Search

Refereed/Peer-reviewed Australian Genomics is a national collaborative partnership of more than 100 organizations piloting a whole-of-system approach to integrating genomics into healthcare, based on federation principles. In the first five years of operation, Australian Genomics has evaluated the outcomes of genomic testing in more than 5,200 individuals across 19 rare disease and cancer flagship studies. Comprehensive analyses of the health economic, policy, ethical, legal, implementation and workforce implications of incorporating genomics in the Australian context have informed evidence-based change in policy and practice, resulting in national government funding and equity of access for a range of genomic tests. Simultaneously, Australian Genomics has built national skills, infrastructure, policy, and data resources to enable effective data sharing to drive discovery research and support improvements in clinical genomic delivery.

Primary mitochondrial diseases are a group of genetically and clinically heterogeneous disorders resulting from oxidative phosphorylation (OXPHOS) defects. COX11 encodes a copper chaperone that participates in the assembly of complex IV and has not been previously linked to human disease. In a previous study, we identified that COX11 knockdown decreased cellular adenosine triphosphate (ATP) derived from respiration, and that ATP levels could be restored with coenzyme Q

Premature ovarian insufficiency (POI) is a common cause of infertility in women, characterised by amenorrhea and elevated FSH under the age of 40 years. In some cases, POI is syndromic in association with other features such as sensorineural hearing loss in Perrault syndrome. POI is a heterogeneous disease with over 80 causative genes known so far; however, these explain only a minority of cases. Using whole-exome sequencing (WES), we identified a MRPL50 homozygous missense variant (c.335T > A; p.Val112Asp) shared by twin sisters presenting with POI, bilateral high-frequency sensorineural hearing loss, kidney and heart dysfunction. MRPL50 encodes a component of the large subunit of the mitochondrial ribosome. Using quantitative proteomics and western blot analysis on patient fibroblasts, we demonstrated a loss of MRPL50 protein and an associated destabilisation of the large subunit of the mitochondrial ribosome whilst the small subunit was preserved. The mitochondrial ribosome is responsible for the translation of subunits of the mitochondrial oxidative phosphorylation machinery, and we found patient fibroblasts have a mild but significant decrease in the abundance of mitochondrial complex I. These data support a biochemical phenotype associated with MRPL50 variants. We validated the association of MRPL50 with the clinical phenotype by knockdown/knockout of mRpL50 in Drosophila, which resulted abnormal ovarian development. In conclusion, we have shown that a MRPL50 missense variant destabilises the mitochondrial ribosome, leading to oxidative phosphorylation deficiency and syndromic POI, highlighting the importance of mitochondrial support in ovarian development and function.

MRPL39 encodes one of 52 proteins comprising the large subunit of the mitochondrial ribosome (mitoribosome). In conjunction with 30 proteins in the small subunit, the mitoribosome synthesizes the 13 subunits of the mitochondrial oxidative phosphorylation or OXPHOS system encoded by mitochondrial DNA. We used multi-omics and gene matching to identify three unrelated individuals with biallelic variants in MRPL39 presenting with multisystem diseases with severity ranging from lethal, infantile onset (Leigh syndrome spectrum) to milder with survival into adulthood. Clinical exome sequencing of known disease genes failed to diagnose these patients; however quantitative proteomics identified a specific decrease in the abundance of large but not small mitoribosomal subunits in fibroblasts from the two patients with severe phenotype. Re-analysis of exome sequencing led to the identification of candidate single heterozygous variants in mitoribosomal genes MRPL39 (both patients) and MRPL15. Genome sequencing identified a shared deep intronic MRPL39 variant predicted to generate a cryptic exon, with transcriptomics and targeted studies providing further functional evidence for causation. The patient with milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detection of protein signatures and in characterization of gene-disease associations in exome-unsolved patients. We describe Relative Complex Abundance analysis of proteomics data, a sensitive method that can identify defects in OXPHOS disorders to a similar or greater sensitivity to the traditional enzymology. Relative Complex Abundance has potential utility for functional validation or prioritization in many hundreds of inherited rare diseases where protein complex assembly is disrupted.

Complex I deficiency, the most common respiratory chain defect, is genetically heterogeneous: mutations in 8 nuclear and 7 mitochondrial DNA genes encoding complex I subunits have been described. However, these genes account for disease in only a minority of complex I–deficient patients. We investigated whether there may be an unknown common gene by performing functional complementation analysis of cell lines from 10 unrelated patients. Two of the patients were found to have mitochondrial DNA mutations. The other 8 represented 7 different (nuclear) complementation groups, all but 1 of which showed abnormalities of complex I assembly. It is thus unlikely that any one unknown gene accounts for a large proportion of complex I cases. The 2 patients sharing a nuclear complementation group had a similar abnormal complex I assembly profile and were studied further by homozygosity mapping, chromosome transfers, and microarray expression analysis. NDUFS6, a complex I subunit gene not previously associated with complex I deficiency, was grossly underexpressed in the 2 patient cell lines. Both patients had homozygous mutations in this gene, one causing a splicing abnormality and the other a large deletion. This integrated approach to gene identification offers promise for identifying other unknown causes of respiratory chain disorders.

BackgroundClinical exome sequencing typically achieves diagnostic yields of 30%–57.5% in individuals with monogenic rare diseases. Undiagnosed diseases programmes implement strategies to improve diagnostic outcomes for these individuals.AimWe share the lessons learnt from the first 3 years of the Undiagnosed Diseases Program-Victoria, an Australian programme embedded within a clinical genetics service in the state of Victoria with a focus on paediatric rare diseases.MethodsWe enrolled families who remained without a diagnosis after clinical genomic (panel, exome or genome) sequencing between 2016 and 2018. We used family-based exome sequencing (family ES), family-based genome sequencing (family GS), RNA sequencing (RNA-seq) and high-resolution chromosomal microarray (CMA) with research-based analysis.ResultsIn 150 families, we achieved a diagnosis or strong candidate in 64 (42.7%) (37 in known genes with a consistent phenotype, 3 in known genes with a novel phenotype and 24 in novel disease genes). Fifty-four diagnoses or strong candidates were made by family ES, six by family GS with RNA-seq, two by high-resolution CMA and two by data reanalysis.ConclusionWe share our lessons learnt from the programme. Flexible implementation of multiple strategies allowed for scalability and response to the availability of new technologies. Broad implementation of family ES with research-based analysis showed promising yields post a negative clinical singleton ES. RNA-seq offered multiple benefits in family ES-negative populations. International data sharing strategies were critical in facilitating collaborations to establish novel disease–gene associations. Finally, the integrated approach of a multiskilled, multidisciplinary team was fundamental to having diverse perspectives and strategic decision-making.

COX16 is involved in the biogenesis of cytochrome‐c‐oxidase (complex IV), the terminal complex of the mitochondrial respiratory chain. We present the first report of two unrelated patients with the homozygous nonsense variant c.244C>T(p. Arg82*) in COX16 with hypertrophic cardiomyopathy, encephalopathy and severe fatal lactic acidosis, and isolated complex IV deficiency. The absence of COX16 protein expression leads to a complete loss of the holo‐complex IV, as detected by Western blot in patient fibroblasts. Lentiviral transduction of patient fibroblasts with wild‐type COX16 complementary DNA rescued complex IV biosynthesis. We hypothesize that COX16 could play a role in the copper delivery route of the COX2 module as part of the complex IV assembly. Our data provide clear evidence for the pathogenicity of the COX16 variant as a cause for the observed clinical features and the isolated complex IV deficiency in these two patients and that COX16 deficiency is a cause for mitochondrial disease., We present the first report of two unrelated patients with the homozygous nonsense variant c.244C>T(p. Arg82*) in COX16 with hypertrophic cardiomyopathy, encephalopathy and severe fatal lactic acidosis, and isolated complex IV deficiency. Our data provide clear evidence for the pathogenicity of the COX16 variant as a cause for the observed clinical features and the isolated complex IV deficiency in these two patients and that COX16 deficiency is a cause for mitochondrial disease.

The diagnosis of Mendelian disorders following uninformative exome and genome sequencing remains a challenging and often unmet need. Following uninformative exome and genome sequencing of a family quartet including two siblings with suspected mitochondrial disorder, RNA sequencing (RNAseq) was pursued in one sibling. Long-read amplicon sequencing was used to determine and quantify transcript structure. Immunoblotting studies and quantitative proteomics were performed to demonstrate functional impact. Differential expression analysis of RNAseq data identified significantly decreased expression of the mitochondrial OXPHOS complex I subunit NDUFB10 associated with a cryptic exon in intron 1 of NDUFB10, that included an in-frame stop codon. The cryptic exon contained a rare intronic variant that was homozygous in both affected siblings. Immunoblot and quantitative proteomic analysis of fibroblasts revealed decreased abundance of complex I subunits, providing evidence of isolated complex I deficiency. Through multi-omic analysis we present data implicating a deep intronic variant in NDUFB10 as the cause of mitochondrial disease in two individuals, providing further support of the gene-disease association. This study highlights the importance of transcriptomic and proteomic analyses as complementary diagnostic tools in patients undergoing genome-wide diagnostic evaluation.

Purpose: The utility of genome sequencing (GS) in the diagnosis of suspected pediatric mitochondrial disease (MD) was investigated. Methods: An Australian cohort of 40 pediatric patients with clinical features suggestive of MD were classified using the modified Nijmegen mitochondrial disease severity scoring into definite (17), probable (17), and possible (6) MD groups. Trio GS was performed using DNA extracted from patient and parent blood. Data were analyzed for single-nucleotide variants, indels, mitochondrial DNA variants, and structural variants. Results: A definitive MD gene molecular diagnosis was made in 15 cases and a likely MD molecular diagnosis in a further five cases. Causative mitochondrial DNA (mtDNA) variants were identified in four of these cases. Three potential novel MD genes were identified. In seven cases, causative variants were identified in known disease genes with no previous evidence of causing a primary MD. Diagnostic rates were higher in patients classified as having definite MD. Conclusion: GS efficiently identifies variants in MD genes of both nuclear and mitochondrial origin. A likely molecular diagnosis was identified in 67% of cases and a definitive molecular diagnosis achieved in 55% of cases. This study highlights the value of GS for a phenotypically and genetically heterogeneous disorder like MD.

A recent report has raised the possibility of biparental mitochondrial DNA (mtDNA) inheritance, which could lead to concerns by health-care professionals and patients regarding investigations and genetic counseling of families with pathogenic mitochondrial DNA variants. Our aim was to examine the frequency of this phenomenon by investigating a cohort of patients with suspected mitochondrial disease. We studied genome sequencing (GS) data of DNA extracted from blood samples of 41 pediatric patients with suspected mitochondrial disease and their parents. All of the mtDNA variants in the probands segregated with their mother or were apparently de novo. There were no variants that segregated only with the father and none of these families showed evidence of biparental inheritance of their mtDNA. Paternal mitochondrial transmission is unlikely to be a common occurrence and therefore at this point we would not recommend changes in clinical practice.

The diagnostic and clinical benefits of genomic sequencing are being increasingly demonstrated across multiple rare genetic conditions. Despite the expanding clinical literature, there is a significant paucity of health economics evidence to inform the prioritization and implementation of genomic sequencing. This study aims to evaluate whether genomic sequencing for pediatric-onset mitochondrial disorders (MDs) is cost-effective and cost-beneficial relative to conventional care from an Australian healthcare system perspective. Two independent and complementary health economic modeling approaches were used. Approach 1 used a decision tree to model the costs and outcomes associated with genomic sequencing and conventional care. Approach 2 used a discrete-event simulation to incorporate heterogeneity in the condition and clinical practice. Deterministic and probabilistic sensitivity analyses were performed. Genomic sequencing was less costly and more effective compared with conventional care, saving AU$1997 (Approach 1) to AU$8823 (Approach 2) per child tested, while leading to an additional 11 (Approach 1) to 14 (Approach 2) definitive diagnoses per 100 children tested. The mean monetary value of the incremental benefits of genomic sequencing was estimated at AU$5890 (95% CI: AU$5730-$6046). Implementation of genomic sequencing for MDs in Australia could translate to an annual cost-saving of up to AU$0.7 million. Genomic sequencing is cost-saving relative to traditional investigative approaches, while enabling more diagnoses to be made in a timely manner, offering substantial personal benefits to children and their families. Our findings support the prioritization of genomic sequencing for children with MDs.

Primary genetic mitochondrial diseases are often difficult to diagnose, and the term ‘possible’ mitochondrial disease is used frequently by clinicians when such a diagnosis is suspected. There are now many known phenocopies of mitochondrial disease. Advances in genomic testing have shown that some patients with a clinical phenotype and biochemical abnormalities suggesting mitochondrial disease may have other genetic disorders. In instances when a genetic diagnosis cannot be confirmed, a diagnosis of ‘possible’ mitochondrial disease may result in harm to patients and their families, creating anxiety, delaying appropriate diagnosis and leading to inappropriate management or care. A categorisation of ‘diagnosis uncertain’, together with a specific description of the metabolic or genetic abnormalities identified, is preferred when a mitochondrial disease cannot be genetically confirmed.

Mitochondrial diseases can be caused by pathogenic variants in nuclear or mitochondrial DNA-encoded genes that often lead to multisystemic symptoms and can have any mode of inheritance. Using a single test, Genome Sequencing (GS) can effectively identify variants in both genomes, but it has not yet been universally used as a first-line approach to diagnosing mitochondrial diseases due to related costs and challenges in data analysis. In this article, we report three patients with mitochondrial disease molecularly diagnosed through GS performed on DNA extracted from blood to demonstrate different diagnostic advantages of this technology, including the detection of a low-level heteroplasmic pathogenic variant, an intragenic nuclear DNA deletion, and a large mtDNA deletion. Current technical improvements and cost reductions are likely to lead to an expanded routine diagnostic usage of GS and of the complementary “Omic” technologies in mitochondrial diseases.

Summary Background In about half of all patients with a suspected monogenic disease, genomic investigations fail to identify the diagnosis. A contributing factor is the difficulty with repetitive regions of the genome, such as those generated by segmental duplications. The ATAD3 locus is one such region in which recessive deletions and dominant duplications have recently been reported to cause lethal perinatal mitochondrial diseases characterized by pontocerebellar hypoplasia or cardiomyopathy, respectively. Methods Whole-exome, whole-genome, and long-read DNA sequencing techniques combined with studies of RNA and quantitative proteomics were used to investigate 17 subjects from 16 unrelated families with suspected mitochondrial disease. Findings We report 6 different de novo duplications in the ATAD3 gene locus causing a distinctive presentation, including lethal perinatal cardiomyopathy, persistent hyperlactacidemia, and frequently, corneal clouding or cataracts and encephalopathy. The recurrent 68-kb ATAD3 duplications are identifiable from genome and exome sequencing but usually missed by microarrays. The ATAD3 duplications result in the formation of identical chimeric ATAD3A/ATAD3C proteins, altered ATAD3 complexes, and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. Conclusions ATAD3 duplications appear to act in a dominant-negative manner and the de novo inheritance infers a low recurrence risk for families, unlike most pediatric mitochondrial diseases. More than 350 genes underlie mitochondrial diseases. In our experience, the ATAD3 locus is now one of the five most common causes of nuclear-encoded pediatric mitochondrial disease, but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies. Funding Australian NHMRC, US Department of Defense, US National Institutes of Health, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance, and Australian Mito Foundation.

Not available.

International audience; Perrault syndrome is a rare heterogeneous condition characterised by sensorineural hearing loss and premature ovarian insufficiency. Additional neuromuscular pathology is observed in some patients. There are six genes in which variants are known to cause Perrault syndrome; however, these explain only a minority of cases. We investigated the genetic cause of Perrault syndrome in seven affected individuals from five different families, successfully identifying the cause in four patients. This included previously reported and novel causative variants in known Perrault syndrome genes, CLPP and LARS2, involved in mitochondrial proteolysis and mitochondrial translation, respectively. For the first time, we show that pathogenic variants in PEX6 can present clinically as Perrault syndrome. PEX6 encodes a peroxisomal biogenesis factor, and we demonstrate evidence of peroxisomal dysfunction in patient serum. This study consolidates the clinical overlap between Perrault syndrome and peroxisomal disorders, and highlights the need to consider ovarian function in individuals with atypical/mild peroxisomal disorders. The remaining patients had variants in candidate genes such as TFAM, involved in mtDNA transcription, replication, and packaging, and GGPS1 involved in mevalonate/coenzyme Q(10) biosynthesis and whose enzymatic product is required for mouse folliculogenesis. This genomic study highlights the diverse molecular landscape of this poorly understood syndrome.

LARS2 variants are associated with Perrault syndrome, characterized by premature ovarian failure and hearing loss, and with an infantile lethal multisystem disorder: Hydrops, lactic acidosis, sideroblastic anemia (HLASA) in one individual. Recently we reported LARS2 deafness with (ovario) leukodystrophy. Here we describe five patients with a range of phenotypes, in whom we identified biallelic LARS2 variants: three patients with a HLASA-like phenotype, an individual with Perrault syndrome whose affected siblings also had leukodystrophy, and an individual with a reversible mitochondrial myopathy, lactic acidosis, and developmental delay. Three HLASA cases from two unrelated families were identified. All were males with genital anomalies. Two survived multisystem disease in the neonatal period; both have developmental delay and hearing loss. A 55-year old male with deafness has not displayed neurological symptoms while his female siblings with Perrault syndrome developed leukodystrophy and died in their 30s. Analysis of muscle from a child with a reversible myopathy showed reduced LARS2 and mitochondrial complex I levels, and an unusual form of degeneration. Analysis of recombinant LARS2 variant proteins showed they had reduced aminoacylation efficiency, with HLASA-associated variants having the most severe effect. A broad phenotypic spectrum should be considered in association with LARS2 variants.

The diagnosis of mitochondrial disorders remains a challenging and often unmet need. We sought to investigate a sibling pair with suspected mitochondrial disease and a clinical presentation notable for global developmental delay, poor growth, sensorineural hearing loss, and brain MRI abnormalities, both with early death. Following uninformative exome and genome sequencing of the family quartet, RNA sequencing was pursued as an orthogonal testing strategy. RNA sequencing of fibroblasts from the older sibling identified the presence of a cryptic exon in intron 1 of NDUFB10, that included an in-frame stop codon. NDUFB10 encodes a subunit of mitochondrial OXPHOS complex I. Differential expression analysis relative to control samples suggested significantly decreased expression. The cryptic exon was found to contain a rare intronic variant, NM_004548.3:c.131-442G>C, that was homozygous in both affected siblings and absent from population allele frequency databases. Immunoblot and quantitative proteomic analysis of fibroblasts from the older sibling revealed decreased abundance of complex I subunits associated with NDUFB10, providing evidence of isolated complex I deficiency. Biallelic variants in NDUFB10 have previously been reported in a single individual with infantile-onset mitochondrial disease. We present data implicating a deep intronic variant in NDUFB10 as the cause of mitochondrial disease in two further individuals. This variant results in loss of expression and overall destabilization of mitochondrial OXPHOS complex I and highlights the importance of RNA sequencing as a complementary diagnostic tool in patients undergoing genome-wide diagnostic evaluation.

The synthesis of all 13 mitochondrial DNA (mtDNA)-encoded protein subunits of the human oxidative phosphorylation (OXPHOS) system is carried out by mitochondrial ribosomes (mitoribosomes). Defects in the stability of mitoribosomal proteins or mitoribosome assembly impair mitochondrial protein translation, causing combined OXPHOS enzyme deficiency and clinical disease. Here we report four autosomal-recessive pathogenic mutations in the gene encoding the small mitoribosomal subunit protein, MRPS34, in six subjects from four unrelated families with Leigh syndrome and combined OXPHOS defects. Whole-exome sequencing was used to independently identify all variants. Two splice-site mutations were identified, including homozygous c.321+1G>T in a subject of Italian ancestry and homozygous c.322−10G>A in affected sibling pairs from two unrelated families of Puerto Rican descent. In addition, compound heterozygous MRPS34 mutations were identified in a proband of French ancestry; a missense (c.37G>A [p.Glu13Lys]) and a nonsense (c.94C>T [p.Gln32∗]) variant. We demonstrated that these mutations reduce MRPS34 protein levels and the synthesis of OXPHOS subunits encoded by mtDNA. Examination of the mitoribosome profile and quantitative proteomics showed that the mitochondrial translation defect was caused by destabilization of the small mitoribosomal subunit and impaired monosome assembly. Lentiviral-mediated expression of wild-type MRPS34 rescued the defect in mitochondrial translation observed in skin fibroblasts from affected subjects, confirming the pathogenicity of MRPS34 mutations. Our data establish that MRPS34 is required for normal function of the mitoribosome in humans and furthermore demonstrate the power of quantitative proteomic analysis to identify signatures of defects in specific cellular pathways in fibroblasts from subjects with inherited disease.

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations.The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change p.R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

The vast majority of cellular ATP is produced by the oxidative phosphorylation (OXPHOS) system, which comprises the four complexes of the electron transfer chain plus the ATP synthase. Complex I is the largest of the OXPHOS complexes, and mutation of the genes encoding either the subunits or assembly factors of Complex I can result in Complex I deficiency, which is the most common OXPHOS disorder. Mutations in the Complex I gene NDUFS4 lead to Leigh syndrome, which is the most frequent presentation of Complex I deficiency in children presenting with progressive encephalopathy shortly after birth. Symptoms include motor and intellectual retardation, often accompanied by dystonia, ataxia, and growth retardation, and most patients die by 3 years of age. To understand the origins of this disease, we have generated a series of mouse embryonic stem cell lines from blastocysts that were wild type, heterozygous, and homozygous for the deletion of the Ndufs4 gene. We have demonstrated their pluripotency and potential to differentiate into all cell types of the body. Although the loss of Ndufs4 did not affect the stability of the mitochondrial and nuclear genomes, there were significant differences in patterns of chromosomal gene expression following both spontaneous differentiation and directed neural differentiation into astrocytes. The defect also affected the potential of the cells to generate beating embryoid bodies. These outcomes demonstrate that defects associated with Complex I deficiency affect early gene expression patterns, which escalate during early and later stages of differentiation and are mediated by the defect and not other chromosomal or mitochondrial DNA defects.

Leigh syndrome is the most common pediatric presentation of mitochondrial disease. This neurodegenerative disorder is genetically heterogeneous, and to date pathogenic mutations in >75 genes have been identified, encoded by 2 genomes (mitochondrial and nuclear). More than one-third of these disease genes have been characterized in the past 5 years alone, reflecting the significant advances made in understanding its etiological basis. We review the diverse biochemical and genetic etiology of Leigh syndrome and associated clinical, neuroradiological, and metabolic features that can provide clues for diagnosis. We discuss the emergence of genotype-phenotype correlations, insights gleaned into the molecular basis of disease, and available therapeutic options.

Leigh syndrome is a mitochondrial disease caused by pathogenic variants in over 85 genes. Whole exome sequencing of a patient with Leigh-like syndrome identified homozygous protein-truncating variants in two genes associated with Leigh syndrome; a reported pathogenic variant in PDHX (NP_003468.2:p.(Arg446*)), and an uncharacterized variant in complex I (CI) assembly factor TIMMDC1 (NP_057673.2:p.(Arg225*)). The TIMMDC1 variant was predicted to truncate 61 amino acids at the C-terminus and functional studies demonstrated a hypomorphic impact of the variant on CI assembly. However, the mutant protein could still rescue CI assembly in TIMMDC1 knockout cells and the patient's clinical phenotype was not clearly distinct from that of other patients with the same PDHX defect. Our data suggest that the hypomorphic effect of the TIMMDC1 protein-truncating variant does not constitute a dual diagnosis in this individual. We recommend cautious assessment of variants in the C-terminus of TIMMDC1 and emphasize the need to consider the caveats detailed within the American College of Medical Genetics and Genomics (ACMG) criteria when assessing variants.

Leigh syndrome (LS) is a progressive mitochondrial neurodegenerative disorder, whose symptoms most commonly include psychomotor delay with regression, lactic acidosis and a failure to thrive. Here we describe three siblings with LS, but with additional manifestations including hypertrophic cardiomyopathy, hepatosplenomegaly, cholestatic hepatitis, and seizures. All three affected siblings were found to be homoplasmic for an m. 5559A>G mutation in the T stem of the mitochondrial DNA-encoded MT-TW by next generation sequencing. The m.5559A>G mutation causes a reduction in the steady state levels of tRNA(Trp) and this decrease likely affects the stability of other mitochondrial RNAs in the patient fibroblasts. We observe accumulation of an unprocessed transcript containing tRNA(Trp), decreased de novo protein synthesis and consequently lowered steady state levels of mitochondrial DNA-encoded proteins that compromise mitochondrial respiration. Our results show that the m.5559A>G mutation at homoplasmic levels causes LS in association with severe multi-organ disease (LS-plus) as a consequence of dysfunctional mitochondrial RNA metabolism.

The oxidative phosphorylation (OXPHOS) system in mitochondria is responsible for the generation of the majority of cellular energy in the form of ATP. Patients with genetic OXPHOS disorders form the largest group of inborn errors of metabolism. Unfortunately, there is still a lack of efficient therapies for these disorders other than management of symptoms. Developing therapies has been complicated because, although the total group of OXPHOS patients is relatively large, there is enormous clinical and genetic heterogeneity within this patient population. Thus there has been a lot of interest in generating relevant mouse models for the different kinds of OXPHOS disorders. The most common treatment strategies tested in these mouse models have aimed to up-regulate mitochondrial biogenesis, in order to increase the residual OXPHOS activity present in affected animals and thereby to ameliorate the energy deficiency. Drugs such as bezafibrate, resveratrol and AICAR target the master regulator of mitochondrial biogenesis PGC-1α either directly or indirectly to manipulate mitochondrial metabolism. This review will summarize the outcome of preclinical treatment trials with these drugs in mouse models of OXPHOS disorders and discuss similar treatments in a number of mouse models of common diseases in which pathology is closely linked to mitochondrial dysfunction. In the majority of these studies the pharmacological activation of the PGC-1α axis shows true potential as therapy; however, other effects besides mitochondrial biogenesis may be contributing to this as well. Linked Articles This article is part of a themed issue on Mitochondrial Pharmacology: Energy, Injury & Beyond. To view the other articles in this issue visit http://dx.doi.org/10.1111/bph.2014.171.issue-8

PNPT1 (PNPase—polynucleotide phosphorylase) is involved in multiple RNA processing functions in the mitochondria. Bi-allelic pathogenic PNPT1 variants cause heterogeneous clinical phenotypes affecting multiple organs without any established genotype−phenotype correlations. Defects in PNPase can cause variable combined respiratory chain complex defects. Recently, it has been suggested that PNPase can lead to activation of an innate immune response. To better understand the clinical and molecular spectrum of patients with bi-allelic PNPT1 variants, we captured detailed clinical and molecular phenotypes of all 17 patients reported in the literature, plus seven new patients, including a 78-year-old male with the longest reported survival. A functional follow-up of genomic sequencing by cDNA studies confirmed a splicing defect in a novel, apparently synonymous, variant. Patient fibroblasts showed an accumulation of mitochondrial unprocessed PNPT1 transcripts, while blood showed an increased interferon response. Our findings suggest that functional analyses of the RNA processing function of PNPase are more sensitive than testing downstream defects in oxidative phosphorylation (OXPHPOS) enzyme activities. This research extends our knowledge of the clinical and functional consequences of bi-allelic pathogenic PNPT1 variants that may guide management and further efforts into understanding the pathophysiological mechanisms for therapeutic development.

International audience; Mitochondrial respiratory chain (RC) disorders are the most prevalent inborn metabolic diseases and remain without effective treatment to date. Up-regulation of residual enzyme activity has been proposed as a possible therapeutic approach in this group of disorders. As resveratrol (RSV), a natural compound, was proposed to stimulate mitochondrial metabolism in rodents, we tested the effect of this compound on mitochondrial functions in control or in Complex I (CI)-or Complex IV (CIV)-deficient patients' fibroblasts. We show that RSV stimulates the expression of a panel of proteins representing structural subunits or assembly factors of the five RC complexes, in control fibroblasts. In moderate RC-deficient patients' cells, RSV treatment increases the amount of mutated proteins and stimulates residual enzyme activities. In these patients' cells, we establish that up-regulation of RC enzyme activities induced by RSV translates into increased cellular O 2 consumption rates and results in the correction of RC deficiencies. Importantly, RSV also prevents the accumulation of lactate that occurred in RC-deficient fibroblasts. Different complementary approaches demonstrate that RSV induces a mitochondrial biogenesis that might underlie the increase in mitochondrial capacities. Finally, we showed that, in human fibroblasts, RSV stimulated mitochondrial functions mainly in a SIRT1-and AMPK-independent manner and that its effects rather involved the estrogen receptor (ER) and estrogen-related receptor alpha (ERRa) signaling pathways. These results represent the first demonstration that RSV could have a beneficial effect on inborn CI and CIV deficiencies from nuclear origin, in human fibroblasts and might be clinically relevant for the treatment of some RC deficiencies.

Many individuals with abnormalities of mitochondrial respiratory chain complex III remain genetically undefined. Here, we report mutations (c.288G>T [p.Trp96Cys] and c.643C>T [p.Leu215Phe]) in CYC1, encoding the cytochrome c1 subunit of complex III, in two unrelated children presenting with recurrent episodes of ketoacidosis and insulin-responsive hyperglycemia. Cytochrome c1, the heme-containing component of complex III, mediates the transfer of electrons from the Rieske iron-sulfur protein to cytochrome c. Cytochrome c1 is present at reduced levels in the skeletal muscle and skin fibroblasts of affected individuals. Moreover, studies on yeast mutants and affected individuals’ fibroblasts have shown that exogenous expression of wild-type CYC1 rescues complex III activity, demonstrating the deleterious effect of each mutation on cytochrome c1 stability and complex III activity.

An ENMC meeting was held in Naarden, the Netherlands (20–22 April 2012) of 17 clinical and basic scientists and a patient representative to discuss the diagnosis and treatment of mitochondrial respiratory chain complex I deficiency. Participants work in six member countries (Finland, France, Germany, Italy, the Netherlands and the UK) and three non-member countries (Australia, Canada and Israel). Mitochondrial complex I (NADH:ubiquinone oxidoreductase) is the largest enzyme in the inner mitochondrial membrane and provides the main entry point into the respiratory chain for electrons derived from fuel oxidation. Complex I deficiency is the most commonly observed biochemical defect in childhood-onset mitochondrial disease, accounting for 25% of cases [1]. The enzyme has essential functions in electron transfer and proton pumping, generating 40% of the proton motive force that is eventually harnessed by complex V to synthesise ATP. However, despite its fundamental role in mitochondrial energy generation, complex I remains the least well understood of the respiratory chain enzyme complexes. Little is known about the function of many of the 45 subunits, and the factors necessary for and

SLC39A8 variants have recently been reported to cause a type II congenital disorder of glycosylation (CDG) in patients with intellectual disability and cerebellar atrophy. Here we report a novel SLC39A8 variant in siblings with features of Leigh-like mitochondrial disease. Two sisters born to consanguineous Lebanese parents had profound developmental delay, dystonia, seizures and failure to thrive. Brain MRI of both siblings identified bilateral basal ganglia hyperintensities on T2-weighted imaging and cerebral atrophy. CSF lactate was elevated in patient 1 and normal in patient 2. Respiratory chain enzymology was only performed on patient 1 and revealed complex IV and II + III activity was low in liver, with elevated complex I activity. Complex IV activity was borderline low in patient 1 muscle and pyruvate dehydrogenase activity was reduced. Whole genome sequencing identified a homozygous Chr4(GRCh37):g.103236869C>G; c.338G>C; p.(Cys113Ser) variant in SLC39A8, located in one of eight regions identified by homozygosity mapping. SLC39A8 encodes a manganese and zinc transporter which localises to the cell and mitochondrial membranes. Patient 2 blood and urine manganese levels were undetectably low. Transferrin electrophoresis of patient 2 serum revealed a type II CDG defect. Oral supplementation with galactose and uridine led to improvement of the transferrin isoform pattern within 14 days of treatment initiation. Oral manganese has only recently been added to the treatment. These results suggest SLC39A8 deficiency can cause both a type II CDG and Leigh-like syndrome, possibly via reduced activity of the manganese-dependent enzymes β-galactosyltransferase and mitochondrial manganese superoxide dismutase.

MPV17 is a mitochondrial inner membrane protein whose dysfunction causes mitochondrial DNA abnormalities and disease by an unknown mechanism. Perturbations of deoxynucleoside triphosphate (dNTP) pools are a recognized cause of mitochondrial genomic instability; therefore, we determined DNA copy number and dNTP levels in mitochondria of two models of MPV17 deficiency. In Mpv17 ablated mice, liver mitochondria showed substantial decreases in the levels of dGTP and dTTP and severe mitochondrial DNA depletion, whereas the dNTP pool was not significantly altered in kidney and brain mitochondria that had near normal levels of DNA. The shortage of mitochondrial dNTPs in Mpv17-/- liver slows the DNA replication in the organelle, as evidenced by the elevated level of replication intermediates. Quiescent fibroblasts of MPV17-mutant patients recapitulate key features of the primary affected tissue of the Mpv17-/- mice, displaying virtual absence of the protein, decreased dNTP levels and mitochondrial DNA depletion. Notably, the mitochondrial DNA loss in the patients’ quiescent fibroblasts was prevented and rescued by deoxynucleoside supplementation. Thus, our study establishes dNTP insufficiency in the mitochondria as the cause of mitochondrial DNA depletion in MPV17 deficiency, and identifies deoxynucleoside supplementation as a potential therapeutic strategy for MPV17-related disease. Moreover, changes in the expression of factors involved in mitochondrial deoxynucleotide homeostasis indicate a remodeling of nucleotide metabolism in MPV17 disease models, which suggests mitochondria lacking functional MPV17 have a restricted purine mitochondrial salvage pathway., Author Summary Mitochondrial DNA depletion syndrome (MDS) is a genetically heterogeneous condition characterized by a decrease of mitochondrial DNA (mtDNA) copy number and decreased activities of respiratory chain enzymes. Depletion of mtDNA has been associated with mutations in several genes, which encode either proteins directly involved in mtDNA replication or factors regulating the homeostasis of the mitochondrial deoxynucleotide pool. However, for some genes the mechanism linking mutations and mtDNA depletion is not known. One such gene is MPV17, whose loss-of-function causes mtDNA abnormalities in human, mouse and yeast. Here we show that MPV17 dysfunction leads to a shortage of the precursors for DNA synthesis in the mitochondria, slowing DNA replication in the organelle. Not only does mtDNA copy number correlate with dNTP pool size in both mouse tissues and human cells, deoxynucleoside supplementation of the growth medium prevents depletion and restores mtDNA copy number in quiescent MPV17-deficient cells. Hence, our study links MPV17 deficiency, insufficiency of mitochondrial dNTPs, and slow replication in mitochondria to depletion of mtDNA manifesting in the human disease, and places MPV17-related disease firmly in the category of mtDNA disorders caused by deoxynucleotide perturbation. The prevention and reversal of mtDNA loss in MPV17 patient-derived cells identifies potential therapeutic strategy for a currently untreatable disease.

Complex I (NADH:ubiquinone oxidoreductase) is the largest multimeric enzyme complex of the mitochondrial respiratory chain, which is responsible for electron transport and the generation of a proton gradient across the mitochondrial inner membrane to drive ATP production. Eukaryotic complex I consists of 14 conserved subunits, which are homologous to the bacterial subunits, and more than 26 accessory subunits. In mammals, complex I consists of 45 subunits, which must be assembled correctly to form the properly functioning mature complex. Complex I dysfunction is the most common oxidative phosphorylation (OXPHOS) disorder in humans and defects in the complex I assembly process are often observed. This assembly process has been difficult to characterize because of its large size, the lack of a high resolution structure for complex I, and its dual control by nuclear and mitochondrial DNA. However, in recent years, some of the atomic structure of the complex has been resolved and new insights into complex I assembly have been generated. Furthermore, a number of proteins have been identified as assembly factors for complex I biogenesis and many patients carrying mutations in genes associated with complex I deficiency and mitochondrial diseases have been discovered. Here, we review the current knowledge of the eukaryotic complex I assembly process and new insights from the identification of novel assembly factors. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Recent advances in next-generation sequencing strategies have led to the discovery of many novel disease genes. We describe here a non-consanguineous family with two affected boys presenting with early onset of severe axonal neuropathy, optic atrophy, intellectual disability, auditory neuropathy and chronic respiratory and gut disturbances. Whole-exome sequencing (WES) was performed on all family members and we identified compound heterozygous variants (c.[760C>A];[1528G>C];p.[(Gln254Lys);(Ala510Pro)] in the polyribonucleotide nucleotidyltransferase 1 (PNPT1) gene in both affected individuals. PNPT1 encodes the polynucleotide phosphorylase (PNPase) protein, which is involved in the transport of small RNAs into the mitochondria. These RNAs are involved in the mitochondrial translation machinery, responsible for the synthesis of mitochondrially encoded subunits of the oxidative phosphorylation (OXPHOS) complexes. Both PNPT1 variants are within highly conserved regions and predicted to be damaging. These variants resulted in quaternary defects in the PNPase protein and a clear reduction in protein and mRNA expression of PNPT1 in patient fibroblasts compared with control cells. Protein analysis of the OXPHOS complexes showed a significant reduction in complex I (CI), complex III (CIII) and complex IV (CIV). Enzyme activity of CI and CIV was clearly reduced in patient fibroblasts compared with controls along with a 33% reduction in total mitochondrial protein synthesis. In vitro rescue experiments, using exogenous expression of wild-type PNPT1 in patient fibroblasts, ameliorated the deficiencies in the OXPHOS complex protein expression, supporting the likely pathogenicity of these variants and the importance of WES in efficiently identifying rare genetic disease genes.

The assembly of complex I (NADH-ubiquinone oxidoreductase) is a complicated process, requiring the integration of 45 subunits encoded by both nuclear and mitochondrial DNAs into a structure of approximately 1 MDa. A number of "assembly factors" that aid complex I biogenesis have recently been described, including C8orf38. This protein was identified as an assembly factor by its evolutionary conservation in organisms containing complex I and by a C8orf38 mutation in a patient presenting with Leigh syndrome and isolated complex I deficiency. In this report, we have undertaken the characterization of C8orf38 and its role in complex I assembly. Analysis of mitochondria from fibroblasts of a patient harboring a C8orf38 mutation showed almost undetectable levels of steady-state complex I and defective biogenesis of the mtDNA-encoded subunit ND1. Complementation with wild-type C8orf38 restored the levels of both ND1 and complex I, confirming the C8orf38 mutation as the cause of the complex I defect in the patient. In the absence of ND1 in patient cells, early- and mid-stage intermediate complexes were still formed; however, assembly of late-stage intermediates was impaired, indicating a convergence point in the assembly process. While C8orf38 appears to behave at a step in complex I biogenesis similar to that of the assembly factor C20orf7, complementation studies showed that both proteins are required for ND1 synthesis/stabilization. We conclude that C8orf38 is a crucial factor required for the translation and/or integration of ND1 into an early-stage assembly intermediate and that mutation of C8orf38 disrupts the initial stages of complex I biogenesis.

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that fulfills the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by mitochondrial methionyl-tRNA formyltransferase (MTFMT) to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)), which is used for translation initiation; however, the requirement of formylation for initiation in human mitochondria is still under debate. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identified compound heterozygous mutations in MTFMT in two unrelated children presenting with Leigh syndrome and combined OXPHOS deficiency. Patient fibroblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous expression of MTFMT. Furthermore, patient fibroblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation profile of mitochondrially translated COX1. Our findings demonstrate that MTFMT is critical for efficient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

Mutations in the polymerase γ (POLG) gene are among the most common causes of mitochondrial disease and more than 160 POLG mutations have been reported. However, a large proportion of patients suspected of having POLG mutations only have one (heterozygous) definitive pathogenic mutation identified. Using oligonucleotide array CGH, we identified a compound heterozygous large intragenic deletion encompassing exons 15–21 of this gene in a child with Alpers syndrome due to mtDNA depletion. This is the first large POLG deletion reported and the findings show the clinical utility of using array CGH in cases where a single heterozygous mutation has been identified in POLG.

Isolated complex I deficiency is the most frequently observed oxidative phosphorylation defect in children with mitochondrial disease, leading to a diverse range of clinical presentations, including Leigh syndrome. For most patients the genetic cause of the biochemical defect remains unknown due to incomplete understanding of the complex I assembly process. Nonetheless, a plethora of pathogenic mutations have been described to date in the seven mitochondrial-encoded subunits of complex I as well as in 12 of the nuclear-encoded subunits and in six assembly factors. Whilst several mitochondrial DNA mutations are recurrent, the majority of these mutations are reported in single families. We have sequenced core structural and functional nuclear-encoded subunits of complex I in a cohort of 34 paediatric patients with isolated complex I deficiency, identifying pathogenic mutations in 6 patients. These included a novel homozygous NDUFS1 mutation in an Asian child with Leigh syndrome, a previously identified NDUFS8 mutation (c.236C>T, p.P79L) in a second Asian child with Leigh-like syndrome and six novel, compound heterozygous NDUFS2 mutations in four white Caucasian patients with Leigh or Leigh-like syndrome. Three of these children harboured an identical NDUFS2 mutation (c.875T>C, p.M292T), which was also identified in conjunction with a novel NDUFS2 splice site mutation (c.866+4A>G) in a fourth Caucasian child who presented to a different diagnostic centre, with microsatellite and single nucleotide polymorphism analyses indicating that this was due to an ancient common founder event. Our results confirm that NDUFS2 is a mutational hotspot in Caucasian children with isolated complex I deficiency and recommend the routine diagnostic investigation of this gene in patients with Leigh or Leigh-like phenotypes.

International audience; Mitochondrial respiratory chain disorders are a heterogeneous group of disorders in which the underlying genetic defect is often unknown. We have identified a pathogenic mutation (c.156C>G [p.F52L]) in YARS2, located at chromosome 12p11.21, by using genome-wide SNP-based homozygosity analysis of a family with affected members displaying myopathy, lactic acidosis, and sideroblastic anemia (MLASA). We subsequently identified the same mutation in another unrelated MLASA patient. The YARS2 gene product, mitochondrial tyrosyl-tRNA synthetase (YARS2), was present at lower levels in skeletal muscle whereas fibroblasts were relatively normal. Complex I, III, and IV were dysfunctional as indicated by enzyme analysis, immunoblotting, and immunohistochemistry. A mitochondrial protein-synthesis assay showed reduced levels of respiratory chain subunits in myotubes generated from patient cell lines. A tRNA aminoacylation assay revealed that mutant YARS2 was still active; however, enzyme kinetics were abnormal compared to the wild-type protein. We propose that the reduced aminoacylation activity of mutant YARS2 enzyme leads to decreased mitochondrial protein synthesis, resulting in mitochondrial respiratory chain dysfunction. MLASA has previously been associated with PUS1 mutations; hence, the YARS2 mutation reported here is an alternative cause of MLASA.

Mitochondrial encephalopathy, the most common neurometabolic disorder, may be caused by mutations in approximately 100 different genes and may present with various symptoms, such as seizures, ataxia, myopathy, cognitive impairment, blindness, and stroke. Fewer than 50% of patients with mitochondrial encephalopathy receive a molecular diagnosis, primarily because of the large degree of clinical and genetic heterogeneity among patients and the limited knowledge of the genes involved in mitochondrial function. Here we review the most recent discoveries of genes associated with mitochondrial disease with variable neuropathology. All these genes have been identified via homozygosity mapping or linkage analysis; however, advances in sequencing technology indicate that the future of genetic diagnosis and disease gene discovery likely lies in high-throughput sequencing.

Inherited disorders of mitochondrial oxidative phosphorylation are the most common group of inborn errors of metabolism and cause a wide range of clinical presentations. Mitochondrial DNA encodes 13 protein subunits required for oxidative phosphorylation plus 22 transfer RNAs and two ribosomal RNAs, and mutations in most of these genes cause human disease. Nuclear genes encode most of the protein subunits and all other proteins required for mitochondrial biogenesis and mitochondrial DNA replication and expression. Mutations in 64 nuclear genes and 34 mitochondrial genes are now known to cause mitochondrial disease and many novel mitochondrial disease genes await discovery. The genetic complexity of oxidative phosphorylation means that maternal, autosomal recessive, autosomal dominant and X-linked modes of inheritance can occur, along with de novo mutations. This complexity presents a challenge in planning efficient molecular genetic diagnosis of patients with suspected mitochondrial disease. In some situations, clinical phenotype can be strongly predictive of the underlying genotype. However, more often this is not the case and it is usually helpful, particularly with pediatric patients, to determine whether the activity of one or more of the individual oxidative phosphorylation enzymes is deficient before proceeding with mutation analysis. In this review we will summarize the genetic bases of mitochondrial disease and discuss some approaches to integrate information from clinical presentation, laboratory findings, family history, and imaging to guide molecular investigation.