Improving Genetic Diagnosis: Why and How Why: Participant Perspectives on Rare Disease Genomic Research How: Analysis of Splicing Coding and Noncoding Genes and Pseudogenes Reveals Novel Gene-Disease Relationships

Abstract

This master’s thesis on improving genetic diagnosis revolves around two projects. Project 1: Participant Perspectives on Rare Disease Genomic Research Abstract Introduction: Although advances in genomic sequencing technology have improved diagnostic capabilities, access to these technologies is variable. Furthermore, novel approaches to genomic analysis may be outside of the scope of usual clinical testing. Rare disease genomic research thus represents a potential pathway to genetic diagnosis. However, factors influencing the decision to participate in a genomic research study remain incompletely understood. We therefore investigated motivators for participation in a rare disease genomic research study and the impact of diagnostic findings. Methods: Prospective quantitative analysis of survey data from participants in the Rare Genomes Project was conducted. Surveys were sent upon enrollment to the study and at 3 and 12 months after result disclosure and included both novel items and the Perceived Stress Scale. Results: 509 participants responded out of 884 targeted surveys, either affected persons (226) or their caregivers (283). For all respondents, diagnostic importance, improved clinical understanding, future preparedness, and treatment optimization were the strongest motivators for seeking a genetic diagnosis. Perceived stress upon entry to the study was significantly higher than population norms (17.61 vs 13, P 0.00001). Variables associated with perceived diagnostic importance are mainly related to the financial burden of health conditions (OR 3.859 (95% CI 1.306–11.404), P = 0.015). At follow-up timepoints, diagnosed participants endorsed statements related to a positive impact of the diagnosis on group connections. Perceived benefit from social connectedness became more prominent over time. Conclusion: We described the psychosocial context of research enrollment for a diverse cohort of rare disease genomic research participants and identified financial problems as the prominent determinant for diagnostic importance perception. We also highlighted a potential positive impact of the genetic diagnosis in reducing the perceived stress and shifting feelings towards genetic diagnosis from vigilance to acceptance. Project 2: Analysis of Splicing Coding and Noncoding Genes and Pseudogenes Reveals Novel Gene-Disease Relationships Abstract Splicing is a complex molecular mechanism occurring in each living human cell where the precursor messenger RNA (pre-mRNA) is processed into the mature messenger RNA (mRNA). It involves more than 300 protein-coding genes (PCGs) and around 43 small nuclear RNA (snRNA) genes. However, fewer than 30 diseases have been described as spliceosomopathies to date. This discrepancy points towards splicing machinery as an underexplored area for genetic discoveries. Furthermore, there are almost 1700 spliceosome-related snRNAs pseudogenes that are overlooked in diagnostic analyses, but recently a novel gene-disease relationship has led to reclassification of a snRNA pseudogene to a gene associated with a developmental and epileptic encephalopathy, highlighting the importance of further evaluation of snRNA pseudogenes. We evaluated both coding and noncoding snRNA genes and their pseudogenes using adapted approaches to each gene category. Analysis strategies involved evaluating heterozygous variants in PCGs with loss of function constraint (pLI > 0.9) and in snRNAs in variant depleted regions in gnomAD, with biallelic variants reviewed throughout both gene sets. For snRNA pseudogenes, we prioritized candidates having similar epigenomic, genomic, and hypermutability features to functional snRNA genes. These signals, located upstream, downstream, and within a gene, indicate the likelihood of it being a functional gene. For around 23,000 families with rare disease sequenced through the GREGoR consortium, we identified 30 variants of interest across 9 PCGs with established gene-disease relationships (GDRs) and 13 genes not yet associated with disease, including one pseudogene. Of note, one gene, CWC25, had candidate variants with dominant and recessive inheritance patterns. For snRNAs genes, we identified 55 variants of interest located in seven established GDR and 11 genes not yet associated with disease, including two pseudogenes, prioritized by one or two of the prioritization strategies. Our results expanded the phenotypic expression of one variant located in the three-way junction in RNU4-2 gene from retinal dystrophy (RD) to RD and Neurodevelopmental disorder (NDD). We also suggested pleiotropy expansion for RNU6 variants with NDD and oculo-muscular fibrosis. Finally, we identified biallelic variants in RNU5 family, suggesting inheritance pattern extension. This study highlighted the importance of splicing-related PCG and snRNA in rare disease diagnosis. The genes prioritized through this research represent selected candidates for future confirmatory studies.