Transmission disequilibrium and haplotype analyses of the G72G30 locus Suggestive linkage to schizophrenia in Palestinian Arabs living in the North of Israel.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:91 –95 (2006) Transmission Disequilibrium and Haplotype Analyses of the G72/G30 Locus: Suggestive Linkage to Schizophrenia in Palestinian Arabs Living in the North of Israel M. Korostishevsky,1 I. Kremer,2 M. Kaganovich,1 A. Cholostoy,1 I. Murad,3 M. Muhaheed,3 I. Bannoura,4 M. Rietschel,5 M. Dobrusin,6 U. Bening-Abu-Shach,1 R.H. Belmaker,6 W. Maier,5 R.P. Ebstein,7,8 and Ruth Navon1* 1 Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel Emek Hospital, Afula, Israel 3 Kemal Psychiatric Hospital, Bethlehem, Palestinian Authority 4 The Palestinian Research Center for Genetics of Mental Disorders, Bethlehem, Palestinian Authority 5 Department of Psychiatry, University of Bonn, Bonn, Germany 6 Beersheva Mental Health Center, Beersheva, Israel 7 Scheinfeld Center of Human Genetics in the Social Sciences, Department of Psychology, The Hebrew University, Jerusalem, Israel 8 Herzog Hospital, Jerusalem, Israel 2 Association of the G72/G30 locus with schizophrenia was recently reported in French Canadian, Russian, and Ashkenazi populations using casecontrol studies. In the present study we hypothesize the existence of a G72/G30 risk allele overtransmitted to affected sibs in Palestinian Arab families. A total of 223 Palestinian Arab families that included an affected offspring and parents were genotyped with 11 SNPs encompassing the G72/G30 genes. The families were recruited from three regions of Israel: 56 from the North (Afula), 136 from the central hill region (Bethlehem, Palestinian Authority), and 31 from the South (Beersheva). Individual SNP analyses disclosed a risk allele in SNP rs3916970 by both haplotype relative risk (HRR: x2 ¼ 5.59, P ¼ 0.018) and transmission disequilibrium test (TDT: x2 ¼ 6.03, P ¼ 0.014) in the Afula families. Follow-up multilocus analysis using family-based association tests (FBAT: z ¼ 2.197, P ¼ 0.028) exposed the adjacent haplotype. SNP rs3916970 is located about 8 kb from the linkage disequilibrium block that was reported to be associated with schizophrenia in Ashkenazi Jews. Excess of similar haplotypes of this region was observed in the Palestinian Arabs and the Ashkenazi patients. These data suggest a common risk factor for schizophrenia susceptibility in the G72/G30 locus among Ashkenazi Jews and Palestinian Arabs. The results strengthen This article contains supplementary material, which may be viewed at the American Journal of Medical Genetics website at http://www.interscience.wiley.com/jpages/1552-4841/suppmat/ index.html. Grant sponsor: Adams Super Center for Brain Studies, Tel Aviv University. *Correspondence to: Ruth Navon, Department of Human Genetics and Molecular Medicine, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel 69978. E-mail: firstname.lastname@example.org Received 24 September 2004; Accepted 17 March 2005 DOI 10.1002/ajmg.b.30212 ß 2005 Wiley-Liss, Inc. previous reports on the role of this locus in the etiology of schizophrenia. ß 2005 Wiley-Liss, Inc. KEY WORDS: 13q32 region; family trios; major psychosis; multi-SNP statistics; founder effect INTRODUCTION Schizophrenia is a major public health problem affecting about 1% of the population worldwide with a devastating effect on patients’ lives. It is characterized by profound disturbances of cognition, emotion, and social functioning. Family, twin and adoption studies consistently show that both hereditary and non-heritable factors influence disease susceptibility [Gottesman, 1991; Andreasen, 1995]. Being a complex disorder, it is accepted that several genes, each with small to modest effect, contribute to the etiology of the disease. The identification of genes for complex disorders like schizophrenia has been difficult as there is no definitive data about which genes are involved. One approach to locating candidate genes is to search for them in regions known for linkage. A region of potential interest in schizophrenia is chromosome 13q, suggested by a number of linkage studies [Lin et al., 1995; Shaw et al., 1998; Levinson et al., 2000]. By testing affected schizophrenic families, Blouin et al.  and Brzustowicz et al.  narrowed the suspected region to 13q32-34 (for review see Christian et al. ). However, no candidate genes were initially observed in this region, considered a ‘‘gene desert,’’ until Chumakov et al.  identified two overlapping genes, G72 and G30, transcribed from opposite DNA strands. Extensive genotyping of single nucleotide polymorphisms (SNPs) in this region, combined with advanced statistical analysis, showed a significant association of the G72/G30 region with schizophrenia in two populations, French Canadians, and Russians. Subsequently, the G72 protein was identified as a D-amino acid oxidase activator (DAOA) [Chumakov et al., 2002]. These findings, together with the observation that oxidation of D-serine by DAAO greatly attenuates N-methyl-D-aspartate (NMDA) receptor-mediated neurotransmission [Mothet et al., 2000], are consistent with the glutamatergic theory of schizophrenia [Konradi and Heckers, 2003]. The G30 gene is predicted to encode a 71 amino acid protein; however, the existence of the protein has not been verified and its function is not yet clear. 92 Korostishevsky et al. TABLE I. General Characteristics of the Patient Samples Sample AF BL BS Sample size (male/female) Age of onset in years (range) 56 (32/24) 136 (106/30) 31 (20/11) 22.0 (14–45) 24.5 (17–32) 23.0 (16–35) Once the G72/G30 region was shown to be associated with schizophrenia [Chumakov et al., 2002], the association had to be replicated on different populations in independent casecontrol and family-based studies. Two independent casecontrol studies of German and Ashkenazi Jew patients replicated the association between the G72/G30 gene locus and schizophrenia [Korostishevsky et al., 2004; Schumacher et al., 2004]. Intriguingly, the association to the G72/G30 gene locus was also reported in two series of independent pedigrees with bipolar disorder [Hattori et al., 2003]. The present study examines the involvement of the G72/ G30 gene locus with schizophrenia in Palestinian Arab families, using transmission disequilibrium and comparative haplotype analyses. We hypothesize the existence of a G72/G30 risk allele over-transmitted to affected sibs in the families. Eleven SNPs in the G72/G30 region were genotyped in the 223 family trios studied. MATERIALS AND METHODS Subjects The study enrolled 223 Palestinian Arab family trios, each consisting of an affected offspring and parents. The families were from three geographic regions of Israel: 56 from the North (AF, Afula region), 136 from the center of the country (BL, Bethlehem, Palestine authority), and 31 Bedouin families from the South (BS, Beersheva region). All patients were diagnosed based on the SCID interview according to DSM-IV criteria for schizophrenia [American Psychiatric Association, 1994]. The protocol for recruiting schizophrenic families was approved by the local Institutional Review Board (IRB) committees. The age of onset and the male/female distribution in the patient samples are presented in Table I. SNP Genotyping Eleven SNPs were genotyped: rs3916965 (M12), rs3916966 (M13), rs3916967 (M14), rs2391191 (M15), rs3918341 (M16), rs947267 (M18), rs778294 (M19), rs3916970 (M20), rs3916971 (M21), rs778293 (M22), and rs3918342 (M23). The designations of the SNPs in parentheses are according to Chumakov et al. . The relative positions of the SNPs in the G72/G30 region are given on web Table V (the supplementary material). SNP genotyping was performed by the high-throughput system of chip-based mass spectrometry, matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) [Sequenom, Inc., San Diego, CA, http://www.sequenom.com/]. Primer sequences and PCR conditions are described in Korostishevsky et al. . Re-genotyping was performed for M20, M22 in BL group and for M22, M23 in BS group, where a deviation from Hardy– Weinberg equilibrium (HWE) was observed. No genotyping errors were disclosed. Statistical Analysis Genotype and allelic distribution at each of the 11 SNP loci were determined by direct counting in the samples. Possible differences in the frequency of each of the SNP genotypes or alleles between the samples were estimated using the w2-test, as described elsewhere [Komlos et al., 1997; Abramson and Gahlinger, 1999]. The Arlequin software package, http:// Lgb.unige.chnarlequin [Schneider et al., 2000] was used to: (1) evaluate the gene diversity index [Nei, 1987], (2) estimate pairwise linkage disequilibrium (LD) between the SNP markers [Slatkin, 1994; Slatkin and Excoffer, 1996], (3) detect significant departure from HWE [Guo and Thomson, 1992], and (4) calculate the maximum likelihood (ML) of haplotype frequencies [Excoffier and Slatkin, 1995]. Based on the ML haplotype frequency estimates, the likelihood ratio test (LRT) [Kendall and Stuart, 1973] for sample differentiation was applied (for details see web Appendix in the supplementary material). Haplotype relative risk statistics (HRR) [Falk and Rubinstein, 1987] and transmission disequilibrium test (TDT) [Spielman et al., 1993] for individual SNPs in family trios were performed as described by Ewens and Spielman . Haplotypic associations were tested using family-based association tests (FBAT) [Horvath et al., 2004]. The minimal Fig. 1. Significance level for pairwise LD in the Palestinian samples. LD significance above the diagonal corresponds to AF patient sample and below the diagonal to the total sample, AF þ BL þ BS (white: P > 0.05; gray: 0.05 < P < 0.005; dark gray: 0.005 < P < 0.0005; black: P < 0.0005). G72 Association to Schizophrenia in Palestinians 93 TABLE II. ML Haplotype Frequencies in the LD Blocks of the G72/G30 Region Haplotypesa LD block number Frequency (%) SNP markers Number Nucleotide sequence AF BL BS Ia M12-M16 Ib M18-M20 II M21-M23 1 2 3 1 2 3 4 1 2 3 4 5 G-A-A-G-A A-C-G-A-G G-A-A-G-G A-G-A C-A-G C-G-G A-G-G C-A-T T-G-C T-A-C C-G-C T-G-T 57.2 35.4 2.8 51.4 15.7 19.8 10.3 58.7 23.6 6.1 4.4 6.2 64.5 25.1 5.1 43.2 21.0 16.8 18.6 47.4 26.9 7.0 10.6 6.8 55.7 30.0 8.6 36.4 22.7 24.2 16.6 38.2 33.1 12.6 8.1 2.1 a Haplotypes with frequencies <5% in each sample are not enumerated; the two most frequent haplotypes in each block are in bold. number of informative families required for a haplotype to be included in the haplotype FBAT-test statistic was set at 10. The Bonferroni corrections were performed by the SISA online procedure (http://home.clara.net/sisa/bonfer.htm). RESULTS Genotype, Allele, and Haplotype Frequencies in Patient’s Samples The patients were categorized into three subgroups according to place of dwelling. Genotype and allele frequencies for patient samples for each SNP are summarized on web Table V (the supplementary material). All 11 SNPs were highly polymorphic, with an overall gene diversity of about 98% in each of the samples (AF: 97.9 0.5, BL: 98.4 0.2, 98.4 0.6). Pairwise LD-test disclosed three LD blocks. The LD-test results for the AF sample and for the total sample (AF þ BL þ BS) are presented in Figure 1. The LD blocks were designated Ia, Ib, and II reflecting a relatively high LD between Ia and Ib. Similar LD blocks have been observed in other populations [Chumakov et al., 2002; Hattori et al., 2003, Korostishevsky et al., 2004]. The haplotype frequency estimates in the patients were performed separately for each block (Table II). Although block Ia contains five SNPs, only two common haplotypes, accounting for more than 90% of all cases, occurred. Block Ib, which contains of three SNPs, represents four haplotypes of frequent occurrence. The two Fig. 2. Scatter-plot of HRR and TDT results. The horizontal line denotes the critical value (¼ 3.841) for 5% test significance. Scatter-plots A, B, C, and D are for AF, BL, BS and the overall family trios, respectively. 94 Korostishevsky et al. TABLE III. Results of HRR and TDT Statistics for Marker M20 in the AF Sample Non-transmitted Allele Transmitted G A Total G A Total 29 38 67 20 21 41 49 59 108 most frequent haplotypes in each block are a mirror image of each other, a phenomenon reported previously for other populations genotyped in the G72/G30 region [Hattori et al., 2003; Korostishevsky et al., 2004]. Family-Based Association Analyses HRR and TDT for individual SNPs were performed in the overall family trios as well as in AF, BL, and BS samples separately, and the results are shown in Figure 2. Both tests disclosed a significant deviation in allele transmission of M20 in the AF sample. An over-transmission of the A versus G nucleotide to affected offspring was found (Table III). Other maximal deviations in each sample, although not significant, were observed for SNPs of block II (AF-M22, BL-M21, BSM23). The combination of all samples reduces HRR and TDT result for M20 (Fig. 2D), while AF and BL combination preserved near-significant values (HRR ¼ 3.69, P ¼ 0.055; TDT ¼ 3.50, P ¼ 0.061). Haplotype association analyses using the haplotype FBAT statistics disclosed significant deviations for the block Ib haplotypes in the AF sample (Table IV). Haplotype Ib-1 was found more frequent in affected family members than expected under the null hypothesis of no linkage and association, while lack of haplotype Ib-4 was observed. Also multi-haplotype FBAT permutation test disclosed significant association of the Ib block in the AF sample (permutation cycles ¼ 31,573, S ¼ 1,073.0, P ¼ 0.034). In BL and BS as well as in the overall families no significant haplotype association was found (data not shown). DISCUSSION The Palestinian Arab families enrolled in this study came from three areas of the State of Israel and the Palestinian Authority. One sample was recruited primarily from Bethlehem (presently part of the Palestinian Authority) in the central-hill region of Israel. The second sample was from the Negev Desert in the Southern part of Israel, the home of the nomadic Bedouins, with Beersheva (BS) the largest city in this region. The third group came from the Northern part of Israel that includes the Jezreel and Galilee Valleys, with Afula TABLE IV. Results of the Haplotype FBAT Statistics in the AF Families LD block Ia Ib II a Haplotype numbera 1 2 1 2 3 4 1 2 3 4 z-value 0.074 0.745 2.174 0.635 0.175 2.694 1.820 0.949 0.258 1.155 P-value 0.941 0.456 0.030 0.525 0.861 0.007 0.069 0.343 0.796 0.248 Haplotype numbers strictly correspond to the ones denoted in Table II. HRR 5.59 P ¼ 0.018 TDT 6.03 P ¼ 0.014 (AF) the largest city. It was reported that Mendelian disorders found in Palestinian Arabs are not uniformly distributed among different geographic regions, ‘‘each of the villages may be considered as a small isolate’’ [Zlotogora, 1997; Zlotogora, 2002]. Notably, with regard to the G72/G30 region, our comparison of the patients from various places of dwelling (AF, BL, and BS) revealed significant differences in the individual SNP genotype/allele frequency as well as in the haplotype frequency (web Table VI, VII respectively; in the supplementary material). The frequencies of individual SNP alleles transmitted and non-transmitted to affected children were compared using HRR and TDT (Fig. 2). TDT estimates based on minimal nuclear families—family trios—offer an efficient way to detect LD between a marker and a disease susceptibility gene. These tests are similar, although the TDT, unlike HRR, focuses on linkage rather than association, while both aspects should be taken into account in research on a complex disorder [Ewens and Spielman, 1995]. The SNPs of blocks Ia and II did not show clear-cut transmission disequilibrium in the family samples, however M20, the telomeric SNP of block Ib, exhibited significant disequilibrium in allelic transmission (over-transmission of the A nucleotide) by both HRR and TDT in the AF sample. The result significance is preserved after Bonferroni correction taking into account the number of SNPs and the inter-SNP correlation level (data not shown). The M20 SNP is located near the 50 end of the G30 gene but outside the G72 gene. The G30 gene may regulate the G72 mRNA, a phenomenon described for other sense/antisense gene pairs [Yelin et al., 2003]. Multilocus association tests were performed using the haplotype FBAT statistics. This family-based statistics is robust to population admixture and can handle unknown haplotype phase in family trios [Horvath et al., 2004]. Haplotype Ib-1, that was found to be associated with schizophrenia in AF family sample, includes the A nucleotide at M20. In addition, there was found an excess of haplotype II-1, in affected family members, although not significantly. This finding can be attributed to LD observed between M20 and the SNPs of block II in the patient samples. Intriguingly, the very same I-1 haplotype is the haplotype associated with schizophrenia in Ashkenazi Jews [Korostishevsky et al., 2004. To elucidate the relation between above mentioned findings we performed the haplotype FBAT statistics for extended M20-II block of SNPs: M20-M21-M22-M23 (data not shown). A single haplotype of this extended block, the A nucleotide at M20 in addition to the II-1 alleles (A-C-A-T), demonstrates significant association in AF families: Z ¼ 2.197, P ¼ 0.028. This implies a common risk factor for schizophrenia susceptibility in the G72/G30 region among Ashkenazi Jews and Arabs. However, the involvement of this factor in schizophrenia susceptibility differs between various ethnic groups of Palestinian Arabs. Our findings may reflect a common history of Jews and Arabs before the dispersion of the Jews following the Roman conquest. Studies of classical genomic markers, HLA, RFLPs, mitochondrial, and Y chromosome markers, as well as disease-related mutations disclosed genetic affinities between these two populations [Hammer et al., 2000; Nebel et al., 2000; Thomas et al., 2000]. G72 Association to Schizophrenia in Palestinians ACKNOWLEDGMENTS This work was supported in part by Adams Super Center for Brain Studies, Tel Aviv University (R.N.). Genotyping was performed at the Genome Knowledge Center at the Weizmann Institute of Science, supported by the Israel Ministry of Science and Crown Human Genome Center. REFERENCES Abramson JH, Gahlinger PM. 1999. Computer program for epidemiologists, PEPI version 3. London: Brixton Books. American Psychiatric Association. 1994. Diagnostic and statistical manual of mental disorders, 4th edn. Washington, DC: American Psychiatric Association. Andreasen NC. 1995. Symptoms, signs, and diagnosis of schizophrenia. Lancet 346:477–481. Blouin JL, Dombroski BA, Nath SK, Lasseter VK, Wolyniec PS, Nestadt G, et al. 1998. Schizophrenia susceptibility loci on chromosomes 13q32 and 8p21. Nat Genet 20:70–73. Brzustowicz LM, Honer WG, Chow EW, Little D, Hogan J, Hodgkinson K, et al. 1999. Linkage of familial schizophrenia to chromosome 13q32. Am J Hum Genet 65:1096–1103. Christian SL, McDonough J, Liu Cy CY, Shaikh S, Vlamakis V, Badner JA, et al. 2002. An evaluation of the assembly of an approximately 15-Mb region on human chromosome 13q32-q33 linked to bipolar disorder and schizophrenia. Genomics 79:635–656. Chumakov I, Blumenfeld M, Guerassimenko O, Cavarec L, Palicio M, Abderrahim H, et al. 2002. Genetic and physiological data implicating the new human gene G72 and the gene for D-amino acid oxidase in schizophrenia. Proc Natl Acad Sci USA 99:13675–13680. Ewens W, Spielman RS. 1995. The transmission/disequilibrium test: History, subdivision, and admixture. Am J Hum Genet 57:455–464. Excoffier L, Slatkin M. 1995. Maximum-likelihood estimation of molecular haplotype frequencies in a diploid population. Mol Biol Evol 12:921– 927. Falk CT, Rubinstein P. 1987. Haplotype relative risks: An easy reliable way to construct a proper control sample for risk calculations. Ann Hum Genet 51:227–233. 95 Komlos L, Korostishevsky M, Livni E, Halbrecht I, Vardimon D, Ben-Rafael Z, et al. 1997. Possible sex-correlated transmission of maternal class I HLA haplotypes. Eur J Immunogenet 24:169–177. Konradi C, Heckers S. 2003. Molecular aspects of glutamate dysregulation: Implications for schizophrenia and its treatment. Pharmacol Ther 97: 153–179. Korostishevsky M, Kaganovich M, Cholostoy A, Ashkenazi M, Ratner Y, Dahary D, et al. 2004. Is the G72/G30 locus associated with schizophrenia? Single nucleotide polymorphisms, haplotypes, and gene expression analysis. Biol Psychiatry 56:169–176. Levinson DF, Holmans P, Straub RE, Owen MJ, Wildenauer DB, Gejman PV, et al. 2000. Multicenter linkage study of schizophrenia candidate regions on chromosomes 5q, 6q, 10p, and 13q: Schizophrenia linkage collaborative group III. Am J Hum Genet 67:652–663. Lin MW, Curtis D, Williams N, Arranz M, Nanko S, Collier D, et al. 1995. Suggestive evidence for linkage of schizophrenia to markers on chromosome 13q14.1-q32. Psychiatr Genet 5:117–126. Mothet JP, Parent AT, Wolosker H, Bradu RO, Linden DJ, Ferris CD, et al. 2000. D-Serine is an endogenous ligand for the glycine site of the N-methyl-D-aspartate receptor. Proc Natl Acad Sci USA 97:4926– 4931. Nebel A, Filon D, Weiss DA, Weale M, Faerman M, Oppenheim A, Thomas MG. 2000. High-resolution Y chromosome haplotypes of Israeli and Palestinian Arabs reveal geographic substructure and substantial overlap with haplotypes of Jews. Hum Genet 107:630–641. Nei M. 1987. Molecular evolutionary genetics. New York, NY, USA: Columbia University Press. Schneider S, Roessli D, Excoffer L. 2000. A software for population genetic data analysis. Manual arlequin, version 2.00. Zwitzerland: University of Geneva. Schumacher J, Jamra RA, Freudenberg J, Becker T, Ohlraun S, Otte AC, et al. 2004. Examination of G72 and D-amino-acid oxidase as genetic risk factors for schizophrenia and bipolar affective disorder. Mol Psychiatry 9:203–207. Shaw SH, Kelly M, Smith AB, Shields G, Hopkins PJ, Loftus J, et al. 1998. A genome-wide search for schizophrenia susceptibility genes. Am J Med Genet 81:364–376. Slatkin M. 1994. Linkage disequilibrium in growing and stable population. Genetics 137:331–336. Gottesman II. 1991. Schizophrenia genesis: The origins of madness. New York: WH. Freeman. Slatkin M, Excoffer L. 1996. Testing for linkage disequilibrium in genotypic data using the EM algorithm. Heredity 76:377–383. Guo S, Thomson E. 1992. Performing the exact test of Hardy–Weinberg proportion for multiple alleles. Biometrics 48:361–372. Spielman RS, McGinnis RE, Ewens WJ. 1993. Transmission test for linkage disequilibrium: The insulin gene region and insulin dependent diabetes mellitus (NDDM). Am J Hum Genet 52:506–516. Hammer MF, Redd AJ, Wood ET, Bonner MR, Jarjanazi H, Karafet T, et al. 2000. Jewish and Middle Eastern non-Jewish populations share a common pool of Y-chromosome biallelic haplotypes. Proc Natl Acad Sci USA 97:6769–6774. Hattori E, Liu C, Badner JA, Bonner TI, Christian SL, Maheshwari M, et al. 2003. Polymorphisms at the G72/G30 gene locus, on 13q33, are associated with bipolar disorder in two independent pedigree series. Am J Hum Genet 72:1131–1140. Horvath S, Xu X, Lake SL, Silverman EK, Weiss ST, Nan M, Laird NM. 2004. Family-based tests for associating haplotypes with general phenotype data: Application to asthma genetics. Genet Epidemiol 26: 61–69. Kendall MG, Stuart A. 1973. The advanced theory of statistics. Vol. 2, London: Griffin and Company Ltd., pp 234–237. Thomas MG, Parfitt T, Weiss DA, Skorecki K, Wilson JF, le Roux M, et al. 2000. Y chromosomes traveling south: The cohen modal haplotype and the origins of the Lemba- the "Black Jews of Southern Africa". Am J Hum Genet 66:674–686. Yelin R, Dahary D, Sorek R, Levanon EY, Goldstein O, Shosan A, et al. 2003. Widespread occurrence of antisense transcription in the human genome. Nat Biotechnol 21:379–386. Zlotogora J. 1997. Autosomal recessive diseases among Palestinian Arabs. J Med Genet 34:765–766. Zlotogora J. 2002. Molecular basis of autosomal recessive diseases among the Palestinian Arabs. Am J Med Genet 109:176–182.