Molecular Psychiatry (2006) 11, 471–478 & 2006 Nature Publishing Group All rights reserved 1359-4184/06 $30.00 www.nature.com/mp ORIGINAL ARTICLE Identification of the semaphorin receptor PLXNA2 as a candidate for susceptibility to schizophrenia S Mah1,4, MR Nelson1,4, LE DeLisi2, RH Reneland1, N Markward1, MR James3, DR Nyholt3, N Hayward3, H Handoko3, B Mowry3, S Kammerer1 and A Braun1 1Sequenom Inc., San Diego, CA, USA; 2Department of Psychiatry, New York University, New York, NY, USA and 3Queensland Institute of Medical Research, Herston, QLD, Australia The discovery of genetic factors that contribute to schizophrenia susceptibility is a key challenge in understanding the etiology of this disease. Here, we report the identification of a novel schizophrenia candidate gene on chromosome 1q32, plexin A2 (PLXNA2), in a genome- wide association study using 320 patients with schizophrenia of European descent and 325 matched controls. Over 25 000 single-nucleotide polymorphisms (SNPs) located within approximately 14 000 genes were tested. Out of 62 markers found to be associated with disease status, the most consistent finding was observed for a candidate locus on chromosome 1q32. The marker SNP rs752016 showed suggestive association with schizo- phrenia (odds ratio (OR) = 1.49, P = 0.006). This result was confirmed in an independent case– control sample of European Americans (combined OR = 1.38, P = 0.035) and similar genetic effects were observed in smaller subsets of Latin Americans (OR = 1.26) and Asian Americans (OR = 1.37). Supporting evidence was also obtained from two family-based collections, one of which reached statistical significance (OR = 2.2, P = 0.02). High-density SNP mapping showed that the region of association spans approximately 60 kb of the PLXNA2 gene. Eight out of 14 SNPs genotyped showed statistically significant differences between cases and controls. These results are in accordance with previous genetic findings that identified chromosome 1q32 as a candidate region for schizophrenia. PLXNA2 is a member of the transmembrane semaphorin receptor family that is involved in axonal guidance during development and may modulate neuronal plasticity and regeneration. The PLXNA2 ligand semaphorin 3A has been shown to be upregulated in the cerebellum of individuals with schizophrenia. These observations, together with the genetic results, make PLXNA2 a likely candidate for the 1q32 schizophrenia susceptibility locus. Molecular Psychiatry (2006) 11, 471–478. doi:10.1038/sj.mp.4001785; published online 10 January 2006 Keywords: single-nucleotide polymorphism; genome-wide association; chromosome 1q32; plexin; semaphorin Introduction 13q34,7 catechol-O-methyltransferase on 22q118 and proline dehydrogenase 1 on 22q11.9 Despite intensive Schizophrenia is one of the most severe and disabling research, results for most of these regions have been of the mental disorders affecting up to 1% of the inconsistent.10,11 world’s population, regardless of gender, race, culture 1 There has been increasing interest in using whole- and class. Family, twin and adoption studies genome association as a method to identify genes strongly suggest a genetic basis for this illness with 2 involved in complex diseases. Genome-wide associa- the heritability estimated at about 70%. Among the tion studies using large numbers of single-nucleotide genes that have been postulated as candidates from polymorphisms (SNPs) have been proposed and linkage and association studies are the regulator of G- 3 successfully applied to find genetic variations asso- protein signaling 4 on 1q22, disrupted in schizo- 12 4 ciated with myocardial infarction, bone mineral phrenia (DISC) 1 and 2 on chromosome 1q42, density13 and breast cancer.14,15 In an effort to identify dystrobrevin-binding protein 1 on 6p22,5 neuregulin 6 genes and variants that influence the risk of schizo- 1 on 8p21, D-amino-acid oxidase activator (G72) on phrenia, we carried out a large-scale SNP association study using a multi-center collection of schizophrenia Correspondence: Dr A Braun, Sequenom Inc., 3595 John Hopkins cases and controls. We identified a gene on chromo- Court, San Diego, CA 92121, USA. some 1q32, plexin A2 (PLXNA2), as a candidate gene E-mail: [email protected] 4These authors contributed equally to this work. for schizophrenia. Several linkage studies previously Received 17 May 2005; revised 19 October 2005; accepted 16 reported schizophrenia susceptibility loci in the November 2005; published online 10 January 2006 1q22-42 region;16–20 other studies, however, have been PLXNA2 as a candidate for susceptibility to schizophrenia S Mah et al 472 unable to reproduce these results.21–23 The findings control individuals in the four ethnic groups was 230, described here might be important for the elucidation 180, 123 and 72, respectively. No detailed information of this controversial schizophrenia locus. on the ancestral origin of the two sample sets collected by PMI was available. Materials and methods Family-based sample 1 Schizophrenia collections A family-based sample was selected from a collection Subjects were selected from four clinical DNA sample of 294 affected sibling pair nuclear families from collections that had been collected independently for throughout the USA (a national sample), and centers genetic research. The appropriate research ethics in the UK (Oxford), Italy (Milan) and Chile (Santiago) committees approved all collections and all subjects recruited between 1985 and 2000.21 The large majority gave their written informed consent to have blood of families (263) were from the USA and the UK and drawn and to participate in genetic studies before were predominantly of Northern European descent. enrollment. Patient information was collected, stored The sample used in this study consisted of 463 and analyzed in accordance with the highest ethical affected and unaffected individuals from 135 families standards. with at least two siblings diagnosed with schizophre- nia or schizoaffective disorder. The two largest ethnic Discovery subjects groups included European Americans (397 indivi- The affected subjects were derived from a large multi- duals from 117 families) and Latin Americans (45 center collection of patients with schizophrenia individuals from 13 families). Diagnoses were made recruited by Precision Medicine Inc. (PMI). All using DSM-III-R criteria on the basis of medical individuals qualified for a clinical trial of the new records and structured interviews performed by atypical antipsychotic medication olanzapine. All trained professionals using validated diagnostic in- subjects had DSM-IV diagnoses of paranoid schizo- struments. phrenia (295.30), disorganized schizophrenia (295.10), residual schizophrenia (295.60), undifferen- Family-based sample 2 tiated schizophrenia (295.90) or schizoaffective dis- A second family-based sample originated from an order (295.70). The majority of subjects were collected Australian high-density family study that consisted of in Los Angeles, with additional subjects coming from 96 recruited families. Each family had to include a Atlanta, Ohio, Philadelphia and Austin. Diagnoses proband with DSM-III-R or DSM-IV schizophrenia were confirmed and documented by the psychiatrist plus at least one other person affected with either in charge of the patient. Additional inclusion criteria schizophrenia or schizoaffective disorder. Eighty-five were > 6 months of olanzapine treatment (before of these families were part of a larger molecular genetic enrollment) and absence of other antipsychotic drug study of schizophrenia, the NIMH GI SZ MGS1, a treatments. The cases selected for our genetic study collaboration of 10 centers (nine US and one Austra- consisted of 110 females and 210 males of European lian).24 Participants were recruited from clinical net- descent, with a median age of 48 and 45, respectively. works and advocacy groups such as Schizophrenia The numbers of affected male (M) and female (F) Fellowship. Individuals (18 years or older) were individuals were as follows: schizoaffective disorder interviewed by trained research clinicians using the 63 M/50 F, paranoid schizophrenia 97 M/39 F, Diagnostic Interview for Genetic Studies 2.0 (DIGS),25 undifferentiated schizophrenia 47 M/19 F, residual a semistructured interview specifically constructed for schizophrenia 1 M/1 F and disorganized schizophre- the assessment of psychotic and major mood dis- nia 2 M/1 F. Three hundred twenty-five controls were orders. Diagnoses were based on the DIGS interview, selected from a repository of healthy blood donors family history information from a reliable informant from the California Blood Bank of San Bernardino and using the Family Interview for Genetic Studies.26,27 Riverside Counties. Subjects with a family history of Two experienced research psychiatrists independently neuropsychiatric disorders were excluded and con- reviewed these data and assigned a diagnosis, subse- trols were sex and age matched to cases. quently meeting to resolve any diagnostic disagree- ments (by reference to collected data or by collection Replication subjects of additional critical clinical data) and thus to assign a The affected subjects for the replication sample primary Best Estimate Final Diagnosis.28 The collec- consisted of 200 European Americans, 157 African tion was composed of 370 individuals, including 177 Americans, 111 Latin Americans and 63 Asian affected and 193 unaffected individuals from 90 Americans derived from a large multi-center