Genetics of amyotrophic lateral sclerosis in the Han Chinese Ji He A thesis submitted for the degree of Master of Philosophy at The University of Queensland in 2015 The University of Queensland Diamantina Institute 1 Abstract Amyotrophic lateral sclerosis is the most frequently occurring neuromuscular degenerative disorders, and has an obscure aetiology. Whilst major progress has been made, the majority of the genetic variation involved in ALS is, as yet, undefined. In this thesis, multiple genetic studies have been conducted to advance our understanding of the genetic architecture of the disease. In the light of the paucity of comprehensive genetic studies performed in Chinese, the presented study focused on advancing our current understanding in genetics of ALS in the Han Chinese population. To identify genetic variants altering risk of ALS, a genome-wide association study (GWAS) was performed. The study included 1,324 Chinese ALS cases and 3,115 controls. After quality control, a number of analyses were performed in a cleaned dataset of 1,243 cases and 2,854 controls that included: a genome-wide association analysis to identify SNPs associated with ALS; a genomic restricted maximum likelihood (GREML) analysis to estimate the proportion of the phenotypic variance in ALS liability due to common SNPs; and a gene- based analysis to identify genes associated with ALS. There were no genome-wide significant SNPs or genes associated with ALS. However, it was estimated that 17% (SE: 0.05; P=6×10-5) of the phenotypic variance in ALS liability was due to common SNPs. The top associated SNP was within GNAS (rs4812037; p =7×10-7). GNAS was also the most associated gene from the gene-based study (p =2×10-5). Based on GWAS data, a fragment-length and repeat-primed PCR was performed to determine GGGGCC copy number and expansion within the C9orf72 gene in the cohorts (1,092 sporadic ALS and 1,062 controls) from China. A haplotype analysis of 23 SNPs within and surrounding the C9orf72 gene was performed. The C9orf72 hexanucleotide (GGGGCC)n repeat expansion (HRE) was found in three sALS patients (0.3%) but not in control subjects (p = 0.25, Fisher’s exact test). Two cases 2 with HRE did not harbor four risk alleles that have previously been determined to be strongly associated with ALS in Caucasian populations. The presence of risk alleles (including rs2814707 and rs384992) of the 20-SNP consensus risk founder haplotype in Caucasians demonstrated that two of the three cases shared a novel haplotype carrying HRE. The low frequency (1.8%) of the 20-SNP consensus risk haplotype and the distinct allele distribution in Chinese ALS patients compared to Caucasian populations indicates that the C9orf72 HRE is not from the same single founder haplotype involved in Caucasian populations. In addition, using next generation sequencing, a population-specific mutational spectrum of ALS demonstrated that mutations in SOD1 are the most common causative mutations in fALS in the Han Chinese population. To identify further genetic associations, a novel strategy based on functional annotations and current understanding of ALS for specifically filtering candidate genes has been suggested. These findings broaden the spectrum of causative mutations in ALS and are essential for optimal design of strategies of mutational analysis and genetic counselling of Han Chinese ALS patients. To conclude, the presented study with the largest non-Caucasian ALS cohorts showed the heterogeneity of ALS in the Chinese Han population and laid the foundation for further genetic studies of ALS in Mainland China. 3 Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the policy and procedures of The University of Queensland, the thesis be made available for research and study in accordance with the Copyright Act 1968 unless a period of embargo has been approved by the Dean of the Graduate School. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. 4 Publications during candidature Amyotrophic Lateral Sclerosis Genetic Studies: From Genome-wide Association Mapping to Genome Sequencing. He J, Mangelsdorf M, Fan D, Bartlett P, Brown MA. Neuroscientist. 2014 Nov 5. pii: 1073858414555404; Contributor Statement of contribution Ji He Data collection and analysis(80%) Wrote the paper (70%) Mangelsdorf M Data collection and analysis(20%) Wrote and edited paper (30%) Fan D, Bartlett P, Brown MA Edited paper (70%) Publications included in this thesis Chapter 1. Literature review: Amyotrophic Lateral Sclerosis Genetic Studies: From Genome-wide Association Mapping to Genome Sequencing. He J, Mangelsdorf M, Fan D, Bartlett P, Brown MA. Neuroscientist. 2014 Nov 5. pii: 1073858414555404; Contributor Statement of contribution Ji He Data collection and analysis(80%) Wrote the paper (70%) Mangelsdorf M Data collection and analysis(20%) Wrote and edited paper (30%) Fan D, Bartlett P, Brown MA Edited paper (70%) 5 Contributions by others to the thesis Dr. Marie Mangelsdorf, Prof. Dongsheng Fan, Prof Peter Visscher, Prof Naomi Wray, Prof. Perry Bartlett and, Prof. Matthew A. Brown were involved in the conception and design of studies in the thesis; and the acquisition, analysis and interpretation of data. Chapter 1: edited by Ji He, Dr. Marie Mangelsdorf, Prof. Dongsheng Fan, Prof. Perry Bartlett and Prof. Matthew A. Brown. Chapter 2: edited by Ji He, Dr. Marie Mangelsdorf, Dr Beben Benyamin, Prof. Dongsheng Fan and Prof. Matthew A. Brown. Katie Cremin, Jessica Haris, Lawrie Wheeler and Brooke Gardiner co-contributed in carrying out microarray genotyping assays. The analysis is also contributed by Dr. Xin Wu, Prof. Huji Xu, Dr. Beben Benyamin, Dr. Paul Leo. Chapter 3: Ji He, Dr. Marie Mangelsdorf, Prof. Dongsheng Fan and Prof. Matthew A. Brown wrote the manuscript. Repeat-Primed PCR study is co- contributed with Lu Tang and Prof. Dongsheng Fan, Haplotype SNP Analysis is co- contributed with Katie Cameron, Jessica Harris, Lawrie Wheeler, Brooke Gardiner, Dr. Xin Wu, Prof. Huji Xu, Dr. Beben Benyamin, Dr. Paul Leo and Professor Matthew A. Brown. Chapter 4: edited by Ji He, Dr. Marie Mangelsdorf, Prof. Dongsheng Fan and Prof. Matthew A. Brown. Whole Exome Sequencing with Next Generation Sequencing is co- contributed by Sharon Song, Lisa Anderson, Janette Edson and Brooke Gardener, variants analysis is co-contributed by Dr. Mhairi Marshall, Dr. Paul Leo and Dr. Marie Mangelsdorf. Chapter 5: edited by Ji He, Dr. Marie Mangelsdorf, Prof. Dongsheng Fan, Prof. Perry Bartlett and Prof. Matthew A. Brown. All authors read and approved the final manuscript. 6 Statement of parts of the thesis submitted to qualify for the award of another degree None 7 Acknowledgements The author gratefully acknowledges the receipt of a University of Queensland Postgraduate Research Scholarship (UQPRS) and MND scholarship (UQ Diamantina Institute) to support the work reported in this thesis. I would like to thank my supervisor Dr.Marie Mangelsdorf, Dr.Beben Benyamin and Prof. Matthew A. Brown for their guidance, support and complimentary insights into scientific and clinical aspects of my project. Also, I would like to thank Dr. Paul Leo for his support in statistic analysis and modelling, Prof. Dongsheng Fan and Prof Huji Xu for their support in the study, writing my thesis and paper. Likewise, I would like to thank the members, present and past, of the University of Queensland Diamantina Institute (UQDI) and Queensland Brain Institute (QBI) for their patience, assistance and friendship. I would like to thank my family, especially my father, mother and brother, and friends for their ongoing support, patience and understanding. This project would not have been possible without the time and blood samples cheerfully donated by amyotrophic lateral sclerosis patients of the Peking University Third Hospital. The people and institutions that made specific contribution to this thesis are acknowledged in the relevant chapters. 8 Keywords Neurodegenerative disease, amyotrophic lateral sclerosis, genetic study, genome-wide association study, haplotype analysis, next-generation sequencing study. Australian and New Zealand Standard Research Classifications (ANZSRC) ANZSRC code: 110904, Neurology and Neuromuscular Diseases, 40%, ANZSRC code: 060102, Bioinformatics, 30% ANZSRC code: 060405, Gene Expression, 30% Fields of Research