Title: Can muscle biopsy be avoided for paediatric patients with suspected mitochondrial disease?
Funder: Department of Health and Social Care

Abstract: Mitochondrial disease is an inborn error of metabolism with no cure; it affects at least 1 in 4300 births. Mitochondria produce cellular energy (ATP), and their dysfunction causes marked clinical heterogeneity, affecting any organ or tissue with energy demands e.g. brain or muscle. Symptoms can arise in adulthood or childhood but age at onset often correlates with severity; children with mitochondrial disease often have a poor prognosis and few survive into adulthood. A rapid genetic diagnosis delivers significant patient impact, facilitating clinical management and counselling for families with otherwise limited options.The diagnosis of mitochondrial disease is hindered by genetic heterogeneity; more than 1100 genes encode the mitochondrial proteome, including 280 known disease genes reported to date. The clinical and genetic heterogeneity necessitates a multidisciplinary approach to diagnosis and this is delivered by the NHS-funded Highly Specialised Mitochondrial Diagnostic Service in Newcastle upon Tyne. The multidisciplinary diagnostic service is complemented by the translational research of the Wellcome Centre for Mitochondrial Research; I recently completed my NIHR-funded doctoral project across both centres. I successfully implemented a rapid targeted next generation sequencing (NGS) test (n=50 genes) that increased the genetic diagnosis rate to 50% (n=35) for a cohort of mitochondrial disease patients. Prenatal testing has already been undertaken for 13 couples as a consequence of my NIHR-funded research, demonstrating the impact of a genetic diagnosis.Our nationally-comissioned Highly Specialised mitochondrial diagnostic service receives 140 paediatric muscle biopsies annually; results from the functional analysis of muscle biopsy remains paramount for selecting appropriate candidate genes for sequencing analysis for genetic diagnosis. Muscle biopsy is invasive, expensive and requires critically ill children to undergo general anaesthetic and upon completion of the candidate gene analysis, many patients still remain without a genetic diagnosis. Our current diagnostic strategy (muscle biopsy proceeded by candidate gene sequencing) fails to genetically diagnose half of our paediatric cohort, and this needs to be addressed. This research proposal aims to do this by implementing whole exome sequencing of trios (affected patients and their parents) as a first line diagnostic genetic test, concurrently removing the requirement for muscle biopsy for these patients.Whole exome sequencing (WES) provides an unbiased strategy, whereby all candidate genes (n=1158) can be screened concurrently; through analysis of trios, phasing of variants and detection of sporadic variants becomes achievable. By using blood DNA, candidate gene selection is bypassed and removes the need for muscle biopsy.Muscle biopsy has historically been critical for functional studies, but emerging cell-based strategies such as CRISPR/Cas9 have demonstrated potential as a surrogate tissue and I propose to harness this technology to generate cellular models of patient mutations to investigate their clinical utility in the diagnostic setting. In the post-genomic era, the issue of classification of variants of uncertain significance (VUS) requires increasing scientist time; development of novel systems to investigate pathogenicity will be paramount and this arm of the project seeks to future-proof our laboratory, ensuring we are prepared to cope with the VUS that emerge from our patients' NGS.

Lay abstract: Childhood mitochondrial disease is a life-limiting condition. It is caused by faulty genes and affects at least 1 in 4,300 births. There is no cure.Mitochondria are the powerhouses of the cell, generating the energy that the body needs. When mitochondria fail, through a faulty gene, any part of the body that requires energy to work can be affected. The parts of the body requiring the most energy are worst affected (e.g. brain and muscle). Mitochondrial disease symptoms can emerge at any age but the earlier they appear, the worse the outcome tends to be. Some babies develop symptoms at birth, whilst others are seemingly healthy until they reach infancy or older. Although they may have attained developmental milestones, such as sitting, they then lose these abilities and this has profound impacts on them and their parents; many affected patients die during early childhood.For many patients, confirming a diagnosis of mitochondrial disease involves taking a piece of muscle, often from their leg. Clearly this is invasive and whilst straightforward for adults who can remain still throughout, children require general anaesthetic which has a risk of complications, particularly when they are already critically ill. Despite the risks, many children with mitochondrial disease undergo muscle biopsy to achieve a diagnosis.Many common genetic disorders (e.g. Duchenne Muscular Dystrophy) are caused by faults in a single gene. Identifying the cause of such disorders is therefore relatively straightforward. Mitochondrial disease is not like this and one of over 1000 potentially faulty genes could be responsible. Muscle tests tell us where we should start looking, but it can take years to find the faulty gene responsible.Most children with mitochondrial disease have healthy parents, but each of them will have one faulty and one normal copy of the gene. These parents have a 1 in 4 chance of having another child with mitochondrial disease, i.e. 25% per pregnancy. However, if we can identify the faulty gene in these couples, we can offer the family options:1) We can test their baby early in pregnancy - if the baby has mitochondrial disease, the parents can decide whether to terminate their pregnancy or prepare for their baby's arrival, who may be very poorly. 2) We can offer IVFbased tests that can allow us to screen couples' embryos at a very early stage of development; only babies with a low risk of mitochondrial disease are transferred to the mother's womb to implant and grow into a healthy child.To be able to do either of the above, we must know what the faulty gene is. If we don't, the only choice for these parents is whether or not to take the risk.My previous NIHR-funded research developed a new 50 gene test which found the first faults in a new mitochondrial gene prompting international media interest that raised awareness of mitochondrial disease. I identified the faulty gene for 35 children and pregnancy testing has already been performed for 13 couples, reinforcing the importance of a genetic diagnosis for these families.Unfortunately, that test still needs clues from muscle to be effective. I want to find the fault without putting a poorly child through general anaesthetic and muscle biopsy. By using blood from the child and both parents I can check every possible gene for faults without the need for patient muscle. I can even use cutting-edge tools to make patient muscle; by creating cells in the lab that behave just like the patient's I can study the effect of the fault without patient muscle. This proposed research will transform the diagnosis of mitochondrial disease for many families.

Research activity codes: 2.1 BIOLOGICAL AND ENDOGENOUS FACTORS;4.2 EVALUATION OF MARKERS AND TECHNOLOGIES
Health categories: CONGENITAL DISORDERS