RANBP1 Haplotype With Smooth Pursuit Eye Movement Abnormality. Am J Med Genet Part B 156:67–71. subjects [Holzman, 2000; Rybakowski et al., 2002]. However, the genetic origin for SPEM abnormality has been poorly understood and only a few researches such as the association studies with COMT and ZDHHC8 polymorphisms had been performed [Park et al., 2009; Vorstman et al., 2009; Shin et al., 2010]. Additional Supporting Information may be found in the online version of this article. Grant sponsor: Korea Science and Engineering Foundation (KOSEF); Grant sponsor: Korean Government (MEST); Grant Number: 20090080157. *Correspondence to: Dr. Sung-Il Woo, Department of Neuropsychiatry, Soonchunhyang University Hospital, 657, Hannam-dong, Yongsan-gu, Seoul 140-743, Republic of Korea. E-mail: email@example.com **Correspondence to: Dr. Hyoung Doo Shin, Department of Life Science, Sogang University, Seoul 121-742, Republic of Korea. E-mail: firstname.lastname@example.org Published online 2 November 2010 in Wiley Online Library (wileyonlinelibrary.com) DOI 10.1002/ajmg.b.31139 67 68 RAN-binding protein 1 (RANBP1; MIM# 601180), a member of proteins regulating the binding and hydrolysis of GTP by Ras-related proteins, regulates nucleotide turnover on RAN resulting from the stimulation of hydrolysis by RANGAP1 and inhibition of RCC1 activity [Bischoff et al., 1995]. RANBP1 is found in complexes with RAN and transport factors [Plafker and Macara, 2002], and it may contribute to the modulation of the assembly and disassembly of these complexes [Chi et al., 1996; Kehlenbach et al., 1999]. RANBP1 is located in the microdeleted region of 22q11 locus, the deletion of which causes the Velo-cardio-facial syndrome (VCFS) characterized by high rates of schizophrenia [Murphy, 2002], and many studies have focused on the associations of the genes on the microdeletion locus of 22q11 with VCFS, and especially with schizophrenia [Badner and Gershon, 2002; Lewis et al., 2003; Mukai et al., 2004; Liu et al., 2007; Kempf et al., 2008; Meltzer et al., 2008; Park et al., 2009; Coman et al., 2010; Shin et al., 2010]. In a previous study [Liu et al., 2002], a RANBP1 polymorphism (rs175163) was associated with schizophrenia vulnerability in non-22q11 deleted individuals. Our previous study also showed that ZDHHC8 SNPs, a nearby (4 kb) gene of RANBP1 were associated with SPEM abnormality in Korean schizophrenia patients [Shin et al., 2010]. Based on these previous studies about schizophrenia and SPEM abnormality, it is hypothesized that RANBP1 plays an important role in the development of schizophrenia. In the present study, we analyzed the association of RANBP1 polymorphisms with the risk of schizophrenia and the quality of SPEM function to find the gene involved in schizophrenia development and SPEM abnormality in the Korean population. MATERIALS AND METHODS Subjects All individuals included in this study were of Korean ethnic origin; they consisted of 354 schizophrenia patients (176 males and 178 females) and 396 control subjects (224 males and 172 females). Schizophrenia patients were recruited from three mental hospitals: Keyo Hospital (Kyunggi-Do, Korea), Jinju Mental Hospital, and Soonyoung Hospital (Gyeongsang Nam Do, Korea). The ages of the schizophrenic group were in the range of 23–76 years (mean age ¼ 44.0 years, SD ¼ 9.3). The schizophrenia diagnoses were based on the DSM-IV criteria [American Psychiatric Association, 1994], which were applied by two trained psychiatrists following a prior clinical diagnosis of schizophrenia. Patients with complicating diagnoses of mental retardation, organic brain damage, drug or alcohol abuse, neurological disorders, autoimmune disorders, and those with low comprehension skills were excluded. Patients with drug side effects such as tardive dyskinesia, extrapyramidal symptoms, and oculogyric crisis were also excluded from the study. The controls were unrelated healthy employees of the Center for Health Promotion of Seoul National University Hospital (Seoul, Korea), with an age range of 28–80 years (mean age ¼ 54.6 years, SD ¼ 9.3). Trained clinicians evaluated each control subject using the Structured Clinical Interview for DSM-IV, non-patient edition (SCID-NP), to ensure that the individual did not have an ongoing or previous psychiatric illness. The institutional review board of AMERICAN JOURNAL OF MEDICAL GENETICS PART B each hospital approved the study, and all subjects provided informed consent. The Measurement of Smooth Pursuit Eye Movement (SPEM) We evaluated SPEM of schizophrenia patients by electrooculographic recordings (EOG) of eye movement and calculated the natural logarithmic values of the signal/noise ratio (Ln S/N ratio). Using this method, the global quantitative SPEM abnormality can be scored and analyzed as the Ln S/N ratio. Subjects were seated in a darkened, quiet room, in front of a 19-in. computer monitor situated at a distance of 40 cm away. Patients were instructed to observe the moving target spot as closely as possible during the pursuit task. The green target spot (0.8 cm 1 cm) appeared at the center of the computer monitor for 0.5 sec and then moved horizontally back and forth into 18.2 of visual arc to each side of the monitor screen at a constant speed of 28.2 /sec. Using Biopac MP150 (BIOPAC Systems, Goleta, CA), the electrophysiologic analog signals of SPEM were amplified and sampled at 400 Hz and converted into digitized files. A measurement time of 15 sec during the SPEM task were resampled at 4 Hz and passed through a 2 Hz low-pass filter. After application of Fast Fourier Transformation (FFT) to the EOG data, the Ln S/N ratio was calculated from analysis of the power spectrum curves. SPEM data were collected in 166 schizophrenic patients (81 males and 85 females) receiving stable doses of typical antipsychotic medications for at least 2 weeks. For additional statistical analysis, patients were divided into three groups with equal number of subjects (good: 4.49 0.25, intermediate: 3.92 0.16, and poor SPEM function: 2.96 0.66) according to their SPEM function using the Ln S/N ratio values of 3.60 and 4.20. Single Nucleotide Polymorphism (SNP) Genotyping Among 61 dbSNPs detected, 7 were reported in 2 Asian groups (Han Chinese and Japanese) in the HapMap database (release #27). Only two polymorphisms (rs2238798 and rs175162) of RANBP1 were selected based on their minor allele frequencies (freq. > 10%) in Asian and LD. Although all of dbSNPs were not considered, the two tagging SNPs were selected to capture all common RANBP1 SNPs in the HapMap database. rs175162 is in absolute LD (linkage disequilibrium) with rs175163 which showed an association with schizophrenia susceptibility in European population [Liu et al., 2002] (Supplementary Fig. 1A,C). SNPs were genotyped using TaqMan [Livak, 1999] assay. Genotyping quality control was performed in 10% of the samples by duplicate checking (rate of concordance in duplicates >99%). Assay IDs of rs2238798 and rs175162 were ‘‘C__16173148_10’’ and ‘‘C___2613312_1_,’’ respectively (Applied Biosystems, Foster City, CA). Statistics We examined Lewontin’s D0 (|D0 |) and the linkage disequilibrium coefficient r2 between all pairs of bi-allelic loci [Hedrick, 1987]. CHEONG ET AL. 69 Haplotypes were inferred using the algorithm developed by the Broad Institute, Haploview [Barrett et al., 2005]. The allelic distributions of polymorphisms and haplotypes among patients with schizophrenia and normal subjects were evaluated by c2 analyses (two-tailed) and logistic regression models calculating ORs, 95% CIs, and corresponding P-values using age and sex as covariates. Multiple regressions were used for association analyses of SPEM reliability adjusting for age and sex as covariates among schizophrenia patients. Statistical power of single associations was calculated with false positive rate of 5%, disease lifetime prevalence of 0.4% [McGrath et al., 2004], given minor allele frequencies and sample sizes, and assuming a relative risk of 1.5, using PGA (Power for Genetic Association Analyses) software [Menashe et al., 2008]. RESULTS Current study examined the association of RANBP1 polymorphisms with the risk of schizophrenia and the SPEM abnormality in Korean population. Two tagging SNPs were selected from International HapMap database and genotyped in Korean schizophrenia patients and controls (Supplementary Fig. 1A). The minor allele frequencies were 0.498 (rs2238798) and 0.132 (rs175162), respectively (n ¼ 750), and genotype distributions were in Hardy–Weinberg equilibrium (P > 0.05) (Supplementary Table I). Measured linkage disequilibrium among two SNPs by calculating Lewontin’s D0 and r2 values (Supplementary Fig. 1C), revealed that these SNPs were in complete LD (|D0 | ¼ 1 and r2 „ 1). Among three haplotypes constructed, only RANBP1-ht2 was used for further analyses because RANBP1-ht1 and ht3 were equivalent to rs2238798 and rs175162, respectively (Supplementary Fig. 1B). Allele frequencies of rs2238798, rs175162, and RANBP1-ht2 were compared among schizophrenia patients and population control groups using c2 analysis. None of them showed a significant association with risk of schizophrenia (statistical powers were above 80%) (Table I). In further analysis, association of RANBP1 polymorphisms with the Ln S/N ratio of SPEM were analyzed using multiple regression models controlling for age and sex as covariates. The obtained P values were corrected for multiple testing by Bonferroni method. After correction for multiple testing, RANBP1 -ht2 was significantly associated with the low Ln S/N ratio of SPEM (Pcorr ¼ 0.002–0.0003) among schizophrenia patients. The Ln S/N ratio of RANBP1-ht2 bearing subjects was lower than that of RANBP1-ht2 non-bearing subjects, for example, 3.58 0.81 of ht2 -bearing subjects versus 3.88 0.75 of other subjects (Table II). When patients were subgrouped into three categories based on SPEM index (equal distribution of the number of subjects), similar results were obtained (Pcorr ¼ 0.0006; Supplementary Table II). DISCUSSION Previously many genetic studies were performed focusing on the association of schizophrenia with genes (COMT, HTF9C, PRODH, RANBP1, SULT4A1, and ZDHHC8) at the microdeletion locus of 22q11 [Liu et al., 2002, 2007; Mukai et al., 2004; Kempf et al., 2008; Meltzer et al., 2008; Park et al., 2009; Coman et al., 2010; Shin et al., 2010]. Among these previous studies, an intronic SNP, rs175163 of RANBP1 was shown to be associated with schizophrenia susceptibility in European population [Liu et al., 2002]. But, in the current study, rs2238798 and rs175162 was not associated with the risk of schizophrenia in the Korean population (rs175162 is in absolute LD with rs175163 in Asian of international HapMap data). The different ethnicity may be a major factor responsible for the discrepancy between the two study populations. In our previous reports about the association of COMT and ZDHHC8 with SPEM abnormality in schizophrenia patients, only ZDHHC8 polymorphisms showed strong association with SPEM abnormality in schizophrenia group [Park et al., 2009; Shin et al., 2010]. To further understand the genetic effects of the nearby gene upon SPEM abnormality in schizophrenia, we carried out an association analysis between RANBP1 and SPEM abnormality among Korean schizophrenic patients. The result of our study of Korean subjects indicated that RANBP1 haplotype might also be the genetic factor for SPEM abnormality in schizophrenia. The mechanisms involved in the association of RANBP1 with SPEM abnormality is not currently understood, but our study clearly indicates that RANBP1 haplotype has an influence on SPEM abnormality in Korean subjects with strong association (Pcorr ¼ 0.002–0.0003). We can hypothesize that haplotypes of polymorphisms in the intron region might impact gene function by affecting the mRNA stability. In addition, although it is true that the haplotype itself may be functional and directly affect the expression of the phenotype, a more likely event is that the allele TABLE I. Association Analysis of RANBP1 Polymorphisms With the Risk of Schizophrenia in Korean Population MAF Loci rs2238798 Position Intron2 rs175162 Intron4 ht2 [G-T] Allele G A T G — ht2 MAF, minor allele frequency; SZO, schizophrenia patient; PC, population control. SZO 345 (49.6%) 351 (50.4%) 610 (87.1%) 90 (12.9%) 449 (63.4%) 259 (36.6%) PC 396 (50.8%) 384 (49.2%) 680 (86.5%) 106 (13.5%) 499 (63.0%) 293 (37.0%) P of chi-square 0.65 Statistical power (%) 97.1 0.72 81.7 0.87 96.9 70 AMERICAN JOURNAL OF MEDICAL GENETICS PART B TABLE II. Regression Analysis of RANBP1 Polymorphisms With Eye-Movement Controlling Age and Sex as Covariates in Korean Population Loci rs2238798 rs175162 ht2 [G-T] C/C 42 (3.61 0.76) 130 (3.69 0.80) 64 (4.04 0.63) C/R 84 (3.76 0.84) 31 (4.05 0.74) 77 (3.60 0.86) R/R 40 (3.93 0.69) 5 (3.88 0.33) 25 (3.55 0.76) Pa 0.05 0.04 0.0006 Pacorr 0.15 0.12 0.002 Pb 0.13 0.02 0.0001 Pbcorr 0.36 0.06 0.0003 Pc 0.11 0.70 0.13 Pccorr 0.33 1 0.36 P-value was calculated from multiple linear regression analyses controlling for age and sex as covariates. Pa, Pb, and Pc refer to P-values of codominant, dominant, and recessive models, respectively. C/C, C/R, and R/R refer to major homozygote, heterozygote, and minor homozygote, respectively. Mean SD of Ln S/N ratio values of each genotypes are shown in parenthesis. Pcorr represents the simple corrected P-value (Bonferroni methods, P-value 3). Significant associations are shown in boldface (P-value 0.05). is in linkage disequilibrium with another allele at a nearby gene, ZDHHC8 which was associated with SPEM abnormality in our previous study [Shin et al., 2010]. Four SNPs (rs175169, rs175174, rs175175, and rs175179) and one haplotype (ht4) of ZDHHC8 were strongly associated with the risk of SPEM abnormality. Linkage disequilibrium analysis suggest that there is one LD block covering a 27 kb region beginning with rs2238798 in RANBP1 and ending with rs2292570 in ZDHHC8 (Supplementary Fig. 1D). This LD structure between RANBP1 and ZDHHC8 reveals evidence for the association between RANBP1 polymorphism and SPEM abnormality. Our method of measuring the SPEM function is a global measurement of the SPEM, which is adequate for genetic analysis. Among schizophrenia patients, we evaluated the degree of SPEM impairment based on the Ln S/N ratio of the SPEM curves. However, we did not perform the association analysis of the SPEM function abnormality with RANBP1 SNPs in normal controls. Considering the low incidence of SPEM impairment <10% in normal controls, relatively small number of subjects and low statistical power, the association analysis in normal control subjects would be not appropriate. In summary, we scrutinized the association of RANBP1 polymorphisms with the risk of schizophrenia and the abnormality of SPEM quality among schizophrenic patients in Korean population. Although no association of RANBP1 polymorphisms with the risk of schizophrenia were observed, we presented the possibility for the genetic association with SPEM abnormality in Korean schizophrenia patients. 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