Human Genetics Page 2 of 51 1 2 3 control critera. 4 5 6 The call rate threshold for samples was ≥90%. We have added a statement to this effect in the 7 Methods. 8 9 4. No rationale provided for selection of individuals for sequencing. For example, why were all 10 11 individuals in Family 1 sequenced but only the offspring in family 3? 12 13 We selected the individuals within each family that were likely to be the most informative in 14 sequencing studies. For example, the phenotypes of the parents in family 3 are unknown, 15 sequencing data is unlikely to be of additional benefit, as we would be unable to determine 16 whether variants of interest co-segregate with the phenotype. We have added a section in the 17 18 Methods outlining theseFor choices. Peer Review 19 20 5. “…several [of the coding variants] were associated with the major haplotype of interest in 21 one of the three families”. It would be helpful to know how many variants were associated 22 with the haplotype of interest, without having to go to the Supplemental Table. 23 24 25 We have added this information in the text of the Results section and have highlighted these 26 variants in grey in Supplemental Table 3. 27 28 We hope that these revisions are satisfactory, and submit the revised manuscript for your 29 consideration for publication. 30 31 32 Thank you very much, 33 34 35 36 37 38 39 40 41 42 Carol A. Mathews, MD 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 Page 3 of 51 Human Genetics 1 2 3 Genomewide Linkage Analysis in Costa Rican Families Implicates Chromosome 15q14 as a 4 5 6 Candidate Region for OCD 7 8 9 Jessica Ross, M.D. M.S. 10 11 12 13 Department of Psychiatry, University of California, San Francisco, San Francisco, California 14 15 16 17 18 For PeerJudith Badner, Review M.D., Ph.D. 19 20 21 Departments of Psychiatry and Human Genetics, University of Chicago, Chicago, Illinois 22 23 24 25 26 27 Helena Garrido, M.A. 28 29 30 31 Departments of Psychiatry and Pediatrics, Hospital Nacional de Niños, San Josè, Costa Rica 32 33 34 35 36 37 Brooke Sheppard, B.S. 38 39 40 Department of Psychiatry, University of California, San Francisco, San Francisco, California 41 42 43 44 45 46 Denise Chavira, Ph.D. 47 48 49 50 Department of Psychiatry, University of California, San Diego, La Jolla, California 51 52 53 54 55 56 Marco Grados, M.D., M.P.H. 57 58 59 60 Human Genetics Page 4 of 51 1 2 3 Department of Psychiatry and Behavioral Sciences, Johns Hopkins School of Medicine, 4 5 6 Baltimore, MD 7 8 9 10 11 12 Jonathan M Woo, M.S. 13 14 15 Genome Core Facility, Institute for Human Genetics, University of California, San Francisco, 16 17 18 San Francisco, CaliforniaFor Peer Review 19 20 21 22 23 24 Pamela Doo, B.A. 25 26 27 Genome Core Facility, Institute for Human Genetics, University of California, San Francisco, 28 29 30 San Francisco, California 31 32 33 34 35 Paula Umaña, B.A. 36 37 38 39 Departments of Psychiatry and Pediatrics, Hospital Nacional de Niños, San Josè, Costa Rica 40 41 42 43 44 45 Eduardo Fournier, M.A. 46 47 48 49 Departments of Psychiatry and Pediatrics, Hospital Nacional de Niños, San Josè, Costa Rica 50 51 52 53 54 Sarah Shaw Murray, Ph.D 55 56 57 58 59 60 2 Page 5 of 51 Human Genetics 1 2 3 Department of Genetics, Scripps Genomic Medicine, La Jolla, California 4 5 6 7 8 9 Carol A. Mathews, M.D 10 11 12 13 Department of Psychiatry, University of California, San Francisco, San Francisco, California 14 15 16 17 18 For Peer Review 19 20 21 22 *Correspondence and reprint requests to: Carol A Mathews, M.D., Department of Psychiatry, 23 24 25 University of California San Francisco, 401 Parnassus Avenue, San Francisco CA 94143-0984; 26 27 e-mail: [email protected] .; phone: 415-476-7702; fax: 415-476-7389 28 29 30 31 32 33 34 35 36 37 38 39 40 Keywords: Obsessive Compulsive Disorder, Genetic Linkage, Genetic Isolate, Genetic Loci, 41 42 43 Humans, Genetic Predisposition to Disease 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 3 Human Genetics Page 6 of 51 1 2 3 ABSTRACT 4 5 6 7 Obsessive compulsive disorder (OCD) has a complex etiology that encompasses both genetic 8 9 and environmental factors. However, to date, despite the identification of several promising 10 11 12 candidate genes and linkage regions, the genetic causes of OCD are largely unknown. The 13 14 objective of this study was to conduct linkage studies of childhood-onset OCD, which is thought 15 16 to have the strongest genetic etiology, in several OCD-affected families from the genetically 17 18 For Peer Review 19 isolated population of the Central Valley of Costa Rica (CVCR). The authors used parametric 20 21 and non-parametric approaches to conduct genome-wide linkage analyses using 5786 single 22 23 24 nucleotide repeat polymorphisms (SNPs) in three CVCR families with multiple childhood-onset 25 26 OCD-affected individuals. We identified areas of suggestive linkage (LOD score ≥2) on 27 28 chromosomes 1p21, 15q14, 16q24, and 17p12. The strongest evidence for linkage was on 29 30 31 chromosome 15q14 (LOD=3.13), identified using parametric linkage analysis with a recessive 32 33 model, and overlapping a region identified in a prior linkage study using a Caucasian population. 34 35 Each CVCR family had a haplotype that co-segregated with OCD across a ~7Mbp interval 36 37 38 within this region, which contains 18 identified brain expressed genes, several of which are 39 40 potentially relevant to OCD. Exonic sequencing of the strongest candidate gene in this region, 41 42 the ryanodine receptor 3 (RYR3), identified several genetic variants of potential interest, 43 44 45 although none cosegregated with OCD in all three families. These findings provide evidence that 46 47 chromosome 15q14 is linked to OCD in families from the CVCR, and supports previous findings 48 49 50 to suggest that this region may contain one or more OCD susceptibility loci. 51 52 53 54 55 56 57 58 59 60 4 Page 7 of 51 Human Genetics 1 2 3 INTRODUCTION 4 5 6 7 Although multiple etiologic factors are important in the development of obsessive- 8 9 compulsive disorder (OCD), there is strong evidence for a genetic contribution, particularly 10 11 12 when symptoms begin in childhood(Pauls 2008). Several promising candidate genes have been 13 14 identified for OCD, including SLC1A1(Dickel et al. 2006; Arnold et al. 2006). However, these 15 16 genes, if confirmed, will only account for a small proportion of the genetic variance of this 17 18 For Peer Review 19 complex disorder. Large-scale genome-wide association studies (GWAS) are currently underway 20 21 for OCD; however, such studies will most likely account for only a proportion of the total 22 23 24 disease variation(Yang et al. 2011; Manolio et al. 2009). For example, the estimated proportion 25 26 of the variance explained by all currently reported genetic loci identified by GWAS for height, a 27 28 complex trait with a heritability of 80-90%, is approximately 10%; this number increases to 45% 29 30 31 with recently developed genomic partitioning approaches, but still does not approach 100%. 32 33 Similar results are seen with categorical traits such as type 2 diabetes and Crohn's disease, where 34 35 published susceptibility loci account for 6% and 20% of the total variance, respectively(Manolio 36 37 38 et al. 2009). Linkage studies represent a complementary approach to candidate gene studies and 39 40 GWAS. Where GWAS are useful for the identification of common variants with relatively small 41 42 effect sizes, linkage studies are useful for the identification of rare variants with larger effect 43 44 45 sizes that are increasingly believed to underlie a substantial proportion of the risk for complex 46 47 disorders(Cirulli and Goldstein 2010). 48 49 50 To date, three primary linkage studies for OCD have been published, along with two 51 52 secondary analyses and one targeted replication; these studies have identified several genomic 53 54 candidate regions for OCD (Supplemental Table 1)(Wang et al. 2009; Samuels et al. 2007; 55 56 57 Hanna et al. 2007; Shugart et al. 2006; Hanna et al. 2002; Willour et al. 2004). None of the 58 59 60 5 Human Genetics Page 8 of 51 1 2 3 studies using OCD as the primary phenotype identified regions meeting genome-wide 4 5 6 significance criteria for linkage, although two re-analyses of a large OCD affected-sib pair 7 8 sample using alternate phenotypes or sample selection did identify significant linkage 9 10 11 regions(Samuels et al. 2007; Wang et al. 2009). The first re-analysis included only families with 12 13 two or more members affected with compulsive hoarding, and identified a region on 14 15 chromosome 14q with a LOD score of 3.7, and the second focused on families with male 16 17 18 probands only, and identifiedFor a region Peer on chromosome Review 11p with an initial LOD score of 2.92 and 19 20 a subsequent LOD score of 5.08 following fine mapping with additional markers(Samuels et al.