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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 (OXPHOS) system encoded by mitochondrial Deoxyribonucleic acid (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 the milder disease was homozygous for a missense variant identified through trio exome sequencing. Our study highlights the utility of quantitative proteomics in detecting protein signatures and in characterizing 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., (© The Author(s) 2023. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

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 six 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, Japanese AMED and JSPS agencies, Australian Genomics Health Alliance and Australian Mito Foundation.

Complement component 1 Q subcomponent-binding protein (C1QBP; also known as p32) is a multi-compartmental protein whose precise function remains unknown. It is an evolutionary conserved multifunctional protein localized primarily in the mitochondrial matrix and has roles in inflammation and infection processes, mitochondrial ribosome biogenesis, and regulation of apoptosis and nuclear transcription. It has an N-terminal mitochondrial targeting peptide that is proteolytically processed after import into the mitochondrial matrix, where it forms a homotrimeric complex organized in a doughnut-shaped structure. Although C1QBP has been reported to exert pleiotropic effects on many cellular processes, we report here four individuals from unrelated families where biallelic mutations in C1QBP cause a defect in mitochondrial energy metabolism. Infants presented with cardiomyopathy accompanied by multisystemic involvement (liver, kidney, and brain), and children and adults presented with myopathy and progressive external ophthalmoplegia. Multiple mitochondrial respiratory-chain defects, associated with the accumulation of multiple deletions of mitochondrial DNA in the later-onset myopathic cases, were identified in all affected individuals. Steady-state C1QBP levels were decreased in all individuals' samples, leading to combined respiratory-chain enzyme deficiency of complexes I, III, and IV. C1qbp -/- mouse embryonic fibroblasts (MEFs) resembled the human disease phenotype by showing multiple defects in oxidative phosphorylation (OXPHOS). Complementation with wild-type, but not mutagenized, C1qbp restored OXPHOS protein levels and mitochondrial enzyme activities in C1qbp -/- MEFs. C1QBP deficiency represents an important mitochondrial disorder associated with a clinical spectrum ranging from infantile lactic acidosis to childhood (cardio)myopathy and late-onset progressive external ophthalmoplegia., (Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2017 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Background: D-bifunctional protein deficiency, caused by recessive mutations in HSD17B4, is a severe, infantile-onset disorder of peroxisomal fatty acid oxidation. Few affected patients survive past two years of age. Compound heterozygous mutations in HSD17B4 have also been reported in two sisters diagnosed with Perrault syndrome (MIM # 233400), who presented in adolescence with ovarian dysgenesis, hearing loss, and ataxia., Case Presentation: An adult male presented with cerebellar ataxia, peripheral neuropathy, hearing loss, and azoospermia. The clinical presentation, in combination with biochemical findings in serum, urine, and muscle biopsy, suggested a mitochondrial disorder. Commercial genetic testing of 18 ataxia and mitochondrial disease genes was negative. Targeted exome sequencing followed by analysis of single nucleotide variants and small insertions/deletions failed to reveal a genetic basis of disease. Application of a computational algorithm to infer copy number variants (CNVs) from exome data revealed a heterozygous 12 kb deletion of exons 10-13 of HSD17B4 that was compounded with a rare missense variant (p.A196V) at a highly conserved residue. Retrospective review of patient records revealed mildly elevated ratios of pristanic:phytanic acid and arachidonic:docosahexaenoic acid, consistent with dysfunctional peroxisomal fatty acid oxidation., Conclusion: Our case expands the phenotypic spectrum of HSD17B4-deficiency, representing the first male case reported with infertility. Furthermore, it points to crosstalk between mitochondria and peroxisomes in HSD17B4-deficiency and Perrault syndrome.

We used exome sequencing to identify mutations in sideroflexin 4 (SFXN4) in two children with mitochondrial disease (the more severe case also presented with macrocytic anemia). SFXN4 is an uncharacterized mitochondrial protein that localizes to the mitochondrial inner membrane. sfxn4 knockdown in zebrafish recapitulated the mitochondrial respiratory defect observed in both individuals and the macrocytic anemia with megaloblastic features of the more severe case. In vitro and in vivo complementation studies with fibroblasts from the affected individuals and zebrafish demonstrated the requirement of SFXN4 for mitochondrial respiratory homeostasis and erythropoiesis. Our findings establish mutations in SFXN4 as a cause of mitochondriopathy and macrocytic anemia., (Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Objective: To evaluate the utility of targeted exome sequencing for the molecular diagnosis of mitochondrial disorders, which exhibit marked phenotypic and genetic heterogeneity., Methods: We considered a diverse set of 102 patients with suspected mitochondrial disorders based on clinical, biochemical, and/or molecular findings, and whose disease ranged from mild to severe, with varying age at onset. We sequenced the mitochondrial genome (mtDNA) and the exons of 1,598 nuclear-encoded genes implicated in mitochondrial biology, mitochondrial disease, or monogenic disorders with phenotypic overlap. We prioritized variants likely to underlie disease and established molecular diagnoses in accordance with current clinical genetic guidelines., Results: Targeted exome sequencing yielded molecular diagnoses in established disease loci in 22% of cases, including 17 of 18 (94%) with prior molecular diagnoses and 5 of 84 (6%) without. The 5 new diagnoses implicated 2 genes associated with canonical mitochondrial disorders (NDUFV1, POLG2), and 3 genes known to underlie other neurologic disorders (DPYD, KARS, WFS1), underscoring the phenotypic and biochemical overlap with other inborn errors. We prioritized variants in an additional 26 patients, including recessive, X-linked, and mtDNA variants that were enriched 2-fold over background and await further support of pathogenicity. In one case, we modeled patient mutations in yeast to provide evidence that recessive mutations in ATP5A1 can underlie combined respiratory chain deficiency., Conclusion: The results demonstrate that targeted exome sequencing is an effective alternative to the sequential testing of mtDNA and individual nuclear genes as part of the investigation of mitochondrial disease. Our study underscores the ongoing challenge of variant interpretation in the clinical setting.

Known disease mechanisms in mitochondrial DNA (mtDNA) maintenance disorders alter either the mitochondrial replication machinery (POLG, POLG2 and C10orf2) or the biosynthesis pathways of deoxyribonucleoside 5'-triphosphates for mtDNA synthesis. However, in many of these disorders, the underlying genetic defect has yet to be discovered. Here, we identify homozygous nonsense and missense mutations in the orphan gene C20orf72 in three families with a mitochondrial syndrome characterized by external ophthalmoplegia, emaciation and respiratory failure. Muscle biopsies showed mtDNA depletion and multiple mtDNA deletions. C20orf72, hereafter MGME1 (mitochondrial genome maintenance exonuclease 1), encodes a mitochondrial RecB-type exonuclease belonging to the PD-(D/E)XK nuclease superfamily. We show that MGME1 cleaves single-stranded DNA and processes DNA flap substrates. Fibroblasts from affected individuals do not repopulate after chemically induced mtDNA depletion. They also accumulate intermediates of stalled replication and show increased levels of 7S DNA, as do MGME1-depleted cells. Thus, we show that MGME1-mediated mtDNA processing is essential for mitochondrial genome maintenance.

The molecular diagnosis of mitochondrial disorders still remains elusive in a large proportion of patients, but advances in next generation sequencing are significantly improving our chances to detect mutations even in sporadic patients. Syndromes associated with mitochondrial DNA multiple deletions are caused by different molecular defects resulting in a wide spectrum of predominantly adult-onset clinical presentations, ranging from progressive external ophthalmoplegia to multi-systemic disorders of variable severity. The mutations underlying these conditions remain undisclosed in half of the affected subjects. We applied next-generation sequencing of known mitochondrial targets (MitoExome) to probands presenting with adult-onset mitochondrial myopathy and harbouring mitochondrial DNA multiple deletions in skeletal muscle. We identified autosomal recessive mutations in the DGUOK gene (encoding mitochondrial deoxyguanosine kinase), which has previously been associated with an infantile hepatocerebral form of mitochondrial DNA depletion. Mutations in DGUOK occurred in five independent subjects, representing 5.6% of our cohort of patients with mitochondrial DNA multiple deletions, and impaired both muscle DGUOK activity and protein stability. Clinical presentations were variable, including mitochondrial myopathy with or without progressive external ophthalmoplegia, recurrent rhabdomyolysis in a young female who had received a liver transplant at 9 months of age and adult-onset lower motor neuron syndrome with mild cognitive impairment. These findings reinforce the concept that mutations in genes involved in deoxyribonucleotide metabolism can cause diverse clinical phenotypes and suggest that DGUOK should be screened in patients harbouring mitochondrial DNA deletions in skeletal muscle.

Advances in next-generation sequencing (NGS) promise to facilitate diagnosis of inherited disorders. Although in research settings NGS has pinpointed causal alleles using segregation in large families, the key challenge for clinical diagnosis is application to single individuals. To explore its diagnostic use, we performed targeted NGS in 42 unrelated infants with clinical and biochemical evidence of mitochondrial oxidative phosphorylation disease. These devastating mitochondrial disorders are characterized by phenotypic and genetic heterogeneity, with more than 100 causal genes identified to date. We performed "MitoExome" sequencing of the mitochondrial DNA (mtDNA) and exons of ~1000 nuclear genes encoding mitochondrial proteins and prioritized rare mutations predicted to disrupt function. Because patients and healthy control individuals harbored a comparable number of such heterozygous alleles, we could not prioritize dominant-acting genes. However, patients showed a fivefold enrichment of genes with two such mutations that could underlie recessive disease. In total, 23 of 42 (55%) patients harbored such recessive genes or pathogenic mtDNA variants. Firm diagnoses were enabled in 10 patients (24%) who had mutations in genes previously linked to disease. Thirteen patients (31%) had mutations in nuclear genes not previously linked to disease. The pathogenicity of two such genes, NDUFB3 and AGK, was supported by complementation studies and evidence from multiple patients, respectively. The results underscore the potential and challenges of deploying NGS in clinical settings.

Background: Mitochondrial diseases comprise a diverse set of clinical disorders that affect multiple organ systems with varying severity and age of onset. Due to their clinical and genetic heterogeneity, these diseases are difficult to diagnose. We have developed a targeted exome sequencing approach to improve our ability to properly diagnose mitochondrial diseases and apply it here to an individual patient. Our method targets mitochondrial DNA (mtDNA) and the exons of 1,600 nuclear genes involved in mitochondrial biology or Mendelian disorders with multi-system phenotypes, thereby allowing for simultaneous evaluation of multiple disease loci., Case Presentation: Targeted exome sequencing was performed on a patient initially suspected to have a mitochondrial disorder. The patient presented with diabetes mellitus, diffuse brain atrophy, autonomic neuropathy, optic nerve atrophy, and a severe amnestic syndrome. Further work-up revealed multiple heteroplasmic mtDNA deletions as well as profound thiamine deficiency without a clear nutritional cause. Targeted exome sequencing revealed a homozygous c.1672C > T (p.R558C) missense mutation in exon 8 of WFS1 that has previously been reported in a patient with Wolfram syndrome., Conclusion: This case demonstrates how clinical application of next-generation sequencing technology can enhance the diagnosis of patients suspected to have rare genetic disorders. Furthermore, the finding of unexplained thiamine deficiency in a patient with Wolfram syndrome suggests a potential link between WFS1 biology and thiamine metabolism that has implications for the clinical management of Wolfram syndrome patients.

Disorders of oxidative phosphorylation (OXPHOS) have a birth prevalence of ∼1/5,000 and are the most common inborn errors of metabolism. The most common OXPHOS disorder is complex I deficiency. Patients with complex I deficiency present with variable symptoms, such as muscle weakness, cardiomyopathy, developmental delay or regression, blindness, seizures, failure to thrive, liver dysfunction or ataxia. Molecular diagnosis of patients with complex I deficiency is a challenging task due to the clinical heterogeneity of patients and the large number of candidate disease genes, both nuclear-encoded and mitochondrial DNA (mtDNA)-encoded. In this review, we have thoroughly surveyed the literature to identify 149 patients described with both isolated complex I deficiency and pathogenic mutations within nuclear genes. In total, 115 different pathogenic mutations have been reported in 22 different nuclear genes encoding complex I subunits or assembly factors, highlighting the allelic and locus heterogeneity of this disorder. Missense mutations predominate in genes encoding core subunits and some assembly factors while null-type mutations are common in the genes encoding supernumerary subunits and other assembly factors. Despite developments in molecular technology, many patients do not receive molecular diagnosis and no gene has yet been identified that accounts for more than 5% of cases, suggesting that there are likely many disease genes that await discovery., (Copyright © 2011 Wiley Periodicals, Inc.)

Discovering the molecular basis of mitochondrial respiratory chain disease is challenging given the large number of both mitochondrial and nuclear genes that are involved. We report a strategy of focused candidate gene prediction, high-throughput sequencing and experimental validation to uncover the molecular basis of mitochondrial complex I disorders. We created seven pools of DNA from a cohort of 103 cases and 42 healthy controls and then performed deep sequencing of 103 candidate genes to identify 151 rare variants that were predicted to affect protein function. We established genetic diagnoses in 13 of 60 previously unsolved cases using confirmatory experiments, including cDNA complementation to show that mutations in NUBPL and FOXRED1 can cause complex I deficiency. Our study illustrates how large-scale sequencing, coupled with functional prediction and experimental validation, can be used to identify causal mutations in individual cases.

For nearly three decades, the sequence of the human mitochondrial genome (mtDNA) has provided a molecular framework for understanding maternally inherited diseases. However, the vast majority of human mitochondrial disorders are caused by nuclear genome defects, which is not surprising since the mtDNA encodes only 13 proteins. Advances in genomics, mass spectrometry, and computation have only recently made it possible to systematically identify the complement of over 1,000 proteins that comprise the mammalian mitochondrial proteome. Here, we review recent progress in characterizing the mitochondrial proteome and highlight insights into its complexity, tissue heterogeneity, evolutionary origins, and biochemical versatility. We then discuss how this proteome is being used to discover the genetic basis of respiratory chain disorders as well as to expand our definition of mitochondrial disease. Finally, we explore future prospects and challenges for using the mitochondrial proteome as a foundation for systems analysis of the organelle.

Complex I (NADH:ubiquinone oxidoreductase) is the first and largest multimeric complex of the mitochondrial respiratory chain. Human complex I comprises seven subunits encoded by mitochondrial DNA and 38 nuclear-encoded subunits that are assembled together in a process that is only partially understood. To date, mutations causing complex I deficiency have been described in all 14 core subunits, five supernumerary subunits, and four assembly factors. We describe complex I deficiency caused by mutation of the putative complex I assembly factor C20orf7. A candidate region for a lethal neonatal form of complex I deficiency was identified by homozygosity mapping of an Egyptian family with one affected child and two affected pregnancies predicted by enzyme-based prenatal diagnosis. The region was confirmed by microcell-mediated chromosome transfer, and 11 candidate genes encoding potential mitochondrial proteins were sequenced. A homozygous missense mutation in C20orf7 segregated with disease in the family. We show that C20orf7 is peripherally associated with the matrix face of the mitochondrial inner membrane and that silencing its expression with RNAi decreases complex I activity. C20orf7 patient fibroblasts showed an almost complete absence of complex I holoenzyme and were defective at an early stage of complex I assembly, but in a manner distinct from the assembly defects caused by mutations in the assembly factor NDUFAF1. Our results indicate that C20orf7 is crucial in the assembly of complex I and that mutations in C20orf7 cause mitochondrial disease.

The majority of inherited mitochondrial disorders are due to mutations not in the mitochondrial genome (mtDNA) but rather in the nuclear genes encoding proteins targeted to this organelle. Elucidation of the molecular basis for these disorders is limited because only half of the estimated 1,500 mitochondrial proteins have been identified. To systematically expand this catalog, we experimentally and computationally generated eight genome-scale data sets, each designed to provide clues as to mitochondrial localization: targeting sequence prediction, protein domain enrichment, presence of cis-regulatory motifs, yeast homology, ancestry, tandem-mass spectrometry, coexpression and transcriptional induction during mitochondrial biogenesis. Through an integrated analysis we expand the collection to 1,080 genes, which includes 368 novel predictions with a 10% estimated false prediction rate. By combining this expanded inventory with genetic intervals linked to disease, we have identified candidate genes for eight mitochondrial disorders, leading to the discovery of mutations in MPV17 that result in hepatic mtDNA depletion syndrome. The integrative approach promises to better define the role of mitochondria in both rare and common human diseases.