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Additional file 4. Review history.

Additional file 2. LostArc Reports.

Additional file 1: Results. Figure S1. Mitochondrial DNA content during the LostArc procedure. Figure S2. Internal controls for validation of the LostArc method. Figure S3. The COX-ve count is poorly explained by deletions in Complex IV genes alone. Figure S4. LostArc Report example #1: weighted mean of three HEK samples. Figure S5. LostArc Report example #2: young Gwt sample M01. Figure S6. Example fits to alternative replication/deletion models. Table S1. Sample list and deletion mapping statistics. Table S2. Patient symptoms and references associated with POLG variant samples [99–104].

Additional file 3. Arc Maps.

Objective Single, large‐scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large‐scale mtDNA deletions in skeletal muscle. Methods We investigated 23 muscle biopsies taken from adult patients (6 males/17 females with a mean age of 43 years) with characterized single, large‐scale mtDNA deletions. Mitochondrial respiratory chain deficiency in skeletal muscle biopsies was quantified by immunoreactivity levels for complex I and complex IV proteins. Single muscle fibers with varying degrees of deficiency were selected from 6 patient biopsies for determination of mtDNA deletion level and copy number by quantitative polymerase chain reaction. Results We have defined 3 “classes” of single, large‐scale deletion with distinct patterns of mitochondrial deficiency, determined by the size and location of the deletion. Single fiber analyses showed that fibers with greater respiratory chain deficiency harbored higher levels of mtDNA deletion with an increase in total mtDNA copy number. For the first time, we have demonstrated that threshold levels for complex I and complex IV deficiency differ based on deletion class. Interpretation Combining genetic and immunofluorescent assays, we conclude that thresholds for complex I and complex IV deficiency are modulated by the deletion of complex‐specific protein‐encoding genes. Furthermore, removal of mt‐tRNA genes impacts specific complexes only at high deletion levels, when complex‐specific protein‐encoding genes remain. These novel findings provide valuable insight into the pathogenic mechanisms associated with these mutations. Ann Neurol 2018;83:115–130

PurposeBiallelic mutations in SCYL1 were recently identified as causing a syndromal disorder characterized by peripheral neuropathy, cerebellar atrophy, ataxia, and recurrent episodes of liver failure. The occurrence of SCYL1 deficiency among patients with previously undetermined infantile cholestasis or acute liver failure has not been studied; furthermore, little is known regarding the hepatic phenotype.MethodsWe aimed to identify patients with SCYL1 variants within an exome-sequencing study of individuals with infantile cholestasis or acute liver failure of unknown etiology. Deep clinical and biochemical phenotyping plus analysis of liver biopsies and functional studies on fibroblasts were performed.ResultsSeven patients from five families with biallelic SCYL1 variants were identified. The main clinical phenotype was recurrent low γ-glutamyl-transferase (GGT) cholestasis or acute liver failure with onset in infancy and a variable neurological phenotype of later onset (CALFAN syndrome). Liver crises were triggered by febrile infections and were transient, but fibrosis developed. Functional studies emphasize that SCYL1 deficiency is linked to impaired intracellular trafficking.ConclusionSCYL1 deficiency can cause recurrent low-GGT cholestatic liver dysfunction in conjunction with a variable neurological phenotype. Like NBAS deficiency, it is a member of the emerging group of congenital disorders of intracellular trafficking causing hepatopathy.Genet Med advance online publication, 8 February 2018; doi:10.1038/gim.2017.260.

Distinct clinical syndromes have been associated with pathogenic MT-ATP6 variants. In this cohort study, we identified 125 individuals (60 families) including 88 clinically affected individuals and 37 asymptomatic carriers. Thirty-one individuals presented with Leigh syndrome and 7 with neuropathy ataxia retinitis pigmentosa. The remaining 50 patients presented with variable nonsyndromic features including ataxia, neuropathy, and learning disability. We confirmed maternal inheritance in 39 families and demonstrated that tissue segregation patterns and phenotypic threshold are variant dependent. Our findings suggest that MT-ATP6-related mitochondrial DNA disease is best conceptualized as a mitochondrial disease spectrum disorder and should be routinely included in genetic ataxia and neuropathy gene panels. ANN NEUROL 2019;86:310-315.

Maternally inherited mitochondrial DNA (mtDNA) mutations cause symptoms of Leber hereditary optic neuropathy (LHON) in ;1 in 30,000 individuals. Most of the affected individuals lack respiratory chain defects1 and there is no proven prophylactic treatment. We identified 2 families (figure 1A) and 1 singleton case (appendix e-1 on the Neurology® Web site at Neurology.org) harboring the m.13051G.A pathogenic mtDNA mutation.2 This mutation was homoplasmic (figure e-1) but no respiratory chain defect was apparent in skeletal muscle (figure e-2, table e-1). Three children were severely affected by lactic acidosis: 2 with Leigh syndrome (patients 1 and 2; figure 1B) and 1 with a Leigh-like phenotype (patient 5). Previous authors have shown that mtDNA and mitochondrial mass are increased in individuals harboring LHON mutations.3 They suggested that an upregulation of mitochondrial biogenesis is protective, as the highest mitochondrial content was found in symptom-free carriers.3 We believe this increase in biogenesis reflects heightened mitochondrial turnover and therefore investigated mitophagy, a cellular mechanism whereby redundant or dysfunctional mitochondria are recycled.

Mitochondrial diseases affect >1 in 7500 live births and may be due to mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Genetic counselling for families with mitochondrial diseases, especially those due to mtDNA mutations, provides unique and difficult challenges particularly in relation to disease transmission and prevention. We have experienced an increasing demand for prenatal diagnostic testing from families affected by mitochondrial disease since we first offered this service in 2007. We review the diagnostic records of the 62 prenatal samples (17 mtDNA and 45 nDNA) analysed since 2007, the reasons for testing, mutation investigated and the clinical outcome. Our findings indicate that prenatal testing for mitochondrial disease is reliable and informative for the nuclear and selected mtDNA mutations we have tested. Where available, the results of mtDNA heteroplasmy analyses from other family members are helpful in interpreting the prenatal mtDNA test result. This is particularly important when the mutation is rare or the mtDNA heteroplasmy is observed at intermediate levels. At least 11 cases of mitochondrial disease were prevented following prenatal testing, 3 of which were mtDNA disease. On the basis of our results, we believe that prenatal testing for mitochondrial disease is an important option for couples where appropriate genetic analyses and pre/post-test counselling can be provided.

Pathogenic mitochondrial tRNA (mt-tRNA) gene mutations represent a prominent cause of primary mitochondrial DNA (mtDNA)-related disease despite accounting for only 5%-10% of the mitochondrial genome.(1,2) Although some common mt-tRNA mutations, such as the m.3243A>G mutation, exist, the majority are rare and have been reported in only a small number of cases.(3) The MT-TP gene, encoding mt-tRNA(Pro), is one of the less polymorphic mt-tRNA genes, and only 5 MT-TP mutations have been reported as a cause of mitochondrial muscle disease to date (table e-1 at Neurology.org/ng, P6-10). We report 5 patients with myopathic phenotypes, each harboring different pathogenic mutations in the MT-TP gene, highlighting the importance of MT-TP mutations as a cause of mitochondrial muscle disease and the requirement to study clinically relevant tissue.

Single, large-scale deletions of mitochondrial DNA are an important cause of mitochondrial disease, with a broad phenotypic spectrum. Grady et al. report that disease severity and progression are correlated with the size of the deletion, its location within the genome, and the deletion heteroplasmy level in skeletal muscle., Single, large-scale deletions of mitochondrial DNA are a common cause of mitochondrial disease and cause a broad phenotypic spectrum ranging from mild myopathy to devastating multi-system syndromes such as Kearns-Sayre syndrome. Studies to date have been inconsistent on the value of putative predictors of clinical phenotype and disease progression such as mutation load and the size or location of the deletion. Using a cohort of 87 patients with single, large-scale mitochondrial DNA deletions we demonstrate that a variety of outcome measures such as COX-deficient fibre density, age-at-onset of symptoms and progression of disease burden, as measured by the Newcastle Mitochondrial Disease Adult Scale, are significantly (P < 0.05) correlated with the size of the deletion, the deletion heteroplasmy level in skeletal muscle, and the location of the deletion within the genome. We validate these findings with re-analysis of 256 cases from published data and clarify the previously conflicting information of the value of these predictors, identifying that multiple regression analysis is necessary to understand the effect of these interrelated predictors. Furthermore, we have used mixed modelling techniques to model the progression of disease according to these predictors, allowing a better understanding of the progression over time of this strikingly variable disease. In this way we have developed a new paradigm in clinical mitochondrial disease assessment and management that sidesteps the perennial difficulty of ascribing a discrete clinical phenotype to a broad multi-dimensional and progressive spectrum of disease, establishing a framework to allow better understanding of disease progression.

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i⁶A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i⁶A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i⁶A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i⁶A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i⁶A37 modification. These data demonstrate that deficiencies of i⁶A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs. publishedVersion