Determining the genetic basis of mitochondrial complex I deficiency in children using massively parallel sequencing

© 2019 Dr. Shalini ThirukeswaranMitochondrial oxidative phosphorylation (OXPHOS) disorders constitute the largest group of inborn errors of metabolism. Complex I (CI) deficiency is the most common enzymatic defect identified within mitochondrial OXPHOS disorders, accounting for ~30% of cases. The genetics and clinical spectrum of CI deficiency are extremely heterogeneous, due to participation of both the nuclear and mitochondrial genomes and involvement of at least 44 subunits and an unknown number of assembly and modification factors. Several inheritance patterns (autosomal recessive, X-linked and maternal through the mitochondrial DNA; mtDNA) have been observed in CI deficient patients. Even though over 40 genes have been found to underlie the cause of CI deficiency, around 40% of patients still remain without a molecular diagnosis. Establishing genetic diagnoses for the remaining proportion of CI deficient patients is essential in terms of delivering prenatal diagnosis, appropriate genetic counselling and to aid in the understanding of this group of disorders to translate research into potential therapies. In 2010, we published a cohort of 103 patients with biochemically defined CI deficiency. At the beginning of this PhD project, this cohort had expanded to 135 patients of whom 65 remained without a molecular diagnosis. This cohort received whole exome sequencing (WES) with additional baits to capture the entire mtDNA. Due to Human Research Ethics Committee (HREC) approval constraints, analysis was limited to a panel of ~2300 genes encoding proteins which are known to make up the mitochondrial proteome, and differential diagnosis genes for overlapping clinical phenotypes. The data also underwent ExomeDepth analysis to predict the presence of copy number variants (CNV). Various DNA, RNA and protein-based approaches were employed to determine the pathogenicity of variants in both known and potential novel disease genes. My PhD study has provided a genetic diagnosis to 32 of the 65 undiagnosed patients (49%) in 20 known disease genes. These include seven with mutations in the CI assembly factor ACAD9, five in different mitochondrial translational proteins and interestingly, four in differential diagnosis genes. It has also expanded the mutational spectrum associated with ACAD9-related disease outside the coding region. This includes identification of a deep-intronic mutation that creates a novel donor splice site motif and leads to aberrant splicing and a complex rearrangement (a paired deletion inversion). The utility of linked-read sequencing technology in generating synthetic long reads to identify and characterise complex genomic rearrangements was also demonstrated by this study. Further, the utility of massively parallel sequencing (MPS) in a clinical diagnostic setting and the sensitivity of respiratory chain enzyme assays on diagnosing CI deficiency were also compared within this cohort study. In total, 76% (102 patients) of our entire CI cohort now have a molecular diagnosis with mutations occurring in 37 different disease genes and another eight patients have had candidate disease-causing variants identified for further follow-up. My results expand upon the knowledge of the molecular basis behind CI deficiency and further emphasise the use of MPS approaches supplemented with relevant follow up studies to obtain maximum diagnostic yield in patients with mitochondrial disorders