Functional Assessment of Human Variants in Noncoding Regulatory Sequences

Abstract

The advancement of next-generation sequencing technology has facilitated the identification of deleterious coding variants that underlie various human diseases. Now, emerging evidence indicates that noncoding variants also cause disease and influence clinical manifestations and outcomes (Harper et al., 2021; Khera et al., 2018; Smemo et al., 2012). However, our understanding of the significance of these noncoding variants is limited due to a lack of functional annotation. The primary objective of my research is to identify and characterize noncoding variants that influence gene expression and contribute to human disease. I present five chapters that focus on specific studies aimed at characterizing the functional roles of noncoding variants. I have developed tools to annotate these variants, and I aim to provide a better understanding of the biology underlying human diseases and the roles of noncoding variants in these diseases. In Chapter 1, I studied the role of the noncoding region associated with microtia, a congenital malformation of the external ear with increased prevalence among Amerindian populations. Within a ∼10-kb microtia-associated locus I defined noncoding sequences that regulate ROBO1 and ROBO2, genes that regulate cell-adhesion and promote cellular migration in development. In Chapter 2, I studied the contribution of noncoding de novo variants (DNVs) in congenital heart disease (CHD). Among noncoding de novo variants that were significantly enriched in congenital heart disease iv probands compared to healthy controls, I defined a subset with functional activities in human embryonic cardiomyocytes derived from induced pluripotent stem cells (hiPSC-CMs). In Chapter 3, I describe a robust pipeline for simultaneous analyses of multiple cultured cell samples using single nucleus (sn)RNA-seq. I harnessed this strategy to study the functional effects of noncoding variants on gene expression in Chapters 4 and 5. In Chapter 4, I characterized a novel enhancer that regulates the expression the gene encoding the massive sarcomere protein titin. Heterozygous truncating variants TTN are the most common genetic cause of dilated cardiomyopathy, which often propels heart failure. Using reporter assays, I provide new insights into the noncoding sequences and interacting transcription factors that critically control cardiomyocyte expression of titin. In Chapter 5, I describe results of genome-wide association studies (GWAS) that identified a chromosome 3p12.3 locus associated with atrial septal defects (ASD). I refined the genetic map of this locus and demonstrated a physical proximity to the ROBO2 promoter and a key CTCF binding site. Within the locus I also identified noncoding variants that regulate ROBO2 transcription that together define critical roles for ROBO2 in human heart development.