Genome Sequencing for Diagnosing Rare Diseases.

BACKGROUND: Genetic variants that cause rare disorders may remain elusive even after expansive testing, such as exome sequencing. The diagnostic yield of genome sequencing, particularly after a negative evaluation, remains poorly defined. METHODS: We sequenced and analyzed the genomes of families with diverse phenotypes who were suspected to have a rare monogenic disease and for whom genetic testing had not revealed a diagnosis, as well as the genomes of a replication cohort at an independent clinical center. RESULTS: We sequenced the genomes of 822 families (744 in the initial cohort and 78 in the replication cohort) and made a molecular diagnosis in 218 of 744 families (29.3%). Of the 218 families, 61 (28.0%) - 8.2% of families in the initial cohort - had variants that required genome sequencing for identification, including coding variants, intronic variants, small structural variants, copy-neutral inversions, complex rearrangements, and tandem repeat expansions. Most families in which a molecular diagnosis was made after previous nondiagnostic exome sequencing (63.5%) had variants that could be detected by reanalysis of the exome-sequence data (53.4%) or by additional analytic methods, such as copy-number variant calling, to exome-sequence data (10.8%). We obtained similar results in the replication cohort: in 33% of the families in which a molecular diagnosis was made, or 8% of the cohort, genome sequencing was required, which showed the applicability of these findings to both research and clinical environments. CONCLUSIONS: The diagnostic yield of genome sequencing in a large, diverse research cohort and in a small clinical cohort of persons who had previously undergone genetic testing was approximately 8% and included several types of pathogenic variation that had not previously been detected by means of exome sequencing or other techniques. (Funded by the National Human Genome Research Institute and others.).

De novo variants in the non-coding spliceosomal snRNA gene RNU4-2 are a frequent cause of syndromic neurodevelopmental disorders.

Around 60% of individuals with neurodevelopmental disorders (NDD) remain undiagnosed after comprehensive genetic testing, primarily of protein-coding genes1. Increasingly, large genome-sequenced cohorts are improving our ability to discover new diagnoses in the non-coding genome. Here, we identify the non-coding RNA RNU4-2 as a novel syndromic NDD gene. RNU4-2 encodes the U4 small nuclear RNA (snRNA), which is a critical component of the U4/U6.U5 tri-snRNP complex of the major spliceosome2. We identify an 18 bp region of RNU4-2 mapping to two structural elements in the U4/U6 snRNA duplex (the T-loop and Stem III) that is severely depleted of variation in the general population, but in which we identify heterozygous variants in 119 individuals with NDD. The vast majority of individuals (77.3%) have the same highly recurrent single base-pair insertion (n.64_65insT). We estimate that variants in this region explain 0.41% of individuals with NDD. We demonstrate that RNU4-2 is highly expressed in the developing human brain, in contrast to its contiguous counterpart RNU4-1 and other U4 homologs, supporting RNU4-2's role as the primary U4 transcript in the brain. Overall, this work underscores the importance of non-coding genes in rare disorders. It will provide a diagnosis to thousands of individuals with NDD worldwide and pave the way for the development of effective treatments for these individuals.

Critical assessment of variant prioritization methods for rare disease diagnosis within the rare genomes project.

BACKGROUND: A major obstacle faced by families with rare diseases is obtaining a genetic diagnosis. The average "diagnostic odyssey" lasts over five years and causal variants are identified in under 50%, even when capturing variants genome-wide. To aid in the interpretation and prioritization of the vast number of variants detected, computational methods are proliferating. Knowing which tools are most effective remains unclear. To evaluate the performance of computational methods, and to encourage innovation in method development, we designed a Critical Assessment of Genome Interpretation (CAGI) community challenge to place variant prioritization models head-to-head in a real-life clinical diagnostic setting. METHODS: We utilized genome sequencing (GS) data from families sequenced in the Rare Genomes Project (RGP), a direct-to-participant research study on the utility of GS for rare disease diagnosis and gene discovery. Challenge predictors were provided with a dataset of variant calls and phenotype terms from 175 RGP individuals (65 families), including 35 solved training set families with causal variants specified, and 30 unlabeled test set families (14 solved, 16 unsolved). We tasked teams to identify causal variants in as many families as possible. Predictors submitted variant predictions with estimated probability of causal relationship (EPCR) values. Model performance was determined by two metrics, a weighted score based on the rank position of causal variants, and the maximum F-measure, based on precision and recall of causal variants across all EPCR values. RESULTS: Sixteen teams submitted predictions from 52 models, some with manual review incorporated. Top performers recalled causal variants in up to 13 of 14 solved families within the top 5 ranked variants. Newly discovered diagnostic variants were returned to two previously unsolved families following confirmatory RNA sequencing, and two novel disease gene candidates were entered into Matchmaker Exchange. In one example, RNA sequencing demonstrated aberrant splicing due to a deep intronic indel in ASNS, identified in trans with a frameshift variant in an unsolved proband with phenotypes consistent with asparagine synthetase deficiency. CONCLUSIONS: Model methodology and performance was highly variable. Models weighing call quality, allele frequency, predicted deleteriousness, segregation, and phenotype were effective in identifying causal variants, and models open to phenotype expansion and non-coding variants were able to capture more difficult diagnoses and discover new diagnoses. Overall, computational models can significantly aid variant prioritization. For use in diagnostics, detailed review and conservative assessment of prioritized variants against established criteria is needed.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Assessment of the evidence yield for the calibrated PP3/BP4 computational recommendations.

Purpose: To investigate the number of rare missense variants observed in human genome sequences by ACMG/AMP PP3/BP4 evidence strength, following the calibrated PP3/BP4 computational recommendations. Methods: Missense variants from the genome sequences of 300 probands from the Rare Genomes Project with suspected rare disease were analyzed using computational prediction tools able to reach PP3_Strong and BP4_Moderate evidence strengths (BayesDel, MutPred2, REVEL, and VEST4). The numbers of variants at each evidence strength were analyzed across disease-associated genes and genome-wide. Results: From a median of 75.5 rare (≤1% allele frequency) missense variants in disease-associated genes per proband, a median of one reached PP3_Strong, 3-5 PP3_Moderate, and 3-5 PP3_Supporting. Most were allocated BP4 evidence (median 41-49 per proband) or were indeterminate (median 17.5-19 per proband). Extending the analysis to all protein-coding genes genome-wide, the number of PP3_Strong variants increased approximately 2.6-fold compared to disease-associated genes, with a median per proband of 1-3 PP3_Strong, 8-16 PP3_Moderate, and 10-17 PP3_Supporting. Conclusion: A small number of variants per proband reached PP3_Strong and PP3_Moderate in 3,424 disease-associated genes, and though not the intended use of the recommendations, also genome-wide. Use of PP3/BP4 evidence as recommended from calibrated computational prediction tools in the clinical diagnostic laboratory is unlikely to inappropriately contribute to the classification of an excessive number of variants as Pathogenic or Likely Pathogenic by ACMG/AMP rules.

Digenic inheritance involving a muscle-specific protein kinase and the giant titin protein causes a skeletal muscle myopathy.

In digenic inheritance, pathogenic variants in two genes must be inherited together to cause disease. Only very few examples of digenic inheritance have been described in the neuromuscular disease field. Here we show that predicted deleterious variants in SRPK3, encoding the X-linked serine/argenine protein kinase 3, lead to a progressive early onset skeletal muscle myopathy only when in combination with heterozygous variants in the TTN gene. The co-occurrence of predicted deleterious SRPK3/TTN variants was not seen among 76,702 healthy male individuals, and statistical modeling strongly supported digenic inheritance as the best-fitting model. Furthermore, double-mutant zebrafish (srpk3-/-; ttn.1+/-) replicated the myopathic phenotype and showed myofibrillar disorganization. Transcriptome data suggest that the interaction of srpk3 and ttn.1 in zebrafish occurs at a post-transcriptional level. We propose that digenic inheritance of deleterious changes impacting both the protein kinase SRPK3 and the giant muscle protein titin causes a skeletal myopathy and might serve as a model for other genetic diseases.

Narrowing the diagnostic gap: Genomes, episignatures, long-read sequencing, and health economic analyses in an exome-negative intellectual disability cohort.

PURPOSE: Genome sequencing (GS)-specific diagnostic rates in prospective tightly ascertained exome sequencing (ES)-negative intellectual disability (ID) cohorts have not been reported extensively. METHODS: ES, GS, epigenetic signatures, and long-read sequencing diagnoses were assessed in 74 trios with at least moderate ID. RESULTS: The ES diagnostic yield was 42 of 74 (57%). GS diagnoses were made in 9 of 32 (28%) ES-unresolved families. Repeated ES with a contemporary pipeline on the GS-diagnosed families identified 8 of 9 single-nucleotide variations/copy-number variations undetected in older ES, confirming a GS-unique diagnostic rate of 1 in 32 (3%). Episignatures contributed diagnostic information in 9% with GS corroboration in 1 of 32 (3%) and diagnostic clues in 2 of 32 (6%). A genetic etiology for ID was detected in 51 of 74 (69%) families. Twelve candidate disease genes were identified. Contemporary ES followed by GS cost US$4976 (95% CI: $3704; $6969) per diagnosis and first-line GS at a cost of $7062 (95% CI: $6210; $8475) per diagnosis. CONCLUSION: Performing GS only in ID trios would be cost equivalent to ES if GS were available at $2435, about a 60% reduction from current prices. This study demonstrates that first-line GS achieves higher diagnostic rate than contemporary ES but at a higher cost.

Inferring compound heterozygosity from large-scale exome sequencing data.

Recessive diseases arise when both copies of a gene are impacted by a damaging genetic variant. When a patient carries two potentially causal variants in a gene, accurate diagnosis requires determining that these variants occur on different copies of the chromosome (that is, are in trans) rather than on the same copy (that is, in cis). However, current approaches for determining phase, beyond parental testing, are limited in clinical settings. Here we developed a strategy for inferring phase for rare variant pairs within genes, leveraging genotypes observed in the Genome Aggregation Database (v2, n = 125,748 exomes). Our approach estimates phase with 96% accuracy, both in trio data and in patients with Mendelian conditions and presumed causal compound heterozygous variants. We provide a public resource of phasing estimates for coding variants and counts per gene of rare variants in trans that can aid interpretation of rare co-occurring variants in the context of recessive disease.

Critical assessment of variant prioritization methods for rare disease diagnosis within the Rare Genomes Project.

Background: A major obstacle faced by rare disease families is obtaining a genetic diagnosis. The average "diagnostic odyssey" lasts over five years, and causal variants are identified in under 50%. The Rare Genomes Project (RGP) is a direct-to-participant research study on the utility of genome sequencing (GS) for diagnosis and gene discovery. Families are consented for sharing of sequence and phenotype data with researchers, allowing development of a Critical Assessment of Genome Interpretation (CAGI) community challenge, placing variant prioritization models head-to-head in a real-life clinical diagnostic setting. Methods: Predictors were provided a dataset of phenotype terms and variant calls from GS of 175 RGP individuals (65 families), including 35 solved training set families, with causal variants specified, and 30 test set families (14 solved, 16 unsolved). The challenge tasked teams with identifying the causal variants in as many test set families as possible. Ranked variant predictions were submitted with estimated probability of causal relationship (EPCR) values. Model performance was determined by two metrics, a weighted score based on rank position of true positive causal variants and maximum F-measure, based on precision and recall of causal variants across EPCR thresholds. Results: Sixteen teams submitted predictions from 52 models, some with manual review incorporated. Top performing teams recalled the causal variants in up to 13 of 14 solved families by prioritizing high quality variant calls that were rare, predicted deleterious, segregating correctly, and consistent with reported phenotype. In unsolved families, newly discovered diagnostic variants were returned to two families following confirmatory RNA sequencing, and two prioritized novel disease gene candidates were entered into Matchmaker Exchange. In one example, RNA sequencing demonstrated aberrant splicing due to a deep intronic indel in ASNS, identified in trans with a frameshift variant, in an unsolved proband with phenotype overlap with asparagine synthetase deficiency. Conclusions: By objective assessment of variant predictions, we provide insights into current state-of-the-art algorithms and platforms for genome sequencing analysis for rare disease diagnosis and explore areas for future optimization. Identification of diagnostic variants in unsolved families promotes synergy between researchers with clinical and computational expertise as a means of advancing the field of clinical genome interpretation.

Inferring compound heterozygosity from large-scale exome sequencing data.

Recessive diseases arise when both the maternal and the paternal copies of a gene are impacted by a damaging genetic variant in the affected individual. When a patient carries two different potentially causal variants in a gene for a given disorder, accurate diagnosis requires determining that these two variants occur on different copies of the chromosome (i.e., are in trans) rather than on the same copy (i.e. in cis). However, current approaches for determining phase, beyond parental testing, are limited in clinical settings. We developed a strategy for inferring phase for rare variant pairs within genes, leveraging genotypes observed in exome sequencing data from the Genome Aggregation Database (gnomAD v2, n=125,748). When applied to trio data where phase can be determined by transmission, our approach estimates phase with 95.7% accuracy and remains accurate even for very rare variants (allele frequency < 1×10-4). We also correctly phase 95.9% of variant pairs in a set of 293 patients with Mendelian conditions carrying presumed causal compound heterozygous variants. We provide a public resource of phasing estimates from gnomAD, including phasing estimates for coding variants across the genome and counts per gene of rare variants in trans, that can aid interpretation of rare co-occurring variants in the context of recessive disease.

Unique Capabilities of Genome Sequencing for Rare Disease Diagnosis.

Background: Causal variants underlying rare disorders may remain elusive even after expansive gene panels or exome sequencing (ES). Clinicians and researchers may then turn to genome sequencing (GS), though the added value of this technique and its optimal use remain poorly defined. We therefore investigated the advantages of GS within a phenotypically diverse cohort. Methods: GS was performed for 744 individuals with rare disease who were genetically undiagnosed. Analysis included review of single nucleotide, indel, structural, and mitochondrial variants. Results: We successfully solved 218/744 (29.3%) cases using GS, with most solves involving established disease genes (157/218, 72.0%). Of all solved cases, 148 (67.9%) had previously had non-diagnostic ES. We systematically evaluated the 218 causal variants for features requiring GS to identify and 61/218 (28.0%) met these criteria, representing 8.2% of the entire cohort. These included small structural variants (13), copy neutral inversions and complex rearrangements (8), tandem repeat expansions (6), deep intronic variants (15), and coding variants that may be more easily found using GS related to uniformity of coverage (19). Conclusion: We describe the diagnostic yield of GS in a large and diverse cohort, illustrating several types of pathogenic variation eluding ES or other techniques. Our results reveal a higher diagnostic yield of GS, supporting the utility of a genome-first approach, with consideration of GS as a secondary or tertiary test when higher-resolution structural variant analysis is needed or there is a strong clinical suspicion for a condition and prior targeted genetic testing has been negative.

Oral Coenzyme Q10 supplementation leads to better preservation of kidney function in steroid-resistant nephrotic syndrome due to primary Coenzyme Q10 deficiency.

Primary Coenzyme Q10 (CoQ10) deficiency is an ultra-rare disorder caused by defects in genes involved in CoQ10 biosynthesis leading to multidrug-resistant nephrotic syndrome as the hallmark kidney manifestation. Promising early results have been reported anecdotally with oral CoQ10 supplementation. However, the long-term efficacy and optimal prescription remain to be established. In a global effort, we collected and analyzed information from 116 patients who received CoQ10 supplements for primary CoQ10 deficiency due to biallelic pathogenic variants in either the COQ2, COQ6 or COQ8B genes. Median duration of follow up on treatment was two years. The effect of treatment on proteinuria was assessed, and kidney survival was analyzed in 41 patients younger than 18 years with chronic kidney disease stage 1-4 at the start of treatment compared with that of an untreated cohort matched by genotype, age, kidney function, and proteinuria. CoQ10 supplementation was associated with a substantial and significant sustained reduction of proteinuria by 88% at 12 months. Complete remission of proteinuria was more frequently observed in COQ6 disease. CoQ10 supplementation led to significantly better preservation of kidney function (5-year kidney failure-free survival 62% vs. 19%) with an improvement in general condition and neurological manifestations. Side effects of treatment were uncommon and mild. Thus, our findings indicate that all patients diagnosed with primary CoQ10 deficiency should receive early and life-long CoQ10 supplementation to decelerate the progression of kidney disease and prevent further damage to other organs.

Variation of the clinical spectrum and genotype-phenotype associations in Coenzyme Q10 deficiency associated glomerulopathy.

Primary Coenzyme Q10 deficiency is a rare mitochondriopathy with a wide spectrum of organ involvement, including steroid-resistant nephrotic syndrome mainly associated with disease-causing variants in the genes COQ2, COQ6 or COQ8B. We performed a systematic literature review, PodoNet, mitoNET, and CCGKDD registries queries and an online survey, collecting comprehensive clinical and genetic data of 251 patients spanning 173 published (47 updated) and 78 new cases. Kidney disease was first diagnosed at median age 1.0, 1.2 and 9.8 years in individuals with disease-causing variants in COQ2, COQ6 and COQ8B, respectively. Isolated kidney involvement at diagnosis occurred in 34% of COQ2, 10.8% of COQ6 and 70.7% of COQ8B variant individuals. Classic infantile multiorgan involvement comprised 22% of the COQ2 variant cohort while 47% of them developed neurological symptoms at median age 2.7 years. The association of steroid-resistant nephrotic syndrome and sensorineural hearing loss was confirmed as the distinctive phenotype of COQ6 variants, with hearing impairment manifesting at average age three years. None of the patients with COQ8B variants, but 50% of patients with COQ2 and COQ6 variants progressed to kidney failure by age five. At adult age, kidney survival was equally poor (20-25%) across all disorders. A number of sequence variants, including putative local founder mutations, had divergent clinical presentations, in terms of onset age, kidney and non-kidney manifestations and kidney survival. Milder kidney phenotype was present in those with biallelic truncating variants within the COQ8B variant cohort. Thus, significant intra- and inter-familial phenotype variability was observed, suggesting both genetic and non-genetic modifiers of disease severity.

Clinical implementation of RNA sequencing for Mendelian disease diagnostics.

BACKGROUND: Lack of functional evidence hampers variant interpretation, leaving a large proportion of individuals with a suspected Mendelian disorder without genetic diagnosis after whole genome or whole exome sequencing (WES). Research studies advocate to further sequence transcriptomes to directly and systematically probe gene expression defects. However, collection of additional biopsies and establishment of lab workflows, analytical pipelines, and defined concepts in clinical interpretation of aberrant gene expression are still needed for adopting RNA sequencing (RNA-seq) in routine diagnostics. METHODS: We implemented an automated RNA-seq protocol and a computational workflow with which we analyzed skin fibroblasts of 303 individuals with a suspected mitochondrial disease that previously underwent WES. We also assessed through simulations how aberrant expression and mono-allelic expression tests depend on RNA-seq coverage. RESULTS: We detected on average 12,500 genes per sample including around 60% of all disease genes-a coverage substantially higher than with whole blood, supporting the use of skin biopsies. We prioritized genes demonstrating aberrant expression, aberrant splicing, or mono-allelic expression. The pipeline required less than 1 week from sample preparation to result reporting and provided a median of eight disease-associated genes per patient for inspection. A genetic diagnosis was established for 16% of the 205 WES-inconclusive cases. Detection of aberrant expression was a major contributor to diagnosis including instances of 50% reduction, which, together with mono-allelic expression, allowed for the diagnosis of dominant disorders caused by haploinsufficiency. Moreover, calling aberrant splicing and variants from RNA-seq data enabled detecting and validating splice-disrupting variants, of which the majority fell outside WES-covered regions. CONCLUSION: Together, these results show that streamlined experimental and computational processes can accelerate the implementation of RNA-seq in routine diagnostics.

DNAJC30 defect: a frequent cause of recessive Leber hereditary optic neuropathy and Leigh syndrome.

The recent description of biallelic DNAJC30 variants in Leber hereditary optic neuropathy (LHON) and Leigh syndrome challenged the longstanding assumption for LHON to be exclusively maternally inherited and broadened the genetic spectrum of Leigh syndrome, the most frequent paediatric mitochondrial disease. Herein, we characterize 28 so far unreported individuals from 26 families carrying a homozygous DNAJC30 p.Tyr51Cys founder variant, 24 manifesting with LHON, two manifesting with Leigh syndrome, and two remaining asymptomatic. This collection of unreported variant carriers confirms sex-dependent incomplete penetrance of the homozygous variant given a significant male predominance of disease and the report of asymptomatic homozygous variant carriers. The autosomal recessive LHON patients demonstrate an earlier age of disease onset and a higher rate of idebenone-treated and spontaneous recovery of vision in comparison to reported figures for maternally inherited disease. Moreover, the report of two additional patients with childhood- or adult-onset Leigh syndrome further evidences the association of DNAJC30 with Leigh syndrome, previously only reported in a single childhood-onset case.

Leigh Syndrome: A Study of 209 Patients at the Beijing Children's Hospital.

OBJECTIVE: Leigh syndrome (LS) is a heterogeneous neurodegenerative disease and the most frequent pediatric manifestation of mitochondrial disease. In the largest patient collection to date, this study aimed to provide new insights into the clinical and genetic spectrum of LS, defect-specific associations, and predictors of disease course and survival. METHODS: Clinical, metabolic, neuroimaging, onset, and survival data were collected from the medical records of 209 patients referred to the Beijing Children's Hospital with symmetrical basal ganglia and/or brainstem neuroimaging changes indicative of LS by 30 centers from the Chinese network of mitochondrial disease (mitoC-NET) between January 2013 and July 2021 for exploratory analysis. RESULTS: Pathogenic variants were identified in 52 genes, most frequently MT-ATP6, SURF1, and PDHA1. Maternally inherited variants accounted for 42% (heteroplasmy level ≥90% in 64%). Phenotypes spanned 92 Human Phenotype Ontology terms. Elevated serum lactate (144/195), global developmental delay (142/209), and developmental regression (103/209) were most frequent. Discriminating neuroimaging and/or clinical features were identified for MT-ATP6 (m.9176T>C), MT-ND5, PDHA1, SUCLG1, and SURF1. Poorest survival was associated with MT-ND5, MT-ATP6 (m.8993T>C and m.9176T>C), SURF1, and ALDH5A1 (≤50% 3 year's survival), in contrast to milder defects with specific treatment (ECHS1 and SLC19A3, 100% 3 year's survival). INTERPRETATION: Our data define phenotype, onset, and survival of LS in a defect-specific manner, identifying features discriminating between genetic defects and predictive of disease outcome. These findings are essential to early diagnosis, in optimizing family counseling, and to the design and monitoring of future clinical trials, the next frontier of LS research. ANN NEUROL 2022;91:466-482.

The genetics of mitochondrial disease: dissecting mitochondrial pathology using multi-omic pipelines.

Mitochondria play essential roles in numerous metabolic pathways including the synthesis of adenosine triphosphate through oxidative phosphorylation. Clinically, mitochondrial diseases occur when there is mitochondrial dysfunction - manifesting at any age and affecting any organ system; tissues with high energy requirements, such as muscle and the brain, are often affected. The clinical heterogeneity is parallel to the degree of genetic heterogeneity associated with mitochondrial dysfunction. Around 10% of human genes are predicted to have a mitochondrial function, and defects in over 300 genes are reported to cause mitochondrial disease. Some involve the mitochondrial genome (mtDNA), but the vast majority occur within the nuclear genome. Except for a few specific genetic defects, there remains no cure for mitochondrial diseases, which means that a genetic diagnosis is imperative for genetic counselling and the provision of reproductive options for at-risk families. Next-generation sequencing strategies, particularly exome and whole-genome sequencing, have revolutionised mitochondrial diagnostics such that the traditional muscle biopsy has largely been replaced with a minimally-invasive blood sample for an unbiased approach to genetic diagnosis. Where these genomic approaches have not identified a causative defect, or where there is insufficient support for pathogenicity, additional functional investigations are required. The application of supplementary 'omics' technologies, including transcriptomics, proteomics, and metabolomics, has the potential to greatly improve diagnostic strategies. This review aims to demonstrate that whilst a molecular diagnosis can be achieved for many cases through next-generation sequencing of blood DNA, the use of patient tissues and an integrated, multidisciplinary multi-omics approach is pivotal for the diagnosis of more challenging cases. Moreover, the analysis of clinically relevant tissues from affected individuals remains crucial for understanding the molecular mechanisms underlying mitochondrial pathology. © 2021 The Authors. The Journal of Pathology published by John Wiley & Sons, Ltd. on behalf of The Pathological Society of Great Britain and Ireland.

Impaired complex I repair causes recessive Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy (LHON) is the most frequent mitochondrial disease and was the first to be genetically defined by a point mutation in mitochondrial DNA (mtDNA). A molecular diagnosis is achieved in up to 95% of cases, the vast majority of which are accounted for by 3 mutations within mitochondrial complex I subunit-encoding genes in the mtDNA (mtLHON). Here, we resolve the enigma of LHON in the absence of pathogenic mtDNA mutations. We describe biallelic mutations in a nuclear encoded gene, DNAJC30, in 33 unsolved patients from 29 families and establish an autosomal recessive mode of inheritance for LHON (arLHON), which to date has been a prime example of a maternally inherited disorder. Remarkably, all hallmarks of mtLHON were recapitulated, including incomplete penetrance, male predominance, and significant idebenone responsivity. Moreover, by tracking protein turnover in patient-derived cell lines and a DNAJC30-knockout cellular model, we measured reduced turnover of specific complex I N-module subunits and a resultant impairment of complex I function. These results demonstrate that DNAJC30 is a chaperone protein needed for the efficient exchange of complex I subunits exposed to reactive oxygen species and integral to a mitochondrial complex I repair mechanism, thereby providing the first example to our knowledge of a disease resulting from impaired exchange of assembled respiratory chain subunits.