Molecular diagnosis in inherited polycystic kidney disease

Polycystic Kidney Disease (PKD) incorporates a number of genetically but not always phenotypically distinct inherited cystic kidney disorders. Autosomal Dominant Polycystic Kidney Disease (ADPKD) is the most frequent, with an estimated prevalence of 1:1000. Though common, there are numerous challenges in clinical and molecular diagnosis of ADPKD due to clinical heterogeneity and sequence specific challenges related to pseudogenes that share >97% homology to the main disease gene (PKD1). This thesis addresses the sequencing and variant interpretation challenges encountered in the molecular diagnosis of PKD. In addition, mechanisms of cyst formation are explored. These studies demonstrate that Whole Genome Sequencing (WGS) is able to overcome pseudogene homology without the prior amplification or capture methods that previous techniques have required. The WGS technique was translated into clinical practice as an accredited diagnostic test after validation studies demonstrated 100% sensitivity and specificity in identifying germline variants in PKD1 and PKD2, the main genes implicated in ADPKD. Data from the first 85 patients to undergo diagnostic sequencing demonstrated that clinically reportable variants were identified in 91% of patients with a typical ADPKD phenotype and in 56% of patients in whom the clinician was unable to make a diagnosis based on phenotype alone. To improve interpretation of the variants identified through diagnostic sequencing, the frequency and pathogenicity of ADPKD-related variants found in disease and reference datasets was analysed. This showed that many previously implicated disease-causing ADPKD variants are more common than disease prevalence would predict, challenging their pathogenicity, penetrance or our estimates of ADPKD prevalence. Finally, massively parallel deep-sequencing was applied to kidney cyst tissue with the aim of identifying second-hit somatic variants that are hypothesised to be implicated in cyst pathogenesis. This analysis highlighted important challenges in interpreting data from high depth sequencing. These related to potential amplification bias and distinguishing true positive from false positive variants. Genetic diagnosis in PKD guides treatment decisions, family planning, cascade screening and assessment of living kidney donors. This thesis describes the first application of WGS in ADPKD and demonstrates the validity and utility of integrating genomics into clinical care for families with this common disease