Investigation of the molecular genetics of renal ciliopathies

Abstract

Primary ciliopathies are a group of inherited diseases caused by dysfunction or absence of the primary cilia. They exhibit genetic heterogeneity, genetic pleiotropy and a wide spectrum of clinical phenotypes, often including renal disease. The main objective of this project is to investigate the underlying molecular disease mechanisms and phenotypic and genotypic complexity of renal ciliopathies, and to increase the genetic diagnosis yield in patients with primary ciliopathies. To achieve this, I used a combination of in vitro and in silico techniques, focussing on a selection of pleiotropic genes associated with overlapping ciliopathy phenotypes. This approach covers a broad perspective to study renal ciliopathies, including: genetic pleiotropy, heterogeneity of clinical phenotypes, genetic diagnosis yield, the use of functional analyses to validate disease-causing variants and gene therapies. The spectrum of clinical phenotypes in primary ciliopathies may reflect the tissue specificity of expression of disease genes. Consequently, gene expression of two pleiotropic genes associated with overlapping rare primary ciliopathies were characterised: CEP120 and ARL3, using the MRC Wellcome Trust Human Developmental Biology Resource (HDBR) and RNAscope, an RNA in situ detection technique. Expression patterns of both genes revealed brain, eye and kidney expression, which correlates with known clinical phenotypes. In silico approaches were evaluated to explore genotype-phenotype correlations in two primary ciliopathy genes: CEP120 and CC2D2A. These in silico approaches allowed to investigate potential exon skipping genetic therapies in these two genes. Approximately 30% of patients with a primary ciliopathy phenotype lack a genetic diagnosis. Using the data available in the Genomics England 100,000 Genomes Project, a novel primary multisystem ciliopathy syndrome in a patient with biallelic pathogenic variants in TOGARAM1 was identified. Also, within this dataset, in patients with cystic kidney disease, underlying disease-causing genetic variants in DNAJB11, ALG5, ALG8 and ALG9 were identified, supporting these genes as ciliopathy genes associated with the phenotypic spectrum of autosomal dominant polycystic kidney and liver disease. I used human urine-derived renal epithelial cells (hURECs) to characterise the ciliary phenotype of a patient with cystic kidney disease and proposed hURECs as a valuable non-invasive source of primary cells to validate candidate genes for novel renal ciliopathies. hURECs may also be used as an ex vivo model to study patient specific genetic variants, disease mechanisms and complement therapeutic studies.