Identification of Candidate Disease-Causing Variants

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Several primary immunodeficiencies, including severe combined immunodeficiency (SCID), are characterized by T cell deficits. Elucidating the etiology of T cell deficits provides opportunities to understand the biology of T cell development and inform diagnosis and treatment of individuals with T cell deficiency disorders. Current exome approaches solve <50% of cases of T cell deficiencies. Whole genome sequencing (WGS) offers an opportunity to develop a more comprehensive approach for variant discovery, with superior coverage of exons, as well as new access to noncoding sequences, allowing for interrogation of genomic regulatory variation. Interpretation of WGS is enabled by known genes and regulatory regions associated with particular disease phenotypes. To this end, in Aim 1 we will predict putative genes and regulatory regions associated with T cell disorders. These will be evaluated by CRISPR screens in Project 2 and more detailed studies in Project 3. The integrated results will provide a resource for the field of immunology, and will directly inform our diagnostic WGS interpretation. For Aim 2, we will identify candidate disease-causing variants in affected individuals, and stratify these for experimental validation and interrogation. Drawing upon the resource in Aim 1, we will establish an analysis pipeline that solves cases from probands' WGS. We will stratify our conclusions based on degree of confidence in the computational studies, in order to inform different experiments for Projects 2 and 3 or Aim 3. This pipeline will have quantitative integrative scoring, and will consider both protein altering and regulatory single nucleotide variants and small indels, as well as structural variations (especially deletions). We will prioritize variants based on severity of predicted effect, gene relevance to T cell deficiencies, and consistency with proband phenotype (from Core C). “Compelling” cases where validation of causative variants is likely, may proceed to investigations of molecular mechanism in Project 3, while “intriguing” cases yielding dozens of variants need testing via medium-scale studies in Project 2. “Mysterious” cases with numerous variants of unknown significance will first be examined through Aim 3 RNA-seq, and then proceed to Projects 2 and 3. For Aim 3, we will integrate trio RNA-seq to identify regulatory splicing and expression variants related to T cell deficiency disease. The impact of most putative regulatory variants cannot be reliably predicted today. However, transcriptome profiling, which reveals both RNA expression level and splicing events, is an effective method to assess the regulatory consequences of genetic variations. Transcriptomes from parents will be studied for inferring those of the probands, when T cells are not available due to the disease. We will identify variants associated with altered splicing, as well as regulatory variants associated with allele-specific altered expression. These data will be incorporated into the Aim 2 genome interpretation pipeline, to identify variants for further investigations in Projects 2 and 3.