UNIVERSITY OF CALIFORNIA Los Angeles Investigation of sex-differential genetic risk factors for autism spectrum disorders A dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in Neuroscience by Donna Marie Werling 2014 © Copyright by Donna Marie Werling 2014 ABSTRACT OF THE DISSERTATION Investigation of sex-differential genetic risk factors for autism spectrum disorders by Donna Marie Werling Doctor of Philosophy in Neuroscience University of California, Los Angeles, 2014 Professor Daniel H. Geschwind, Chair Autism spectrum disorders (ASDs) are pervasive neurodevelopmental disorders that affect more males than females, and the mechanisms responsible for increasing males’ risk or protecting females are not understood. This sex biased prevalence is consistent across time and populations, suggesting that an understanding of the processes driving sex-differential risk would likely be informative of fundamental pathophysiology in ASD. One known component of ASD risk is genetic variation. Thus, here I apply several approaches that leverage current knowledge of ASD genetics to investigate the role and mechanisms of sex-differential biology in ASD risk. First, I evaluate a cohort of families with more than one autistic child for evidence of sex- differential, familial risk variation. Second, I use genetic linkage analysis to identify sex- differential risk loci in families from the same multiplex cohort. Third, I characterize gene expression patterns in typical human neocortex to identify points of interaction between typical sexual dimorphism and genes known to carry risk variants for ASD. ii I find that recurrence rates for ASD diagnoses in multiplex families are consistent with a female protective model, in which females require more deleterious genetic variation to be affected with ASD and this greater genetic load is shared with females’ siblings. I also identify several chromosomal loci with evidence of genetic linkage in families either with (chromosome 8p21.2 and 8p12), or without (chromosome 1p31.3), an autistic female. No significant common variants are found in either region that can account for this linkage; these loci will be further investigated by targeted sequencing to identify rare risk variants. Gene expression analyses show that known ASD risk genes are not differentially expressed in males or females in the prenatal or adult human neocortex. However, astrocyte markers and gene sets implicated in immune function and inflammatory processes are expressed at higher levels in males. This suggests that sex-differential factors may operate downstream from, or interact with, ASD risk genes, as opposed to directly regulating the expression of these genes. Overall, findings from these multiple approaches provide valuable context for the function of sex-differential biology in ASD etiology, and suggest promising directions for future research. iii The dissertation of Donna Marie Werling is approved. Arthur P. Arnold Rita M. Cantor Nelson B. Freimer Daniel H. Geschwind, Committee Chair University of California, Los Angeles 2014 iv TABLE OF CONTENTS Chapter 1: Sex differences in autism spectrum disorders......................................................... 1 1.1: Introduction.................................................................................................................. 2 1.2: ASD prevalence in males and females........................................................................ 3 1.3: Presentation of ASD symptoms and related phenotypes in males and females.......... 5 1.4: Sex differences in genetic contributions to ASD risk.................................................. 7 1.5: Sex hormonal contributors to ASD risk....................................................................... 12 1.6: Conclusions.................................................................................................................. 16 Chapter 2: Recurrence rates provide evidence for sex-differential, familial genetic liability for autism spectrum disorders in multiplex families and twins...................................................... 18 2.1: Abstract........................................................................................................................ 19 2.2: Background.................................................................................................................. 20 2.3: Materials and methods................................................................................................. 24 2.3.a: Subjects............................................................................................................... 24 2.3.b: Sex ratios and recurrence risk............................................................................. 26 2.3.c: Quantitative phenotypes..................................................................................... 28 2.3.d: Concordance in twin pairs.................................................................................. 29 2.4: Results.......................................................................................................................... 30 2.5: Discussion.................................................................................................................... 39 2.6: Conclusions.................................................................................................................. 47 Chapter 3: Identification of suggestive sex-differential risk loci and replication of linkage at chromosome 20p13 for autism spectrum disorder.................................................................... 49 3.1: Abstract........................................................................................................................ 50 3.2: Background.................................................................................................................. 51 3.3: Materials and methods................................................................................................. 55 3.3.a: Subjects and genotyping..................................................................................... 55 3.3.b: Linkage analyses using all families.................................................................... 58 3.3.c: Sex-stratified linkage.......................................................................................... 59 3.3.d: Imputation........................................................................................................... 61 v 3.3.e: Linkage-directed association testing................................................................... 62 3.4: Results.......................................................................................................................... 63 3.4.a: Linkage in all families........................................................................................ 63 3.4.b: Sex-stratified linkage.......................................................................................... 66 3.4.c: Linkage-directed association.............................................................................. 72 3.5: Discussion.................................................................................................................... 73 3.6: Conclusions.................................................................................................................. 78 Chapter 4: Strategies for the identification of functional autism risk variants in linkage regions . .............................................................................................................................. 79 4.1: Linkage signals in autism spectrum disorders............................................................. 80 4.2: Common variant association........................................................................................ 82 4.3: Rare variant association............................................................................................... 84 4.4: Potential implications.................................................................................................. 89 Chapter 5: Gene expression implicates pathways at the interface between sexual dimorphisms and genetic risk variants for autism spectrum disorders........................................................... 90 5.1: Abstract........................................................................................................................ 91 5.2: Background.................................................................................................................. 91 5.3: Materials and methods................................................................................................. 96 5.3.a: Gene expression data from human brain tissue.................................................. 96 5.3.a.i: Adult BrainSpan sample............................................................................ 96 5.3.a.ii: Adult replication sample............................................................................ 97 5.3.a.iii: Prenatal BrainSpan sample....................................................................... 99 5.3.b: Differential expression analysis.......................................................................... 101 5.3.c: Annotation gene sets........................................................................................... 101 5.3.c.i: Autism spectrum disorder risk genes......................................................... 102 5.3.c.ii: Gene expression