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

Binnenmilieuadviezen voor nieuw te bouwen scholen : Informatieblad

Bij het bouwen van een nieuwe school kunnen GGD-medewerkers adviseren over een gezond binnenmilieu. Dit informatieblad geeft aanbevelingen hiervoor. De laatste tijd is er toenemende aandacht voor het binnenmilieu van scholen, vooral omdat uit onderzoek blijkt dat het binnenmilieu van veel bestaande basisscholen niet gunstig is. Hierdoor krijgen GGD'en vaak van schoolbesturen en -directies de vraag om informatie te leveren ten behoeve van een gezonde nieuwbouwschool. Met dit informatieblad kunnen GGD-medewerkers een bijdrage leveren in een vroege fase van het bouwproces. Voor verschillende aspecten van het binnenmilieu zijn er aanbevelingen geformuleerd, zoals voor de binnenluchtkwaliteit, thermisch comfort, schoonmaak en onderhoud. Gekozen is voor een hoog ambitieniveau om condities na te streven die zo gunstig mogelijk zijn. Dat betekent een schoolgebouw waarin het behaaglijk en efficiënt leren is met zo weinig mogelijk gezondheidsrisico's. Dit informatieblad heeft het RIVM samen met de GGD Groningen opgesteld.VW

Paternal transmission of mitochondrial DNA in interspecific crosses of Drosophila simulans

Arguably, the three central pillars mitochondrial DNA (mtDNA) evolution that make it a powerful tool for evolutionary studies are maternal inheritance, lack ofrecombination and high copy number. While it is likely that these three pillars cooccurin most higher eukaryotes it is important to consider mechanisms causing thesemainstays to fail. One such mechanism causing failure of strict maternal inheritance ispaternal leakage. The occurrence of paternal leakage followed by its transmission tooffspring may bias the interpretation of mtDNA as a molecular marker by introducingadditional haplotypes into the mtDNA pool of a single population and createindividuals with more than one type of mtDNA (heteroplasmy). The presence ofpaternal mtDNA can potentially affect a variety of disciplines including evolutionarygenetics, molecular ecology, biogeography, mitochondrial medicine, and forensicscience. Here, I examined the frequency of paternal mtDNA transmission inintraspecific crosses of Drosophila simulans harbouring distinct mtDNA haplotypes.First I optimized two primer sets. In initial optimization studies I could detect as littleas 0.1% paternal mtDNA in an individual. Second I assayed a total of 33 individualsfrom each of the 62 intraspecific crosses. Two crosses and six individuals presentstrong evidence for mtDNA paternal leakage indicating the paternal leakage was inthe order of 0.3%. The main limitations of this study were the detection levels of thespecific primers and the need to complete the reciprocal cross to corroborate theresults presented. This experiment clearly showed the notable contribution of paternalmtDNA leakage to the inheritance of mitochondria. Further study regardingestimation of sperm/oocyte content and the mechanisms leading to elimination ofpaternal mtDNA such as ubiquitination of sperm mitochondria in different speciesmay lead to better understanding of this mechanism

Tread carefully: a functional variant in the human NADPH oxidase 4 (NOX4) is not disease causing

In this study, we report a paediatric patient with a lethal phenotype of respiratory distress, failure to thrive, pancreatic insufficiency, liver dysfunction, hypertrophic cardiomyopathy, bone marrow suppression, humoral and cellular immune deficiency. To identify the genetic basis of this unusual clinical phenotype and potentially make available the option of future prenatal testing, whole exome sequencing (WES) was used followed by functional studies in a bid to confirm pathogenicity. The WES we identified a homozygous novel variant, AK298328; c.9_10insGAG; p.[Glu3dup], in NOX4 in the proband, and parental heterozygosity for the variant (confirmed by Sanger sequencing). NADPH Oxidase 4 NOX4 (OMIM 605261) encodes an enzyme that functions as the catalytic subunit of the NADPH oxidase complex. NOX4 acts as an oxygen sensor, catalysing the reduction of molecular oxygen, mainly to hydrogen peroxide (H2O2). However, although, our functional data including 60% reduction in NOX4 protein levels and a 75% reduction in the production of H2O2 in patient fibroblast extracts compared to controls was initially considered to be the likely cause of the phenotype in our patient, the potential contribution of the NOX4 variant as the primary cause of the disease was clearly excluded based on following pieces of evidence. First, Sanger sequencing of other family members revealed that two of the grandparents were also homozygous for the NOX4 variant, one of who has fibromuscular dysplasia. Second, re-evaluation of more recent variant databases revealed a high allele frequency for this variant. Our case highlights the need to re-interrogate bioinformatics resources as they are constantly evolving, and is reminiscent of the short-chain acyl-CoA dehydrogenase deficiency (SCADD) story, where a functional defect in fatty acid oxidation has doubtful clinical ramifications

Compound heterozygous mutations in glycyl-tRNA synthetase (GARS) cause mitochondrial respiratory chain dysfunction

Glycyl-tRNA synthetase (GARS; OMIM 600287) is one of thirty-seven tRNA-synthetase genes that catalyses the synthesis of glycyl-tRNA, which is required to insert glycine into proteins within the cytosol and mitochondria. To date, eighteen mutations in GARS have been reported in patients with autosomal-dominant Charcot-Marie-Tooth disease type 2D (CMT2D; OMIM 601472), and/or distal spinal muscular atrophy type V (dSMA-V; OMIM 600794). In this study, we report a patient with clinical and biochemical features suggestive of a mitochondrial respiratory chain (MRC) disorder including mild left ventricular posterior wall hypertrophy, exercise intolerance, and lactic acidosis. Using whole exome sequencing we identified compound heterozygous novel variants, c.803C > T; p.(Thr268Ile) and c.1234C > T; p.(Arg412Cys), in GARS in the proband. Spectrophotometric evaluation of the MRC complexes showed reduced activity of Complex I, III and IV in patient skeletal muscle and reduced Complex I and IV activity in the patient liver, with Complex IV being the most severely affected in both tissues. Immunoblot analysis of GARS protein and subunits of the MRC enzyme complexes in patient fibroblast extracts showed significant reduction in GARS protein levels and Complex IV. Together these studies provide evidence that the identified compound heterozygous GARS variants may be the cause of the mitochondrial dysfunction in our patient

Dipstick MRC enzyme data from cultured fibroblasts.

<p>Enzyme activity data are expressed as % residual activity relative to protein (% protein).</p

Compound heterozygous mutations in glycyl-tRNA synthetase (<i>GARS</i>) cause mitochondrial respiratory chain dysfunction - Fig 1

<p><b>A</b>) Sanger sequencing profile of <i>GARS</i> from the proband and parents showing c.803C>T; p.(Thr268Ile) variant is heterozygous in the proband and the father. <b>B)</b> Sanger sequencing profile of <i>GARS</i> from the proband and parents showing c.1234C>T; p.(Arg412Cys) variant is heterozygous in the proband and the mother. <b>C)</b> Evolutionary sequence conservations of the altered amino acid residues p.Thr268 and p. Arg412 are denoted in bold red in boxes.</p

Spectrophotometric MRC enzyme diagnostic data in skeletal muscle, liver and fibroblasts.

<p>Patient liver and muscle samples. Data are expressed as activity relative to protein and as % CS ratio, which represents % of the normal control mean value when expressed relative to Citrate Synthase. Bold characters indicate clinically significant abnormal values (H–high, L–low). Complex I (CI), NADH-coenzyme Q1 oxidoreductase; Complex II (CII), succinate-coenzyme Q1 oxidoreductase; Complex III (CIII), decylbenzylquinol-cytochrome c oxidoreductase; Complex IV, cytochrome c oxidase (CIV).</p

<i>In silico</i> analyses of the GARS variants identified in this study.

<p><i>In silico</i> analyses of the GARS variants identified in this study.</p