Characterization of novel genetic variants causing mitochondrial disease

Background. The motivation for this thesis is the desire to improve diagnostics, to increase knowledge about mitochondrial diseases and thus be able to help more patients. To give the patients an explanation for their illness, in some cases offering treatment and in other cases offering prenatal diagnostics to families who have lost a child is incredibly rewarding. Over the years, a lot of unclear variants have been detected in clinical genetic diagnostics at my workplace, Centre for Inherited Metabolic Diseases (CMMS) at Karolinska University Hospital. With the introduction of next-generation sequencing (NGS) about 12 years ago, the number of solved cases has increased exponentially but this is also true for the number of unclear variants detected. In order to find out whether a variant is disease-causing in the patient, the following questions must be addressed. Is the variant rare enough? Is the inheritance pattern as expected? What does the genetic variant lead to at the mRNA and/or protein level? What is the disease mechanism? Can the patient's clinical and biochemical picture be linked to the specific gene? In this thesis, we validate the pathogenicity of some of these variants and report the molecular, clinical, and biochemical findings in the patients.

Methods. This work had not been possible without Clinical Genomics at Science for Life Laboratory at Karolinska Institutet that has the NGS platforms for research and diagnostics and that, in close collaboration with CMMS and Clinical Genetics and Genomics at the hospital, has developed all necessary bioinformatic tools needed for handling the enormous amount of data generated and the annotation and sorting of detected variants. The collaboration is called Genomic Medicine Center Karolinska (GMCK) and has the mission to enable the implementation of genomics-based diagnostics into Swedish healthcare. For validation we have used Drosophila melanogaster as an animal model, primary cells (fibroblasts and myoblasts) from the patients, different in-vitro and biochemical assays, immunostaining, Western and Southern blots, Blue Native Gel electrophoresis and RNA sequencing.

Results. A novel variant, p.Tyr955His, in the mitochondrial DNA polymerase POLYA was shown to have a dominant negative effect on mtDNA synthesis and ligation and was found to cause an early-onset severe disease. The mtDNA variant m.10372G>A in the gene for mitochondrially encoded NADH: ubiquinone oxidoreductase core subunit 3 (MT-ND3) caused a severe complex I deficiency in skeletal muscle and led to an adult-onset polyneuropathy. In addition we describe a new disease gene, NDUFB7, in this thesis. The causative variant was an intronic variant that resulted in abnormal splicing of mRNA and absence of the protein in cells from the patient. Rescue experiments were performed to connect the complex I deficiency to NDUFB7. In the last study several synonymous and intronic variants in the genes PDHA1, PDHX and TPK1 were shown to lead to exon skipping or intronic inclusions and to cause primary or secondary pyruvate dehydrogenase deficiency in the patients.

Significance. For the patients and the families included in the studies the importance is to get a genetic diagnosis, to obtain a better prognosis for the course of the disease and specific genetic counseling. The genetic diagnosis can guide the treatment, e.g. ketogenic diet in patients with pyruvate dehydrogenase deficiency, thiamine supplementation in thiamine pyrophosphokinase deficiency but also the choice of antiepileptic drugs and to enable prenatal diagnostics in future pregnancies. In a larger context the thesis contributes to increase knowledge about novel variants, disease genes, disease mechanisms, and phenotypes associated with mitochondrial disorders.

List of scientific papers

I. A multi-systemic mitochondrial disorder due to a dominant p.Y955H disease variant in DNA polymerase gamma. Siibak T, Clemente P, Bratic A, Bruhn H, Kauppila TES, Macao B, Schober FA, Lesko N, Wibom R, Naess K, Nennesmo I, Wedell A, Peter B, Freyer C, Falkenberg M, Wredenberg A Hum Mol Genet. 2017 Jul 1;26(13):2515-2525
https://doi.org/10.1093/hmg/ddx146

II. Novel Mutation m.10372A>G in MT-ND3 Causing Sensorimotor Axonal Polyneuropathy Bruhn H, Samuelsson K, Schober FA, Engvall M, Lesko N, Wibom R, Nennesmo I, Calvo-Garrido J, Press R, Stranneheim H, Freyer C, Wedell A, Wredenberg A Neurol Genet. 2021 Mar 15;7(2):e566.
https://doi.org/10.1212/nxg.0000000000000566

III. Severe congenital lactic acidosis and hypertrophic cardiomyopathy caused by an intronic variant in NDUFB7. Correia SP, Moedas MF, Naess K, Bruhn H, Maffezzini C, Calvo- Garrido J, Lesko N, Wibom R, Schober FA, Jemt A, Stranneheim H, Freyer C, Wedell A, Wredenberg A Hum Mutat. 2021 Apr;42(4):378-384.
https://doi.org/10.1002/humu.24173

IV. Novel Synonymous and Deep Intronic Variants Causing Primary and Secondary Pyruvate Dehydrogenase Complex Deficiency. Bruhn H, Naess K, Ygberg S, Peña-Pérez L, Lesko N, Wibom R, Freyer C, Stranneheim H, Wedell A, Wredenberg A Hum Mutat. 2024
https://doi.org/10.1155/2024/1611838