Gene Discovery for Genetic Disorders using Next Generation Sequencing and Functional Genomics

The focus of this thesis was the identification of the genetic bases of Mendelian and mitochondrial respiratory chain disorders in a cohort of paediatric patients, to better understand their pathogenesis. Genetic disorders are caused by mutations in the mitochondrial or nuclear genomes and may be influenced to a lesser or greater degree by environmental factors. To date, nearly 3000 genes have been implicated in ~ 4,400 Mendelian phenotypes. However, despite this, the genetic bases for almost 50% of all known Mendelian phenotypes remains to be definitively elucidated. Mitochondrial respiratory chain disorders are the most common group of inborn errors of metabolism and can be caused by mutations in either mitochondrial DNA or nuclear DNA. The genetic heterogeneity of these disorders makes diagnosis challenging, adding distress to families already dealing with the trauma of an extremely ill family member. Mutations in mitochondrial DNA or nuclear DNA genes can result in impaired function of the respiratory chain causing broad symptoms including neuropathy, cardiomyopathy, muscle weakness, fatigue, cognitive impairment, visual and auditory impairment, to name a few. Despite the advances in gene screening techniques, the genetic bases of many respiratory chain disorders remains unidentified. This study had two phases: identification of the likely disease-causing variants in paediatric patients with suspected Mendelian or mitochondrial inherited disorders due to mitochondrial or nuclear DNA mutations, and implementation of functional studies to confirm pathogenicity and gain insights into possible disease mechanisms of the identified variants. Nine patients with suspected Mendelian disorders and two patients with a suspected mitochondrial disorder were studied in this project. Using whole exome sequencing (WES) in collaboration with other institutes or groups within Australia and overseas, we were able to efficiently identify the genetic basis of Mendelian and mitochondrial respiratory chain disorders in the majority of the paediatric patients studied in this project. In collaboration with bioinformaticians and clinician colleagues, we implemented sophisticated filtering pipelines, with candidate causative variants being narrowed down from the very expansive WES data. We then performed functional assays to determine the functional impact of the identified variants. These functional assays included immunoblotting and blue native polyacrylamide gel electrophoresis to measure protein expression and assembly. Further, in the case of the mitochondrial respiratory chain disorders, we measured the effect of the variant on the protein levels of mitochondrial respiratory chain complexes in patient fibroblast samples. We also measured respiratory chain complex enzyme activities using dipstick assays (for complex I and complex IV) or traditional spectrophotometric assays (for complexes I, II, III, and IV). In this PhD project, we have successfully identified four disease variants in NDUFV1 (OMIM: 161015), RARS (OMIM: 107820), GARS (OMIM: 600287), and PIGN (OMIM: 606097) and a novel variant NOX4 (OMIM: 605261) that may act as modifier in causing death in the proband. NDUFV1 encodes a 51 kDa subunit of the NADH: ubiquinone oxidoreductase complex I and was the cause of Leigh disease in one patient. Both RARS and GARS are part of the aminoacyl-tRNA synthetase family, encoding arginyl-tRNA synthetase and glycyl-tRNA synthetase proteins respectively, with mutations in the former causing a hypomyelination disorder very similar to Pelizaeus–Merzbacher disease in three patients, while the latter caused a mitochondrial respiratory chain disorder in one patient. PIGN encodes a protein that is involved in glycosylphosphatidylinositol (GPI)-anchor biosynthesis and was the cause of a neurological disorder in two patients. The NOX4 gene encodes the catalytic subunit of the NADPH oxidase complex that catalyses the reduction of molecular oxygen, mainly to hydrogen peroxide. It is possible that the NOX4 genotype we have identified may act as a modifier for the, as yet, unidentified primary genetic cause in our patient. The findings of this thesis highlight the importance of a multidisciplinary and multipronged approach to the identification of causative variants in patients with suspected Mendelian or mitochondrial respiratory chain disorders. These approaches include careful delineation of the clinical features, biochemical testing, histological analysis, and genetic investigations including WES, coupled to laboratory-based functional studies. Identification of the underlying genetic causes and understanding the resulting pathogenesis of these disorders may point to existing therapies or the development of novel therapies, and provide critical information to genetic counsellors allowing them to more effectively advise the parents of affected individuals for future family planning