Claudio Toma, PhD; Alex D. Shaw, PhD; Anna Heath BSc; Kerrie D. Pierce, BSc; Philip B. Mitchell, MBBS, MD; Peter R. Schofield, PhD, DSc; Janice M. Fullerton, PhD
Background: Bipolar disorder is a highly heritable psychiatric condition for which specific genetic factors remain largely unknown. In the present study, we used combined whole-exome sequencing and linkage analysis to identify risk loci and dissect the contribution of common and rare variants in families with a high density of illness.
Methods: Overall, 117 participants from 15 Australian extended families with bipolar disorder (72 with affective disorder, including 50 with bipolar disorder type I or II, 13 with schizoaffective disorder–manic type and 9 with recurrent unipolar disorder) underwent whole-exome sequencing. We performed genome-wide linkage analysis using MERLIN and conditional linkage analysis using LAMP. We assessed the contribution of potentially functional rare variants using a genebased segregation test.
Results: We identified a significant linkage peak on chromosome 10q11-q21 (maximal single nucleotide polymorphism = rs10761725; exponential logarithm of the odds [LODexp] = 3.03; empirical p = 0.046). The linkage interval spanned 36 protein-coding genes, including a gene associated with bipolar disorder, ankyrin 3 (ANK3). Conditional linkage analysis showed that common ANK3 risk variants previously identified in genome-wide association studies — or variants in linkage disequilibrium with those variants — did not explain the linkage signal (rs10994397 LOD = 0.63; rs9804190 LOD = 0.04). A family-based segregation test with 34 rare variants from 14 genes under the linkage interval suggested rare variant contributions of 3 brain-expressed genes: NRBF2 (p = 0.005), PCDH15 (p = 0.002) and ANK3 (p = 0.014).
Limitations: We did not examine non-coding variants, but they may explain the remaining linkage signal.
Conclusion: Combining family-based linkage analysis with next-generation sequencing data is effective for identifying putative disease genes and specific risk variants in complex disorders. We identified rare missense variants in ANK3, PCDH15 and NRBF2 that could confer disease risk, providing valuable targets for functional characterization.
Submitted Apr. 29, 2020; Revised Sept. 18, 2020; Accepted Oct. 12, 2020
Acknowledgments: We are grateful to all participants and their families, as well as clinical collaborators who were originally involved in collecting and phenotyping these families. We would also thank Richard J. N. Allcock from the University of Western Australia (Australia) for WES library preparation and analytic support. This study was funded by the Australian National Medical and Health Research Council (NHMRC) project grants 1063960 and 1066177, and program grant 1037196. We gratefully acknowledge the Janette Mary O’Neil Research Fellowship (to JMF) and Mrs. Betty Lynch (dec.) for supporting this work and our team. DNA was extracted by Genetic Repositories Australia, an Enabling Facility that was supported by NHMRC Enabling Grant 401184. Samples were sequenced at the Lottery State Biomedical Genomics Facility, University of Western Australia, and genotyped on Illumina PsychChip at the Australian Genome Research Facility. This research was undertaken with the assistance of resources from the National Computational Infrastructure (NCI), which is supported by the Australian Government.
Affiliations: From Neuroscience Research Australia, Sydney, Australia (Toma, Shaw, Heath, Pierce, Schofield); the School of Medical Sciences, University of New South Wales, Sydney, Australia (Toma, Shaw, Schofield, Fullerton); the Centro de Biología Molecular ‘Severo Ochoa‘, Universidad Autónoma de Madrid/CSIC, Madrid, Spain (Toma); the School of Psychiatry, University of New South Wales, Sydney, Australia (Mitchell); and the Black Dog Institute, Prince of Wales Hospital, Sydney, Australia (Mitchell).
Competing interests: P. Mitchell reports personal fees from Sanofi (Hangzhou) and Janssen-Cilag, outside the submitted work.
Contributors: C. Toma, A. Shaw, P. Mitchell, P. Schofield and J. Fullerton designed the study. All authors acquired the data, which C. Toma, A. Shaw, P. Mitchell, P. Schofield and J. Fullerton analyzed. C. Toma and J. Fullerton wrote the article, which all authors reviewed. All authors approved the final version to be published and can certify that no other individuals not listed as authors or in the acknowledgments have made substantial contributions to the paper.
Content licence: This is an Open Access article distributed in accordance with the terms of the Creative Commons Attribution (CC BY-NC-ND 4.0) licence, which permits use, distribution and reproduction in any medium, provided that the original publication is properly cited, the use is non-commercial (i.e. research or educational use), and no modifications or adaptations are made. See: https://creativecommons.org/licenses/by-nc-nd/4.0/
Correspondence to: J. M. Fullerton, Neuroscience Research Australia, Margarete Ainsworth Building, Barker Street, Randwick, Sydney, NSW 2031, Australia, firstname.lastname@example.org; C. Toma, Centro de Biología Molecular “Severo Ochoa,” Universidad Autónoma de Madrid/CSIC, C/Nicolás Cabrera, 1, Campus UAM, 28049 Madrid, Spain, email@example.com