Improving the genetic diagnosis of mitochondrial disease using custom next-generation sequencing strategies /

Abstract

Mitochondrial disease represents one of the most common inborn errors of metabolism with a minimum disease prevalence of ~12.5 per 100,000 (Gorman et al., 2015) in adults and ~4.7 per 100,000 in children (Skladal et al., 2003). Mitochondrial disease affects people of all ages and, although symptoms can sometimes be treated or ameliorated, there is no cure (McFarland et al., 2010). Mitochondrial disease is associated with a diverse spectrum of clinical presentations ranging from isolated symptoms such as seizures or cardiomyopathy, to severe neurological, syndromic presentations such as Leigh syndrome or Mitochondrial Encephalomyopathy, Lactic Acidosis and Stroke-like episodes (MELAS). The underlying genetic defect can be within either the mitochondria's own genome (mtDNA) - a 16.6kb DNA molecule encoding 37 genes (Anderson et al., 1981) - or the nuclear genome, which encodes ~1150 mitochondrial proteins (Calvo et al., 2016). In view of this complexity, the care and diagnostic investigation of mitochondrial patients is part of the NHS Highly Specialised Services for Mitochondrial Diseases of Adults and Children within the UK. The vast genotypic and phenotypic heterogeneity means that the diagnostic algorithm is often complicated; for a large number of patients, the underlying genetic defect remains unknown often in spite of an established respiratory chain enzyme defect. For example, half of all paediatric patients with an isolated complex I deficiency, the most common respiratory chain defect, lack a genetic diagnosis (Swalwell et al., 2011). There is little that can be offered to these families in terms of counselling and recurrence risk prediction for future pregnancies. High-throughput sequencing offers real hope for families affected by massively heterogeneous conditions; the application of these emerging technologies to "undiagnosed" patients maximises the chance of elucidating the underlying genetic defect. My research proposal focuses on improving the genetic diagnostic pathway for predominantly paediatric patients with suspected mitochondrial disease with a view to enabling their parents’ access to reproductive options for future pregnancies. I plan to expand the scope of genetic testing in our Highly Specialised Mitochondrial Diagnostic Service Laboratory by employing three approaches - Sanger sequencing of new disease genes where there are clear genotypephenotype correlations, targeted next-generation sequencing of panels of candidate genes and unbiased whole exome sequencing. Our current strategy permits analysis of <10% of all candidate genes, whereas the system detailed in my proposal will raise this percentage significantly, with scope to incorporate new disease genes as they are described. ii Collaborative research projects with international academic research institutes have meant that a number of NHS patients have already undergone exome sequencing and a molecular diagnosis has been made via this research-reliant pathway. I believe it is vital that we can provide a similar service within the NHS diagnostic framework and my proposal will move conventional diagnostics using Sanger sequencing of single genes to a futureproof high-throughput strategy. I have access to a paediatric patient cohort (n=60) with a proven biochemical defect in muscle and many more with suspected mitochondrial disease for whom our current strategy has failed to identify the causative genetic defect; this proposal aims to obtain a genetic diagnosis for these patients. There will also be a prospective element as the new high-throughput screening strategy will be applied to each new paediatric patient with a biochemical diagnosis of isolated complex I deficiency (n=30/year). Where novel mutations in known disease genes, or novel mutations in unreported candidate genes are identified, functional investigations will be undertaken to establish their pathogenicity. In the absence of a cure for mitochondrial disease, the provision of reproductive counselling is a vital resource for families affected by inherited mitochondrial disease and the current repertoire includes prenatal genetic diagnosis (chorionic villus biopsy; amniocentesis; non-invasive screening) or pre-implantation genetic diagnosis during in vitro fertilisation procedures. Moreover, determining the phenotypic effect of these genetic mutations on mitochondrial function will be vital to understanding the underlying mechanisms and pathways involved. In addition to providing a genetic diagnosis for patients (and their families), it is hoped that advances in knowledge relating to mitochondrial pathology may lead to new treatments.