Mechanisms Underlying Phenotypic Heterogeneity in Simplex Autism Spectrum Disorders Andrew H. Chiang Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2021 © 2021 Andrew H. Chiang All Rights Reserved Abstract Mechanisms Underlying Phenotypic Heterogeneity in Simplex Autism Spectrum Disorders Andrew H. Chiang Autism spectrum disorders (ASD) are a group of related neurodevelopmental diseases displaying significant genetic and phenotypic heterogeneity. Despite recent progress in ASD genetics, the nature of phenotypic heterogeneity across probands is not well understood. Notably, likely gene- disrupting (LGD) de novo mutations affecting the same gene often result in substantially different ASD phenotypes. We find that truncating mutations in a gene can result in a range of relatively mild decreases (15-30%) in gene expression due to nonsense-mediated decay (NMD), and show that more severe autism phenotypes are associated with greater decreases in expression. We also find that each gene with recurrent ASD mutations can be described by a parameter, phenotype dosage sensitivity (PDS), which characteriZes the relationship between changes in a gene’s dosage and changes in a given phenotype. Using simple linear models, we show that changes in gene dosage account for a substantial fraction of phenotypic variability in ASD. We further observe that LGD mutations affecting the same exon frequently lead to strikingly similar phenotypes in unrelated ASD probands. These patterns are observed for two independent proband cohorts and multiple important ASD-associated phenotypes. The observed phenotypic similarities are likely mediated by similar changes in gene dosage and similar perturbations to the relative expression of splicing isoforms. We also identify patterns of developmental and cell type-specific expression that additionally contribute to the variability of several autism phenotypes. Table of Contents List of Charts, Graphs, Illustrations ................................................................................................ 7 Introduction ................................................................................................................................... 10 1.1 Thesis overview ............................................................................................................ 10 1.2 Background ................................................................................................................... 11 Epidemiology and genetics of ASD ...................................................................................... 11 Phenotypic diversity in ASD ................................................................................................ 14 Simplex ASD as a relatively simple model of genetic disease ............................................. 16 2 Gene dosage changes and phenotype severity in ASD ......................................................... 18 2.1 Introduction ................................................................................................................... 18 2.2 Results ........................................................................................................................... 21 Variability in NMD-induced dosage decreases for LGD mutations in different exons ....... 21 Variability in the sensitivity of phenotypes to changes in the dosage of different genes ..... 30 Relationships between gene dosage changes and the severity of ASD phenotypes ............. 31 Inference of quantitative phenotypes based on changes in gene dosage .............................. 35 2.3 Methods......................................................................................................................... 41 SSC sequencing and phenotype data .................................................................................... 41 VIP sequencing and phenotype data ..................................................................................... 41 Phenotype scores analyzed in SSC and VIP ......................................................................... 42 GTEx genotype and expression data ..................................................................................... 42 4 Quantification of allele-specific expression (AE) ................................................................. 43 Gene expression changes due to LGD variants in GTEx ..................................................... 44 BrainSpan expression data .................................................................................................... 52 Dosage-based model of phenotypic effect ............................................................................ 52 Linear model-based predictions of the effects of LGD mutations ........................................ 54 3 Autism phenotypes and the exon-intron structure of genes .................................................. 56 3.1 Introduction ................................................................................................................... 56 3.2 Results ........................................................................................................................... 57 Exon-specific phenotypes for de novo LGD mutations in ASD ........................................... 57 Exon-specific gene- and isoform-level dosage changes due to LGD variants ..................... 68 Phenotypic consequences of LGD mutations across gene and protein sequences ............... 73 Phenotypic consequences of LGDs affecting functional coding sequences ......................... 81 3.3 Methods......................................................................................................................... 87 Genetic and phenotypic data ................................................................................................. 87 NormaliZation of phenotypic scores ..................................................................................... 87 Registration of splice site mutations to exons ....................................................................... 88 Comparison of phenotype variability .................................................................................... 88 Chromosomal distance between mutations ........................................................................... 89 Protein sequence analyses ..................................................................................................... 89 Distance-matched permutation tests ..................................................................................... 90 Identifying protein domains affected by LGD mutations ..................................................... 91 Enrichment of mutations in developmentally biased exons ................................................. 92 Analysis of NMD induced by LGD variants in GTEx ......................................................... 93 5 Isoform-specific expression changes due to LGD variants .................................................. 93 4 Properties of exons and genes harboring LGD mutations in ASD ....................................... 95 4.1 Introduction ................................................................................................................... 95 4.2 Results ........................................................................................................................... 97 Developmental expression of exons harboring LGD mutations ........................................... 97 Cell type-specific expression biases in ASD-associated genes ............................................ 99 Phenotypes associated with expression biases toward neuronal cell types ........................ 105 Combined effects of dosage changes and cell-type specificity .......................................... 106 4.3 Methods....................................................................................................................... 111 Enrichment of mutations in developmentally biased exons ............................................... 111 Cell-type specific expression biases ................................................................................... 111 Grouping ASD-associated genes by biological function .................................................... 112 StandardiZation of ASD phenotypes ................................................................................... 114 Calculation of aggregated phenotype scores ....................................................................... 114 Conclusion .................................................................................................................................. 117 References ................................................................................................................................... 120 6 List of Charts, Graphs, Illustrations Figure 0.1 Diverse genes affected by de novo LGDs and CNVs in ASD .................................... 13 Figure 0.2 Expression bias of ASD genes towards brain regions ................................................. 14 Figure 0.3 Variability of phenotypes for probands with LGDs in the same gene. ....................... 16 Figure 0.4 Probability of observing different numbers of LGD mutations .................................. 17 Figure 1.1 Biological processes considered in modeling of NMD ............................................... 21 Figure