Mutations in DNA2 Link Progressive Myopathy to Mitochondrial DNA Instability

Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance. Syndromes associated with multiple mtDNA deletions are due to different molecular defects that can result in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia (PEO) to multisystemic disorders of variable severity. The autosomal-dominant form of PEO is genetically heterogeneous. Recently, causative mutations have been reported in several nuclear genes that encode proteins of the mtDNA replisome machinery (POLG, POLG2, and C10orf2) or that are involved in pathways for the synthesis of deoxyribonuclotides (ANT1 and RRM2B). Despite these findings, putative mutations remain unknown in half of the subjects with PEO. We report the identification, by exome sequencing, of mutations in DNA2 in adult-onset individuals with a form of mitochondrial myopathy featuring instability of muscle mtDNA. DNA2 encodes a helicase/nuclease family member that is most likely involved in mtDNA replication, as well as in the long-patch base-excision repair (LP-BER) pathway. In vitro biochemical analysis of purified mutant proteins revealed a severe impairment of nuclease, helicase, and ATPase activities. These results implicate human DNA2 and the LP-BER pathway in the pathogenesis of adult-onset disorders of mtDNA maintenance. mtDNA is replicated and maintained in multiple copies in each cell by a specific nuclear-gene-encoded set of proteins.1Wanrooij S. Falkenberg M. The human mitochondrial replication fork in health and disease.Biochim. Biophys. Acta. 2010; 1797: 1378-1388Crossref PubMed Scopus (83) Google Scholar Although some members of the replication machinery, such as the mtDNA polymerase POLG2Stumpf J.D. Copeland W.C. Mitochondrial DNA replication and disease: Insights from DNA polymerase γ mutations.Cell. Mol. Life Sci. 2011; 68: 219-233Crossref PubMed Scopus (77) Google Scholar and the hexameric ring-shaped helicase C10orf2 (also known as Twinkle),3Sen D. Nandakumar D. Tang G.Q. Patel S.S. Human mitochondrial DNA helicase TWINKLE is both an unwinding and annealing helicase.J. Biol. Chem. 2012; 287: 14545-14556Crossref PubMed Scopus (47) Google Scholar have been identified, the roles of other proteins thought to be relevant for mtDNA synthesis and repair are still unclear. Inherited disorders of mtDNA maintenance are associated with most deleterious mitochondrial diseases, which affect people of all ages. In adults, defects in proteins involved in mtDNA replication, deoxyribonucleotide metabolism, and mitochondrial fusion can lead to the accumulation of multiple mtDNA deletions in postmitotic tissues, such as skeletal muscle. Most affected individuals develop progressive external ophthalmoplegia (PEO [MIM 157640, 609283, 609286, and 610131]), which is often accompanied by ptosis and mitochondrial myopathy; multisystemic involvement is not uncommon.4Copeland W.C. Defects in mitochondrial DNA replication and human disease.Crit. Rev. Biochem. Mol. Biol. 2012; 47: 64-74Crossref PubMed Scopus (120) Google Scholar Despite the discovery of causative mutations in several genes, a firm diagnosis can be achieved, at best, in half of patients.5Virgilio R. Ronchi D. Hadjigeorgiou G.M. Bordoni A. Saladino F. Moggio M. Adobbati L. Kafetsouli D. Tsironi E. Previtali S. et al.Novel Twinkle (PEO1) gene mutations in mendelian progressive external ophthalmoplegia.J. Neurol. 2008; 255: 1384-1391Crossref PubMed Scopus (39) Google Scholar To identify genetic defects in individuals who showed signs of mtDNA instability, we performed whole-exome next-generation sequencing6Gilissen C. Hoischen A. Brunner H.G. Veltman J.A. Unlocking Mendelian disease using exome sequencing.Genome Biol. 2011; 12: 228Crossref PubMed Scopus (208) Google Scholar in a family in which two siblings (probands 1 [P1] and 2 [P2]) developed progressive myopathy with evidence of muscle mitochondrial dysfunction(definition), as disclosed by histochemical analysis of skeletal muscle biopsy (Figure S1, available online). P1 is a 62-year-old male who first came to our attention when he was 49 years old, when we diagnosed a preexisting suspected myopathy. He had always been thin with a harmonically decreased muscle bulk and reported that his muscle strength had declined progressively over the last 20 years. A neurological examination revealed an alert person with normal cognitive function, decreased facial expressions, moderate diffuse muscle atrophy, relatively mild weakness with a predominant lower-limb-girdle distribution, an anserine gait with dorsolumbar hyperlordosis, and a positive Gowers’ sign. At the age of 54 years, he developed exertional dyspnea with obstructive sleep apnea and a patent foramen ovale with minimal shunt. Serum creatine-