Suggestive Evidence for Linkage of Schizophrenia to Chromosome 8p21-12 in Multiplex Korean Families

Suggestive Evidence for Linkage of Schizophrenia to Chromosome 8p21-12 in Multiplex Korean Families Eun Young Cho, M.S. 1, Yu Sang Lee, M.D. 2, Kyeong Sook Choi, M.D. 3, 6, Yong Lee Jang, M.D. 4, Seung Hee Cho, M.S. 5, Hyun Ok Jeun, M.S. 3, Jong Won Kim, M.D., Ph.D. 5, Kyung Sue Hong, M.D., Ph.D. 1, 3 1 Center of Clinical Research, Samsung Biomedical Research Institute, Seoul, Korea 2 Yong-In Mental Hospital, Yong-in, Gyeonggi-do, Korea 3 Department of Psychiatry, Sungkyunkwan University School of Medicine, Samsung Seoul Hospital, Seoul, Korea 4 National Chuncheon Hospital, Chuncheon, Kangwon-Do, Korea 5 Department of Laboratory Medicine, Sungkyunkwan University School of Medicine, Samsung Seoul Hospital, Seoul, Korea 6 Department of Psychiatry, Eulji University School of Medicine, Daejeon, Korea Correspondence: Kyung Sue Hong, M.D., Ph.D. Department of Psychiatry #50, Ilwon-dong, Kangnam-gu, Seoul, Korea (135-710) Tel: +82-2-3410-3584, Fax: +82-2-3410-0050 E-mail: kshong@smc.samsung.co.kr Abstract Objectives Chromosome 8p21-12 has been identified as a susceptibility locus for schizophrenia based on several genomewide linkage scans with Caucasian families. The purpose of this study is to investigate the linkage of this locus to schizophrenia i n Korean families. Materials and Methods We recruited ninety-one family members from twenty-seven multiplex schizophrenia families. Fifty-nine of them were affected individuals. Seven microsatellite markers of this region with 3cM intervals were genotyped. Non-parametric linkage analysis was performed by evaluating the levels of allele sharing between the affected relative pairs. Results In the multi-point analysis, all the points tested within this area showed positive but non-significant non-parametric lod (NPL) scores with the peak occurring between D8S1820 and D8S1769. In the single point analysis, statistically significant allele sharing was observed at D8S1769 (NPL=1.65, p=0.049). Higher levels of NPL scores (the highest single point NPL=1.98, p=0.025) were observed when the same analyses were applied to a subgroup of families in which all of the affected individuals showed auditory hallucination. Conclusions These findings support the previous evidence from Caucasian families for a locus predisposing to schizophrenia at 8p21-12. Further studies designed to screen positional candidate genes and their SNPs at this locus are warranted, in order to identify the specific causative genetic variation of schizophrenia. Key Words: Schizophrenia, Susceptibility loci, Linkage analysis, 8p21-12 Psychiatr Investig 2004;1(1):61-67 Introduction Evidence from genetic epidemiological studies has suggested that genetic factors play a major role in determining the susceptibility to schizophrenia. According to well-designed epidemiological studies published since the 1980s, the recurrence risk ratio of the first degree relatives of schizophrenia patients is almost 10 and the estimated heritability is between 0.6 and 0.7 1, 2. However, the aggregation pattern of this illness within the families does not fit into the Mendelian mode of transmission. Thus, like other common diseases, schizophrenia revealed a complex genetic and multifactorial etiology, rather than a monogenic one 3, 4. Linkage analysis is a method of mapping and detecting disease genes, without any knowledge of the true pathophysiology or proteins involved. If two genetic loci are close enough to each other, the alleles compositions of these loci on individual chromosomes tend to be preserved during meiosis, because of the low probability of the recombination between the two loci. If linkage is established between a disease and a marker with a known chromosomal location, then the approximate position of the disease gene can be inferred, and the causative genetic variation can ultimately be isolated in this locus 5. This approach has been successfully used to identify several monogenic neuropsychiatric diseases, i.e., Huntington's disease, familial Alzheimer's disease, fragile X syndrome, etc. 61

PSYCHIATRY INVESTIGATION When applied to complex genetic diseases, however, classical parametric linkage analysis did not generate consistent results, because this method depends on the specification of various genetic parameters such as the mode of inheritance, gene frequencies and genotype penetrances, which cannot be reliably determined for polygenic or multifactorial diseases. To circumvent these problems, non-parametric methods, i.e., the affected sib-pair method and the affected relative pair method, which don't require any prior assumptions to be made about such parameters, have been developed and applied to schizophrenia since the early 1990s 6. The rapid increase in the number of microsatellite markers available for linkage analysis has made the whole genome scan possible, in which linkage analyses are performed for hundreds of markers spanning all twenty-two autosomes and the X chromosome. The recent development of automated laboratory techniques for genotyping these markers has also greatly contributed to the large scale linkage studies of the last decade. Numerous genomewide linkage scans have been reported for schizophrenia. There is modest evidence for linkage with several loci, including 1q21-q22, 1q32-q42 6p22-p24, 8p21- p22, 15q13-q15, and 22q11 7-8. The families recruited for these studies were mostly Caucasians from North America and Europe, Jewish, or a few isolated ethnic groups. Chromosome 8p21-p12 has shown suggestive linkage to schizophrenia in several different populations. Pulver et al 9. first reported linkage of this locus to schizophrenia in fifty-seven Maryland highdensity families. This research group added fifty-four more US pedigrees with mixed ethnicity and reported higher maximum lod scores at a nearby marker of the same locus 10 The maximum lod scores, which were higher than 3.0, were independently replicated in Irish 11, Canadian 12, and English 13 families. Schizophrenia linkage collaborative group for chromosome 3, 6, and 8, a fourteen groups collaborative effort, also generated quite significant lod scores within this area 14. Stefansson et al 15. performed a series of fine mapping processes through haplotyping with additional microsatellite and single nucleotide polymorphism (SNP) markers. They narrowed down this locus to a region of less than 1Mb. They identified Neuregulin 1 (NRG1) within the core haplotype area maximally shared by the affected individuals of families showing high lod scores. This gene is known to be involved in the glutamate pathway of the central nervous system 16. Given that the glutamate pathway is one of the major neurotransmitter systems in the biochemical hypothesis of schizophrenia, this gene could be both a locational and functional candidate gene for schizophrenia. Recently, positive associations with schizophrenia in the general population were also reported for several SNPs within this gene 17-19. The probability of detecting any true linkage of a disease to a specific chromosomal locus depends on the genetic-epidemiological characteristics of the population to which the families involved in the study belong. The population-related factors, which have the most effect on the result of the linkage analysis, are the level of genetic heterogeneity of the disease within each population and the patterns of linkage disequilibrium for the specific chromosomal segments. Thus, testing the linkage for each susceptibility locus should be attempted in various populations with different ethnicity. Neither a genomewide scan nor a large scale linkage analysis for a single locus has been performed in Korean families. This is mainly because of the extensive infrastructures needed for genotyping the microsatellite markers and large scale family recruitment that would be required. The purpose of this study is to test the linkage of the 8p21-12 locus to schizophrenia in multiplex Korean families. We performed a non-parametric linkage analysis, using the affected relative pair method, for twenty-seven families, using seven microsatellite markers within this region. The same analysis was applied to a potentially more homogenous subgroup of families, in which all of the affected members showed auditory hallucination. Materials and Methods Pedigree ascertainment We identified multiplex families containing more than two affected individuals within the second degree relatives through proband screening performed at Samsung Seoul Hospital, Yong- In Mental Hospital, National Chuncheon Hospital, Eumsung Mental Hospital and Saint Andrew Neuropsychiatric Hospital. Twenty-eight families were identified from March 2002 to June 2003 and one family showing the bilineal transmission of psychoses was excluded from the analysis. We limited the diagnosis of the probands to schizophrenia according to the DSM-IV criteria 20. Other affected individuals were diagnosed as having schizophrenia or schizoaffective disorder according to DSM-IV. In the case of schizoaffective disorder, only cases in which long-term maintenance with antipsychotics was needed were included. All of the available first degree relatives of the affected individuals were recruited. Clinical interviews and blood sampling for genotyping were done for ninety-one subjects, including fifty-nine affected cases. This study was approved by the institutional review boards (IRBs) of Samsung Seoul Hospital, Yong-In Mental Hospital and Saint Andrew Neuropsychiatric Hospital. Informed consent was obtained from all of the subjects who participated in the interviews and sampling. Clinical assessment methods Direct interviews using the Korean version of the 'Diagnostic Interview for Genetic Studies (DIGS)' 21 were performed by three psychiatrists and a psychiatric research nurse who had participated in several training sessions regarding the administration of DIGS. This semi-structured interview schedule contains diagnostic algorithms for several major criteria systems of schizophrenia. The individual symptoms are comprehensively recorded on a lifetime basis, as well as being anchored to the current or most recent episode. A best estimate diagnosis for each patient was independently made by two psychiatrists based on the information obtained from the direct interview, hospital records and interviews with the relatives or the psychiatrists who referred the patient. The final diagnosis was decided by consensus. If there was any disagreement, the two diagnosticians met to resolve their differences. The few cases in which consensus could not be reached were excluded from the analysis. For the affected individuals, the Krawieka Rating Scale 22 was also administered for the evaluation of the lifetime characteristics of their psychopathology. Markers and maps Seven microsatellite markers within 8p21-p12 were selected for genotyping based on their heterozygosities and distances from each other (Table 1). Five of them are telomeric and one of them is centromeric to Neuregulin 1 (NRG1). D8S1810 is located on the first intron of NRG 1. The markers had an average spacing of 3.22cM according to the genetic map of the Marshfield Clinic (Center for Medical Genetics) and of 2.35Mb according to the physical map of the human genome provided 62

TABLE 1 Locations and heterozygosities of the microsatellite markers TABLE 2 Results of multi-point linkage analysis: position, nonparametric lod (NPL) score and p-value for each point Marker Cytogenic Map Size of Intervals Genetic (cm) a Physical (Mb) b Number of alleles Heterozygosity in Korean population D8S258 8p21.3 4 0.74 4.71 4.71 D8S1734 8p21.3 4 0.54 3.79 3.79 D8S1771 8p21.2 5 0.70 5.99 5.99 D8S1820 8p21.1 8 0.39 4.30 4.30 D8S1769 8p12 8 0.58 0 0 D8S1810 8p12 5 0.81 c 0.53 0.53 D8S505 8p12 7 0.78 a. distances based on the genetic map obtained from the center for medical genetics, marshfield clinic b. distances based on the physical map from the human genome resources of the national center for Biotechnology Information c. heterozygosity data from the center for medical genetics, marshfield clinic by the National Center for Biotechnology Information (NCBI). The heterozygosities presented in Table 1, except for D8S1810, were calculated from the genotype data of one-hundred normal Korean persons generated by another project of one of the authors of the current study. For D8S1810, the heterozygosity score was provided by the Marshfield Clinic. Genotyping DNA was extracted directly from 10ml of peripheral blood using a WizardTM Genomic DNA purification kit (Promega Madison, USA). In the polymerase chain reaction (PCR) analysis, we used primer sets provided by the ABI PRISM Linkage Mapping Set v2.5 (ABI,CA,USA) for six markers. For D8S1810, we designed the primer set using a primer-designing software, Primer 3 (ABI,CA,USA). The size of the PCR product for each marker was between 100 and 400bp, including the polymorphic CA (dinucleotide) repeat sequences. The PCR reaction for 30ng of genomic DNA template contained 5 pmols of fluorescence-labeled forward primer, 5 pmols of unlabeled reverse primer, 0.6 units of Taq DNA polymerase (BMS,USA), 0.25mMs of each dntp (BMS,USA), 2.5mMs of MgCl2 (BMS, USA) and 1X polymerase buffer. We performed touchdown PCR using two successive annealing temperatures to promote both the specificity and productivity of the amplification. The genotype was analyzed by means of an ABI prism 3100 Genetic Analyzer. This automated genotyping machine has a capillary running system for the electrophoresis of the PCR products. The scoring of the genotypes was performed using the ABI prism software package, Genotyper 3.6 NT. Statistical analysis The model-free (non-parametric) linkage analysis was performed using Genehunter 2.1_r4 beta 23. This program calculates both the single point and multi-point NPL (nonparametric lod) scores through an affected-only allele-sharing method. We employed the NPL all statistic which examines all individuals simultaneously and assigns a high score when more of them share the same allele by descent. This program requires the prior specification of the inter-marker genetic distances and Positions a All families (N of families=26) Families with hallucination (N of families=15) 0.00 0.41637 0.335972 1.17866 0.119920 0.53 0.47016 0.321583 1.18247 0.119920 1.07 0.52406 0.302218 1.18670 0.119920 1.60 0.57810 0.285202 1.19134 0.119920 2.14 0.68669 0.263428 1.19640 0.119920 2.67 0.69121 0.247339 1.20188 0.116021 3.20 0.69611 0.241334 1.16138 0.121797 3.74 0.70138 0.241334 1.12155 0.135637 4.27 0.70703 0.241334 1.08237 0.147977 4.81 0.71306 0.239199 1.04381 0.155774 5.34 0.70055 0.236741 1.00588 0.157838 6.00 0.68928 0.241334 1.01736 0.157838 6.66 0.67924 0.243396 1.02990 0.155774 7.32 0.67044 0.247339 1.04352 0.155774 7.99 0.66290 0.249715 1.05822 0.147977 8.65 0.72399 0.249715 1.07401 0.147977 8.76 0.78527 0.231949 1.15964 0.121797 8.87 0.84675 0.217779 1.24551 0.104478 8.98 0.90843 0.200567 1.33161 0.089310 9.09 0.97030 0.180041 1.41794 0.081492 9.20 0.94364 0.165871 1.50452 0.072621 9.78 0.91757 0.171969 1.46711 0.075717 10.35 0.89208 0.180041 1.43050 0.079615 10.93 0.86715 0.189372 1.39467 0.081492 11.51 0.84278 0.195453 1.35961 0.089154 12.08 0.73422 0.201083 1.32529 0.089310 12.59 0.62605 0.231949 1.26282 0.100580 13.11 0.51822 0.304676 1.20096 0.116021 13.62 0.30968 0.339577 1.13969 0.134467 14.13 0.41069 0.30341 1.07897 0.147977 14.65 0.30341 0.382048 1.01879 0.157838 a. Positions with intervals of 0.53cM between p-ter side of marker (D8S258) and centromere side of marker (D8S505) on chromosome 8, starting from p-ter side of marker (D8S258). TABLE 3 Positions a D8S258 D8S1734 D8S1771 D8S1820 D8S1769 D8S1810 D8S505 Results of single point linkage analysis: non-parametric lod (NPL) score and p-value for each marker All families (N of families=26) 0.14199 0.81680 0.85219-0.20845 1.65332 0.30689-0.70195 0.442329 0.208705 0.199819 0.577880 0.048610 0.377316 0.758121 Families with hallucination (N of families=15) 0.25042 0.86000 0.26830 0.16627 1.98272 0.57674-0.06090 0.410005 0.192259 0.393111 0.429479 0.025205 0.285145 0.523910 63

PSYCHIATRY INVESTIGATION allele frequencies of each marker. For the genetic distance, we made the simplifying assumption that 1cM is equal to 1Mb, based on the distances in the physical map provided by NCBI. The marker allele frequencies in the Korean population were estimated from the same genotype data used for the calculation of the heterozygosities except for D8S1810. Because we didn't have the genotype data for the normal Korean population for D8S1810, we calculated the allele frequencies from the genotypes of unaffected founders recruited for the current analysis. As the purpose of this study is to test the linkage for a specific chromosomal locus instead of the whole genome, we adopted the pointwise p-value to evaluate the statistical significance. Results Among the twenty-seven families genotyped, five families contained three affected individuals and all of the others contained two affected members. The mean age of the affected individuals was 37 ( 10.5) years and 53% of them were males. The mean age of onset of any psychotic symptom was 26 ( 8.6) years. The most prevalent subtype diagnosis was paranoid type based on DSM-IV (64.4%). The others were classified into the disorganized (10.2%) and undifferentiated (25.4%) types. The detailed clinical characteristics of the affected individuals and the patterns of transmission of the disease within the families were described in the report by Kim et al 24. The shapes of the allele peaks generated from the capillary running of the PCR products using the 3100 Genetic Analyzer are presented in Figure 1. Mendelian inconsistency was detected in only one family and this family was excluded from the final statistical analysis. Multi-point NPL scores were calculated at thirty-one positions with intervals of 0.53cM between the most telomeric (D8S258) and the most centromeric (D8S505) markers (Table 2). No statistically significant linkage was found at any of these positions. However, a very broad peak of positive NPL scores was observed with the highest score (NPL=0.97) at 9.2cM points from D8S258. Single point analysis showed a significant linkage (NPL=1.65, p=0.048) at D8S1769 (Table 3). According to the previous analysis of the intra-familial associations of the clinical characteristics in these families 25, auditory hallucination was the only symptom which showed significant concordance within the affected relative-pairs. Assuming that this symptom has more homogenous genetic basis, we performed the same linkage analyses with only fifteen families, in which all of the affected individuals showed definite auditory hallucination. The results are also summarized in Tables 2 and 3. In the multipoint analysis, the same pattern of a positive peak was observed and the maximum NPL score was slightly higher (NPL=1.50, p=0.073) than that of the whole families. The single point NPL score was increased to 1.98 (p=0.025) for the same marker (D8S1769). Discussion Following up on prior reports of significant linkage to schizophrenia in European and American familes 9-15, we examined the chromosomal region, 8p21-p12, in multiplex Korean schizophrenia families. The results provided further evidence for the existence of a schizophrenia vulnerability locus in this region. The linkage was more prominent in a specific subgroup of families in which all of the affected members showed auditory hallucination. For complex genetic traits of unknown pathophysiology, such as schizophrenia, the genomewide linkage scan is one of the best approaches to use to find the susceptibility genes. Once a certain number of linked loci have been identified through the whole genome scans, the following step is the narrowing down and fine mapping of these loci using linkage analysis and haplotyping with additional markers. Several positional candidate genes can be identified within these small segments of the chromosomes. The final step is to determine any genetic variation in these genes that is associated with schizophrenia. A few leading research groups in this field have narrowed down the schizophrenia loci to a few megabases, and are now focusing on the screening of SNPs in the candidate genes of their own loci. Given that schizophrenia is under polygenic or multifactorial genetic control, multiple susceptibility loci need to be identified through linkage analyses. Moreover, we can also speculate that the combinations of genetic variations, which make the major contribution to the vulnerability of schizophrenia, might differ slightly from population to population. Thus, independent linkage screening of the whole genome is warranted for various populations. In addition, each population has its own pattern of linkage disequilibrium for specific chromosomal regions. This characteristic can greatly affect the power of the linkage analysis and, in turn, affect the susceptibility to detect the linkage for each locus. Some susceptibility loci of schizophrenia could be identified more successfully through the analysis of the Korean families. In fact, we are currently in the process of performing a large scale whole genome scan with Korean families and this study is an initial part of that project. The suggestive linkage of 8p21-p12 to schizophrenia has been repeatedly replicated starting from the 1990s. Pulver et al 9. first reported evidence of linkage in fifty-seven Maryland high-density families with schizophrenia (non-parametric p=0.00004 and maximum lod score of 2.36 under dominant model). The same research group found a much higher lod score for this region in the analysis of another fifty-four pedigrees with mixed ethnicity 10. Results suggestive of linkage 11 were also reported by Kendler et al. in their large scale genome scan with 265 Irish pedigrees. Maximum lod scores of higher than 3.0 were also reported for twenty-one Canadian families 12 and thirteen English families 13 multiply affected with schizophrenia. In a collaborative effort of fourteen research groups (Schizophrenia Linkage Collaborative Group for Chromosome 3, 6, and 8) a maximum lod score of 2.22 was observed under a recessive model 14. Recently, Stefansson et al 15. performed a large scale genomewide scan of schizophrenia families in Iceland and their results supportedthe previous works showing that schizophrenia maps to chromosome 8p21-12. Extensive finemapping of this locus and haplotype-association analysis, supplemented by a transmission disequilibrium test, identified Neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, showed a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice 15, 16. Steffanson et al 15. also demonstrated that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine. D8S1769, which showed significant linkage to schizophrenia in the single point analysis of the current study, is located at 64

(A) (B) (C) (D) (E) (F) (G) FIGURE 1 Characteristics of genotype peak for each microsatellite marker generated from the capillary running of PCR products using ABI prism 3100 genetic analyzer. panel 1 contains D8S1734 (A), D8S1769 (B), D8S1771 (C), D8S258 (D) and panel 2 contains D8S1820 (E), D8S505 (F), and D8S1810 (G) 65

PSYCHIATRY INVESTIGATION 0.35Mb upstream of NRG1. It is also located within the at-risk haplotypes which showed maximal haplotype sharing among the affected members of the linkage families from Iceland 15. Despite the proximity of the location to D8S1769, D8S1820 showed a fairly neutral NPL score, neither supporting nor excluding linkage to schizophrenia. This discrepancy might originate from the low informativeness of D8S1820 in the analysis. According to the public data base provided by Marshfield, the heterozygosity of this marker was 0.75. However, the heterozygosities of the normal Korean population and our family samples are 0.39 and 0.38, respectively. This marker might also contribute to the lower NPL scores of the multi-point analysis compared to the single point analysis. Even though the maximum NPL score of this study was not high enough to confirm the linkage, it is sufficiently high to be suggestive of linkage, considering the number and size of the families analyzed. We evaluated the significance according to the pointwise p value instead of the genome-wide p value, because all seven markers were selected for testing a single locus, 8p21-12. Based on the previous results of the clinical analysis with these families, we isolated a subgroup of schizophrenia associated with auditory hallucination 25. Given that the affected relative-pair method of the current analysis uses allele sharing statistics only among the affected family members, and that auditory hallucination tends to be measured false negatively instead of false positively, the increased linkage scores in this subgroup could be regarded as quite a solid finding, suggesting a more homogenous genetic basis for this subgroup. This study is the first large scale linkage analysis in Korean schizophrenia families. And it is also the first report of linkage of 8p21-12 to schizophrenia in an Asian population. 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