Genetic and Functional Studies of Human Ciliary Syndromes

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DESCRIPTION (provided by applicant): The ciliopathies are an emerging group of clinically overlapping disorders, hallmarked by features that include retinal degeneration, renal cystic disease, and central and peripheral nervous system defects. The unification of diverse clinical phenotypes such as nephronophthisis, Bardet-Biedl, Meckel-Gruber and Jeune syndromes is driven by both common cellular basis (defects at the primary cilium) and genetic overlap, where mutations in the same genes can both cause discrete phenotypes and contribute modifying mutations that modulate the penetrance and expressivity of primary genetic lesions. These observations have led to the idea that the total mutational load in the primary cilium is a strong phenotypic determinant. Moreover, robust, physiologically relevant in vitro and in vivo assays that report on organelle output affords us the opportunity to study the nature and properties of second-site modifiers in humans. During the first funding period, we established the ciliopathies as a unified entity, identified several causal and modifying genes and alleles, and developed assays to evaluate their effect. We now propose to expand on this work and, for the first time, assess the total mutational load in a discrete, biochemically characterized macromolecular system ciliary functional system. We will 1) sequence a large cohort of patients across the severity spectrum for all known intraflagellar transport genes and ask how phenotypic severity might track with genetic lesions; 2) we will functionally assay all discovered alleles using a combination of in vivo complementation and in vitro protein stability and localization assays in ciliated cells; 3) finally, we will begin to interrogate the significance of such alleles in mammals. As part of our Preliminary Data, we have identified a missense allele, P209L, in IFT139 that appears genetically sufficient to cause cystic renal disease but also interacts with alleles in other ciliopathy genes as a potential cystogenic modifier. We will therefore engineer a knock-in mouse and ask a) whether homozygosity for this mutation is sufficient for renal cyst formation; and b) whether introduction of 209L in a genetically sensitized background can exacerbate or potentiate cystic renal disease. Our studies will yield additional loci that cause ciliary disease and expand our knowledge base of second-site modifiers. Moreover, we anticipate that understanding of the genetic attributes of epistasis has the potential to inform a broad range of disorders and improve the clinical utility of genetic information. PUBLIC HEALTH RELEVANCE: Although individually rare, the ciliopathies collectively represent a significant health burden and their genetic dissection will expedite prognosis, management, and treatment. Moreover, this group of disorders is emerging as an exceptionally useful system to study the relationship between genetic load in a functional system and clinical variability, offering a unique opportunity to understand pathology and disease progression at the level of the individual patient.