BRIEF REPORT Print ISSN 1738-3684 / On-line ISSN 1976-3026 http://dx.doi.org/10.4306/pi.2015.12.3.402 OPEN ACCESS CDH13 and HCRTR2 May Be Associated with Hypersomnia Symptom of Bipolar Depression: A Genome-Wide Functional Enrichment Pathway Analysis Chul-Hyun Cho1, Heon-Jeong Lee1 , Hyun Goo Woo2, Ji-Hye Choi2, Tiffany A. Greenwood3, and John R. Kelsoe3,4 1Department of Psychiatry, Korea University College of Medicine, Seoul, Republic of Korea 2Department of Physiology, Ajou University School of Medicine, Suwon, Republic of Korea 3Department of Psychiatry, University of California San Diego, La Jolla, CA, USA 4San Diego VA Healthcare System, San Diego, CA, USA Although bipolar disorder is highly heritable, the identification of specific genetic variations is limited because of the complex traits un- derlying the disorder. We performed a genome-wide association study of bipolar disorder using a subphenotype that shows hypersom- nia symptom during a major depressive episode. We investigated a total of 2,191 cases, 1,434 controls, and 703,012 single nucleotide poly- morphisms (SNPs) in the merged samples obtained from the Translational Genomics Institute and the Genetic Association Information Network. The gene emerging as the most significant by statistical analysis was rs1553441 (odds ratio=0.4093; p=1.20×10-5; Permuted p=6.0×10-6). However, the 5×10-8 threshold for statistical significance required in a genome-wide association study was not achieved. The functional enrichment pathway analysis showed significant enrichments in the adhesion, development-related, synaptic transmis- sion-related, and cell recognition-related pathways. For further evaluation, each gene of the enriched pathways was reviewed and matched with genes that were suggested to be associated with psychiatric disorders by previous genetic studies. We found that the cadherin 13 and hypocretin (orexin) receptor 2 genes may be involved in the hypersomnia symptom during a major depressive episode of bipolar disorder. Psychiatry Investig 2015;12(3):402-407 Key Wordsaa Genome-wide association study, Bipolar disorder, Hypersomnia, Functional enrichment pathway analysis, Bipolar depression. INTRODUCTION proach for future genetic studies in BD.5 Several studies suggested that dysregulation of the circadi- Bipolar disorder (BD) patients suffer from both a low phys- an rhythm is associated with the pathophysiology of BD.6,7 In ical quality and low mental quality of life, even during an eu- general, BD patients have common specific circadian rhythm- thymic period.1 BD is known to be highly heritable; however, related clinical manifestations such as: diurnal mood varia- the identification of specific genetic variations resulted in lim- tion, periodicity of manic-depressive episodes, activity changes, ited findings.2,3 One reason for this low success rate in identi- alteration of the secretion of hormones and other endogenous fying genetic variations relies in the nature and complex traits substances, and sleep disturbances.8-10 Sleep disturbances may of BD.4 As a consequence, dividing BD into subgroups ac- have a negative effect on emotional regulation.11 According to cording to clinical subphenotypes is a reliable alternative ap- these results, it can be assumed that circadian rhythm dysreg- ulation is very closely associated with BD.12 Received: August 21, 2014 Revised: November 17, 2014 We hypothesized that a subphenotype group that shows Accepted: November 17, 2014 Available online: July 6, 2015 Correspondence: Heon-Jeong Lee, MD, PhD hypersomnia symptom during a major depressive episode of Department of Psychiatry, Korea University College of Medicine, 73 Inchon- bipolar disorder may represent a genetically distinct subtype ro, Seongbuk-gu, Seoul 136-705, Republic of Korea Tel: +82-2-920-6721, Fax: +82-2-929-7679, E-mail: [email protected] of BD. We tested this hypothesis using a genome-wide asso- cc This is an Open Access article distributed under the terms of the Creative Commons ciation (GWA) analysis between subjects that showed hyper- Attribution Non-Commercial License (http://creativecommons.org/licenses/by- nc/3.0) which permits unrestricted non-commercial use, distribution, and reproduc- somnia symptom during a major depressive episode of bipo- tion in any medium, provided the original work is properly cited. lar disorder (hypersomnia symptom of bipolar depression, 402 Copyright © 2015 Korean Neuropsychiatric Association CH Cho et al. HBD) and subjects that did not show hypersomnia symptom Phenotypes during a major depressive episode of bipolar disorder (non- As part of the DIGS interview, bipolar I disorder subjects hypersomnia symptom of bipolar depression, NHBD). The were queried as to whether they had a hypersomnia symptom subject samples were of European ancestry and were geno- during major depressive episodes. According to the answer, typed as part of the Genetic Association Information Network subjects were categorized either into the HBD or the NHBD (GAIN) by the Bipolar Genome Study (BiGS). group. Those who answered ‘Unknown’ to the question about hypersomnia symptom were categorized as the missing group. METHODS After filtering, there were 263 BD subjects in the HBD group and 112 subjects in the NHBD group. Subject ascertainment Prior to genotyping, which was part of the BiGS, the unre- Association analyses lated bipolar I disorder subjects of European ancestry were To assess genetic factors contributing to HBD, we perform- selected from those collected by the National Institute of Men- ed a genome-wide case-only analysis of HBD versus NHBD. tal Health Genetics Initiative for Bipolar Disorder. All subjects This association analysis was performed using a logistic re- provided written informed consent in accordance to proto- gression using PLINK16 with a covariance adjustment for sex cols from local institutional review boards. The subjects were and age. Adaptive permutations were performed to find the interviewed using protocols from the Diagnostic Interview empirical significance of the results using PLINK.16 for Genetic Studies (DIGS).13 Information was obtained from family informants and medical records. The information was SNP imputation reviewed along with the interview by a panel of experienced Missing SNPs were imputed using the IMPUTE2 tool17 and clinicians to obtain a final best-estimate diagnosis. Control the CEU panel of HapMap 3+1,000 Genomes Pilot haplotypes subjects were selected after they were certified through a Na- as a reference. The imputed SNPs were used for the functional tional Institute of Mental Health-supported contract between enrichment pathway analysis. Dr. Pablo Gejman and Knowledge Networks, Inc.14 All sub- jects donated a blood sample and were given medical ques- Functional enrichment pathway analysis tionnaires. The selected controls were matched for gender and Gene ontology analysis was performed on the gene sets har- ethnicity with the BD cases. Control subjects who had a his- boring the identified SNPs (p<0.005) evaluated using the DA- tory of BD, psychosis, or recurrent major depression were ex- VID software.18,19 The enriched gene functions were identi- cluded from our study. The subject samples as part of the GAIN fied from the HBD versus NHBD analysis using an enrichment were obtained by Dr. Kelsoe who is a member of the BiGS. score. To go further in our investigation, we made a list of genes of the enriched pathways and compared them with various Genotyping and cleaning genes that were suggested to be related to psychiatric disorders The first set of samples was genotyped at the Broad Insti- by previous genetic studies. We searched the available data- tute, as part of GAIN, using the Affymetrix SNP Array 6.0 bases in PubMed (http://www.ncbi.nlm.nih.gov/pubmed/) and (Affymetrix; Santa Clara, CA, US) 1M SNP array. We obtained in the National Human Genome Research Institute (http:// a total of 1,001 BD cases, 1,033 controls, and 724,067 single www.genome.gov/) for genes of enriched pathways. Some of nucleotide polymorphisms (SNPs). These were available for the search terms included: the name of each gene of enriched analysis following an extensive quality control (QC) process.15 pathways and “psychiatric disorder or depression or bipolar The QC process eliminated all individuals with >10% miss- disorder or schizophrenia or circadian rhythm”. After the re- ing data, SNPs with poor allele clustering, duplicate errors, mi- view and matching, we selected genes from those of enriched nor allele frequencies <0.05, and significant deviation from pathways that were described as susceptible genes in psychi- Hardy-Weinberg equilibrium at p<10-6. The second set of atric disorders by previous genetic studies. samples was genotyped similarly to the first set of samples at the Translational Genomics Institute (TGEN) and underwent RESULTS a comparable QC. From this set of samples we obtained 1,190 BD cases, 401 controls, and 728,187 SNPs available for analy- Eighty associated SNPs with p<10-4 were identified in this sis. An additional round of QC was performed on the merged present genome-wide case-only analysis. We found that samples from GAIN and TGEN. This merge resulted in a set rs1553441 is the gene that appeared to be the most significant of 703,012 SNPs that passed the imposed QC process. after statistical analysis (odds ratio=0.4093, p value of 1.20×10-5, Permuted p=6.0×10-6) (Supplementary Figure 1 in the online- www.psychiatryinvestigation.org 403 GWAS for Hypersomnia in Bipolar Disorder only Data Supplement). This SNP is located within a region gories that showed significant enrichment by comparison to of the gene encoding the phospholipase D family, member 5 the results of previous genetic studies. We selected the genes (PLD5) on chromosome 1q43. However, the most significant of enriched pathways that matched those that have been sug- gene of this study did not reach the p value of 5×10-8, which gested in the literature to be related to psychiatric disorders is the threshold for statistical significance in a GWA.16 (Table 1).