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Mitochondrial disorders are clinically heterogeneous and comprise over 350 different genetic conditions. However, the molecular diagnosis is unknown in ~50% of cases, partly due to some genomic regions being refractory to standard genomic analysis. One such region is the ATAD3 locus consisting of 3 highly homologous tandemly arrayed genes (ATAD3C, ATAD3B and ATAD3A) encoding mitochondrial proteins implicated in processes including cholesterol metabolism, and mitochondrial replication, dynamics and morphology. Recessive deletions and dominant duplications in this locus have recently been reported to cause rare, lethal perinatal mitochondrial disorders characterised by pontocerebellar hypoplasia or cardiomyopathy, respectively. We report 17 subjects from 16 unrelated families with cardiomyopathy, persistent hyperlactacidemia, encephalopathy and frequently corneal clouding or cataracts due to de novo ATAD3 duplications. The six different 68 Kb duplications were consistently identifiable from whole genome and exome sequencing, but usually missed on microarray. The duplications all resulted in the formation of an identical chimeric ATAD3A/ATAD3C fusion protein, which appears to act in a dominant manner causing altered ATAD3 complexes and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. In our experience, the ATAD3 locus is one of the five most common causes of nuclear-encoded paediatric mitochondrial disease but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies.

Mitochondrial disorders are clinically heterogeneous and comprise over 350 different genetic conditions. However, the molecular diagnosis is unknown in ~50% of cases, partly due to some genomic regions being refractory to standard genomic analysis. One such region is the ATAD3 locus consisting of 3 highly homologous tandemly arrayed genes (ATAD3C, ATAD3B and ATAD3A) encoding mitochondrial proteins implicated in processes including cholesterol metabolism, and mitochondrial replication, dynamics and morphology. Recessive deletions and dominant duplications in this locus have recently been reported to cause rare, lethal perinatal mitochondrial disorders characterised by pontocerebellar hypoplasia or cardiomyopathy, respectively. We report 17 subjects from 16 unrelated families with cardiomyopathy, persistent hyperlactacidemia, encephalopathy and frequently corneal clouding or cataracts due to de novo ATAD3 duplications. The six different 68 Kb duplications were consistently identifiable from whole genome and exome sequencing, but usually missed on microarray. The duplications all resulted in the formation of an identical chimeric ATAD3A/ATAD3C fusion protein, which appears to act in a dominant manner causing altered ATAD3 complexes and a striking reduction in mitochondrial oxidative phosphorylation complex I and its activity in heart tissue. In our experience, the ATAD3 locus is one of the five most common causes of nuclear-encoded paediatric mitochondrial disease but the repetitive nature of the locus means ATAD3 diagnoses may be frequently missed by current genomic strategies.

Asparagine-linked glycosylation 13 homolog (ALG13) encodes a nonredundant, highly conserved, X-linked uridine diphosphate (UDP)-N-acetylglucosaminyltransferase required for the synthesis of lipid linked oligosaccharide precursor and proper N-linked glycosylation. De novo variants in ALG13 underlie a form of early infantile epileptic encephalopathy known as EIEE36, but given its essential role in glycosylation, it is also considered a congenital disorder of glycosylation (CDG), ALG13-CDG. Twenty-four previously reported ALG13-CDG cases had de novo variants, but surprisingly, unlike most forms of CDG, ALG13-CDG did not show the anticipated glycosylation defects, typically detected by altered transferrin glycosylation. Structural homology modeling of two recurrent de novo variants, p.A81T and p.N107S, suggests both are likely to impact the function of ALG13. Using a corresponding ALG13-deficient yeast strain, we show that expressing yeast ALG13 with either of the highly conserved hotspot variants rescues the observed growth defect, but not its glycosylation abnormality. We present molecular and clinical data on 29 previously unreported individuals with de novo variants in ALG13. This more than doubles the number of known cases. A key finding is that a vast majority of the individuals presents with West syndrome, a feature shared with other CDG types. Among these, the initial epileptic spasms best responded to adrenocorticotropic hormone or prednisolone, while clobazam and felbamate showed promise for continued epilepsy treatment. A ketogenic diet seems to play an important role in the treatment of these individuals.Copyright © 2020 SSIEM

Introduction: Implementation of rapid genomic testing in neonatal and paediatric intensive care units (NICUs/PICUs) is gathering momentum, and requires the development of systems capable of consistent delivery across multi-site networks. Method(s): We developed a rapid genomic diagnosis program involving 10 Australian hospitals and two laboratories with the aim of providing test results in <5 days for acutely unwell paediatric patients with suspected monogenic disorders. Rapid exome sequencing (rES) was performed as trios when possible, and analysis utilised multidisciplinary expertise. Experience was shared between clinical sites, laboratories, and professional groups to enable collective learning. Result(s): The program considered 123 patients for rES over 10 months, and approved 114 (93%). Five families declined testing (4.4%), and nine (7.9%) were withdrawn due to change in clinical circumstances. Of 100 patients tested, 51 received a diagnosis. Eleven of the diagnoses (21%) were made using approaches augmenting standard ES analysis: mitochondrial genome sequencing, ES-based copy number analysis, matchmaking of emerging genes, reverse phenotyping and RNA analysis. Median time from hospital admission to consent was 6 days (range 0-64 days); median time from sample receipt to clinical ES report was 3 days (range 2-7 days). The total cost of testing was AU$1,123,000/701,638 (AU$11,230/7,016 per case). Changes in management following a result occurred in 77% of diagnosed patients and 10% of undiagnosed patients. Conclusion(s): We demonstrate the feasibility of a national, highly integrated clinical-laboratory approach to rapid genomic diagnosis, which delivers results within a timeframe relevant to acute paediatrics, while optimising clinical utility and resource allocation.

Introduction: Standard rapid genomic testing techniques analyse nuclear DNA variants using exome and/or genome sequencing (ES/GS). Rapid mtDNA analysis is not routinely available, particularly in centres performing ES, which does not deliver clinical-grade mtDNA sequencing. We describe our experience using rapid mtDNA sequencing in tandem with an ES-based rapid genomic diagnosis program as part of the Australian Genomics Acute Care flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid ES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): ES was non-diagnostic in both infants. mtDNA sequencing identified a single large mtDNA deletion in both infants, diagnostic of Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 hours 55 minutes and 54 hours 25 minutes, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with ES results in additional diagnoses in seriously ill children with suspected mitochondrial pathology, suggesting that ES alone may be insufficient in this setting. When designing rapid genomic diagnosis programs, centres should consider incorporating mtDNA amplification and analysis in individuals with suspected mitochondrial pathology, by combining ES and mtDNA sequencing in tandem, or analysing mtDNA data from GS, which captures the mitochondrial genome.

Rapid genomic diagnosis programs are transforming rare disease diagnosis in paediatric acute care. Determining the pathogenicity of variants of uncertain significance within clinically relevant timeframes remains challenging. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing, identifying a novel homozygous ASNS splice-site variant (NM_133436.3:c.1476+1G>A) of uncertain significance in 75.5 hours. Rapid ASNS splicing studies using blood-derived mRNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies were reported within 10 days and led to reclassification of c.1476+1G>A as pathogenic at age 27 days, diagnosing asparagine synthetase deficiency (MIM 615574). Intensive care was redirected towards palliation. Time from initial exome sequencing report to variant reclassification was 22 days. Cost analysis based on hospitalisation length of stay for the proband and his similarly affected deceased sibling demonstrated that early diagnosis led to a reduction in hospitalisation costs by AU$117,800. We highlight diagnostic benefits of adjunct RNA testing to confirm pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine.Copyright © 2020

Asparagine-linked glycosylation 13 homolog (ALG13) encodes a nonredundant, highly conserved, X-linked uridine diphosphate (UDP)-N-acetylglucosaminyltransferase required for the synthesis of lipid linked oligosaccharide precursor and proper N-linked glycosylation. De novo variants in ALG13 underlie a form of early infantile epileptic encephalopathy known as EIEE36, but given its essential role in glycosylation, it is also considered a congenital disorder of glycosylation (CDG), ALG13-CDG. Twenty-four previously reported ALG13-CDG cases had de novo variants, but surprisingly, unlike most forms of CDG, ALG13-CDG did not show the anticipated glycosylation defects, typically detected by altered transferrin glycosylation. Structural homology modeling of two recurrent de novo variants, p.A81T and p.N107S, suggests both are likely to impact the function of ALG13. Using a corresponding ALG13-deficient yeast strain, we show that expressing yeast ALG13 with either of the highly conserved hotspot variants rescues the observed growth defect, but not its glycosylation abnormality. We present molecular and clinical data on 29 previously unreported individuals with de novo variants in ALG13. This more than doubles the number of known cases. A key finding is that a vast majority of the individuals presents with West syndrome, a feature shared with other CDG types. Among these, the initial epileptic spasms best responded to adrenocorticotropic hormone or prednisolone, while clobazam and felbamate showed promise for continued epilepsy treatment. A ketogenic diet seems to play an important role in the treatment of these individuals.Copyright © 2020 SSIEM

Introduction: Implementation of rapid genomic testing in neonatal and paediatric intensive care units (NICUs/PICUs) is gathering momentum, and requires the development of systems capable of consistent delivery across multi-site networks. Method(s): We developed a rapid genomic diagnosis program involving 10 Australian hospitals and two laboratories with the aim of providing test results in <5 days for acutely unwell paediatric patients with suspected monogenic disorders. Rapid exome sequencing (rES) was performed as trios when possible, and analysis utilised multidisciplinary expertise. Experience was shared between clinical sites, laboratories, and professional groups to enable collective learning. Result(s): The program considered 123 patients for rES over 10 months, and approved 114 (93%). Five families declined testing (4.4%), and nine (7.9%) were withdrawn due to change in clinical circumstances. Of 100 patients tested, 51 received a diagnosis. Eleven of the diagnoses (21%) were made using approaches augmenting standard ES analysis: mitochondrial genome sequencing, ES-based copy number analysis, matchmaking of emerging genes, reverse phenotyping and RNA analysis. Median time from hospital admission to consent was 6 days (range 0-64 days); median time from sample receipt to clinical ES report was 3 days (range 2-7 days). The total cost of testing was AU$1,123,000/701,638 (AU$11,230/7,016 per case). Changes in management following a result occurred in 77% of diagnosed patients and 10% of undiagnosed patients. Conclusion(s): We demonstrate the feasibility of a national, highly integrated clinical-laboratory approach to rapid genomic diagnosis, which delivers results within a timeframe relevant to acute paediatrics, while optimising clinical utility and resource allocation.

Introduction: Standard rapid genomic testing techniques analyse nuclear DNA variants using exome and/or genome sequencing (ES/GS). Rapid mtDNA analysis is not routinely available, particularly in centres performing ES, which does not deliver clinical-grade mtDNA sequencing. We describe our experience using rapid mtDNA sequencing in tandem with an ES-based rapid genomic diagnosis program as part of the Australian Genomics Acute Care flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid ES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): ES was non-diagnostic in both infants. mtDNA sequencing identified a single large mtDNA deletion in both infants, diagnostic of Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 hours 55 minutes and 54 hours 25 minutes, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with ES results in additional diagnoses in seriously ill children with suspected mitochondrial pathology, suggesting that ES alone may be insufficient in this setting. When designing rapid genomic diagnosis programs, centres should consider incorporating mtDNA amplification and analysis in individuals with suspected mitochondrial pathology, by combining ES and mtDNA sequencing in tandem, or analysing mtDNA data from GS, which captures the mitochondrial genome.

Rapid genomic diagnosis programs are transforming rare disease diagnosis in paediatric acute care. Determining the pathogenicity of variants of uncertain significance within clinically relevant timeframes remains challenging. A ventilated newborn with cerebellar hypoplasia underwent rapid exome sequencing, identifying a novel homozygous ASNS splice-site variant (NM_133436.3:c.1476+1G>A) of uncertain significance in 75.5 hours. Rapid ASNS splicing studies using blood-derived mRNA from the family trio confirmed a consistent pattern of abnormal splicing induced by the variant (cryptic 5' splice-site or exon 12 skipping) with absence of normal ASNS splicing in the proband. Splicing studies were reported within 10 days and led to reclassification of c.1476+1G>A as pathogenic at age 27 days, diagnosing asparagine synthetase deficiency (MIM 615574). Intensive care was redirected towards palliation. Time from initial exome sequencing report to variant reclassification was 22 days. Cost analysis based on hospitalisation length of stay for the proband and his similarly affected deceased sibling demonstrated that early diagnosis led to a reduction in hospitalisation costs by AU$117,800. We highlight diagnostic benefits of adjunct RNA testing to confirm pathogenicity of splicing variants identified via rapid genomic testing pipelines for precision and preventative medicine.Copyright © 2020

Background/Aim: Implementation of rapid genomic testing in neonatal and pediatric intensive care units (NICUs/PICUs) is gathering momentum, and requires the development of systems capable of consistent delivery across multiple sites. Method(s): We developed a rapid genomic diagnosis program involving 10 Australian hospitals and two laboratories with the aim of providing test results in <5 days for acutely unwell pediatric patients with suspected monogenic disorders. Rapid exome sequencing (rES) was performed as trios when possible, and analysis utilized multidisciplinary expertise. Experience was shared between clinical sites, laboratories, and professional groups to enable collective learning. Result(s): The program considered 123 patients for rES over 10 months, and approved 114 (93%). Five families declined testing (4.4%), and nine (7.9%) were withdrawn due to change in clinical circumstances. Of 100 patients tested, 53 received a diagnosis. Twelve of the diagnoses (23%) were made using approaches augmenting standard ES analysis: mitochondrial genome sequencing, ES-based copy number analysis, matchmaking of emerging genes, reverse phenotyping and RNA analysis. Median time from hospital admission to consent was 6 days (range 0-64 days); median time from sample receipt to clinical ES report was 3 days (range 2-7 days). The total cost of testing was AU $1,123,000 (AU$11,230 per case). Changesin management following a result occurred in 77% of diagnosed patients and 10% of undiag-nosed patients. Conclusion(s): We demonstrate the feasibility of a national, highly integrated clinical-laboratory approach to rapid genomic diagnosis, which delivers results within a timeframe relevant to acute pediatrics, while optimizing clinical utility and resource allocation.

Introduction: Rapid genomic diagnosis programs are revolutionizing pediatric and neonatal intensive care. Determining pathogenicity of variants of uncertain significance within clinically relevant timeframes remains challenging. We describe rapid mRNA splicing analysis of a variant of uncertain significance detected by rapid exome sequencing (rES). Case: A male newborn ventilated for apnoea with microcephaly and cerebellar hypoplasia, whose sibling died in similar circumstances, underwent rES, identifying anovel homozygous ASNS splicing variant of uncertain significance in 75.5 h (Chr7(GRCh37):g.97482371C>T; NM-133436.3(ASNS):c.1476+1G>A). Method(s): ASNS splicing was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing of mRNA from whole blood collected from the proband and parents, and cryopreserved fibroblasts from the deceased sibling, with simultaneous Sanger sequencing-based segregation of the variant in the deceased sibling. Hospitalization costs were estimated based on average daily NICU costs and compared to the deceased sibling. Result(s): mRNA splicing analysis in the proband and deceased sibling, also homozygous for the variant, confirmed abnormal ASNS splicing with no residual normal splicing. The variant was reclassified as pathogenic at age 27 days, diagnosing asparagine synthetase deficiency (MIM 615574). Intensive care was redirected towards palliation. Time from initial rES report to variant reclassification was 22 days. Based on the difference in length of stay, early diagnosis reduced hospitalization costs by $85,500. Conclusion(s): Rapid mRNA splicing analysis allowed prompt variant reclassification, redirecting intensive care within a clinically relevant timeframe. This case highlights the benefits of integrating studies to determine pathogenic-ity of variants of uncertain significance into rapid genomic diagnosis programs.

Objective: While standard rapid genomic testing techniques analyse nuclear DNA variants using whole exome (WES) and/or whole genome (WGS) sequencing, rapid mtDNA analysis is not usually performed. We describe our experience using rapid mtDNA sequencing in tandem with WES in the Australian Genomics Acute Care Genomics flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid WES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): WES was non-diagnostic. mtDNA sequencing identified a single large-scale mtDNA deletion in both infants, consistent with Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 hours 55 minutes and 54 hours 25 minutes, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with WES Results in additional diagnoses in seriously ill children with suspected mitochondrial disease, suggesting that WES alone may be insufficient in this setting. When designing rapid genomic diagnostic programs, centres should consider mtDNA analysis, combining WES and mtDNA sequencing in tandem, or analysing mtDNA data from WGS, which captures the mitochondrial genome.Copyright © 2018

Introduction: Standard rapid genomic testing techniques analyze nuclear DNA variants using exome and/or genome sequencing (ES/GS). Rapid mtDNA analysis is not routinely available, particularly in centres performing ES, which does not deliver clinical-grade mtDNA sequencing. We describe our experience using rapid mtDNA sequencing in tandem with an ES-based rapid genomic diagnosis program as part of the Australian Genomics Acute Care flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid ES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): ES was non-diagnostic in both infants. mtDNA sequencing identified a single large mtDNA deletion in both infants, diagnostic of Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 h 55 min and 54 h 25 min, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with ES results in additional diagnoses in seriously ill children with suspected mitochondrial pathology, suggesting that ES alone may be insufficient in this setting. When designing rapid genomic diagnosis programs, centres should consider incorporating mtDNA amplification and analysis in individuals with suspected mitochondrial pathology, by combining ES and mtDNA sequencing in tandem, or analysing mtDNA data from GS, which captures the mitochondrial genome.

Background/Aim: Implementation of rapid genomic testing in neonatal and pediatric intensive care units (NICUs/PICUs) is gathering momentum, and requires the development of systems capable of consistent delivery across multiple sites. Method(s): We developed a rapid genomic diagnosis program involving 10 Australian hospitals and two laboratories with the aim of providing test results in <5 days for acutely unwell pediatric patients with suspected monogenic disorders. Rapid exome sequencing (rES) was performed as trios when possible, and analysis utilized multidisciplinary expertise. Experience was shared between clinical sites, laboratories, and professional groups to enable collective learning. Result(s): The program considered 123 patients for rES over 10 months, and approved 114 (93%). Five families declined testing (4.4%), and nine (7.9%) were withdrawn due to change in clinical circumstances. Of 100 patients tested, 53 received a diagnosis. Twelve of the diagnoses (23%) were made using approaches augmenting standard ES analysis: mitochondrial genome sequencing, ES-based copy number analysis, matchmaking of emerging genes, reverse phenotyping and RNA analysis. Median time from hospital admission to consent was 6 days (range 0-64 days); median time from sample receipt to clinical ES report was 3 days (range 2-7 days). The total cost of testing was AU $1,123,000 (AU$11,230 per case). Changesin management following a result occurred in 77% of diagnosed patients and 10% of undiag-nosed patients. Conclusion(s): We demonstrate the feasibility of a national, highly integrated clinical-laboratory approach to rapid genomic diagnosis, which delivers results within a timeframe relevant to acute pediatrics, while optimizing clinical utility and resource allocation.

Introduction: Rapid genomic diagnosis programs are revolutionizing pediatric and neonatal intensive care. Determining pathogenicity of variants of uncertain significance within clinically relevant timeframes remains challenging. We describe rapid mRNA splicing analysis of a variant of uncertain significance detected by rapid exome sequencing (rES). Case: A male newborn ventilated for apnoea with microcephaly and cerebellar hypoplasia, whose sibling died in similar circumstances, underwent rES, identifying anovel homozygous ASNS splicing variant of uncertain significance in 75.5 h (Chr7(GRCh37):g.97482371C>T; NM-133436.3(ASNS):c.1476+1G>A). Method(s): ASNS splicing was assessed by reverse transcriptase-polymerase chain reaction (RT-PCR) and Sanger sequencing of mRNA from whole blood collected from the proband and parents, and cryopreserved fibroblasts from the deceased sibling, with simultaneous Sanger sequencing-based segregation of the variant in the deceased sibling. Hospitalization costs were estimated based on average daily NICU costs and compared to the deceased sibling. Result(s): mRNA splicing analysis in the proband and deceased sibling, also homozygous for the variant, confirmed abnormal ASNS splicing with no residual normal splicing. The variant was reclassified as pathogenic at age 27 days, diagnosing asparagine synthetase deficiency (MIM 615574). Intensive care was redirected towards palliation. Time from initial rES report to variant reclassification was 22 days. Based on the difference in length of stay, early diagnosis reduced hospitalization costs by $85,500. Conclusion(s): Rapid mRNA splicing analysis allowed prompt variant reclassification, redirecting intensive care within a clinically relevant timeframe. This case highlights the benefits of integrating studies to determine pathogenic-ity of variants of uncertain significance into rapid genomic diagnosis programs.

Objective: While standard rapid genomic testing techniques analyse nuclear DNA variants using whole exome (WES) and/or whole genome (WGS) sequencing, rapid mtDNA analysis is not usually performed. We describe our experience using rapid mtDNA sequencing in tandem with WES in the Australian Genomics Acute Care Genomics flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid WES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): WES was non-diagnostic. mtDNA sequencing identified a single large-scale mtDNA deletion in both infants, consistent with Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 hours 55 minutes and 54 hours 25 minutes, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with WES Results in additional diagnoses in seriously ill children with suspected mitochondrial disease, suggesting that WES alone may be insufficient in this setting. When designing rapid genomic diagnostic programs, centres should consider mtDNA analysis, combining WES and mtDNA sequencing in tandem, or analysing mtDNA data from WGS, which captures the mitochondrial genome.Copyright © 2018

Introduction: Standard rapid genomic testing techniques analyze nuclear DNA variants using exome and/or genome sequencing (ES/GS). Rapid mtDNA analysis is not routinely available, particularly in centres performing ES, which does not deliver clinical-grade mtDNA sequencing. We describe our experience using rapid mtDNA sequencing in tandem with an ES-based rapid genomic diagnosis program as part of the Australian Genomics Acute Care flagship. Method(s): Two infants presenting with persistent lactic acidosis and bone marrow failure were recruited for rapid genomic testing. With clinical suspicion of mitochondrial disease, both infants underwent rapid ES and mtDNA sequencing in tandem, the latter using Nextera libraries from a full length mtDNA amplicon. Result(s): ES was non-diagnostic in both infants. mtDNA sequencing identified a single large mtDNA deletion in both infants, diagnostic of Pearson syndrome (MIM 557000). Diagnostic reports were issued within 73 h 55 min and 54 h 25 min, respectively. Both infants avoided invasive bone marrow biopsies and a range of other investigations. Conclusion(s): Rapid mtDNA sequencing in tandem with ES results in additional diagnoses in seriously ill children with suspected mitochondrial pathology, suggesting that ES alone may be insufficient in this setting. When designing rapid genomic diagnosis programs, centres should consider incorporating mtDNA amplification and analysis in individuals with suspected mitochondrial pathology, by combining ES and mtDNA sequencing in tandem, or analysing mtDNA data from GS, which captures the mitochondrial genome.

Background: Mitochondrial ribosomes (mitoribosomes) synthesize 13 mitochondrial DNA encoded proteins essential for cellular energy generation via oxidative phosphorylation (OXPHOS). Mitoribosomes are composed of ~80 proteins, with biallelic mutations in 11 of these thus far demonstrated to cause severe recessive multisystemic mitochondrial disorders. We have identified MRPL39, encoding a protein component of the mitoribosomal large subunit, as a novel disease gene associated with Leigh and Leigh-like syndrome. Methods and Results: Two unrelated patients with Leigh or Leigh-like syndrome each had single rare heterozygous variants (c.912+5G>A and c.526delT) identified by exome sequencing in MRPL39, predicted to result in premature stop codons and nonsense mediated decay (NMD). Subsequent whole genome sequencing (WGS) uncovered a rare heterozygous deep intronic variant (c.589-924G>A) present in both patients, which created a cryptic exon and also underwent NMD. The variants were confirmed to be biallelic by segregation analysis. RNA sequencing (RNAseq) and full length cDNA analysis of patient fibroblasts established aberrant splicing and aberrant expression corresponding to each of the mutations identified. Steady state levels of large mitoribosome subunit proteins (including MRPL39) and OXPHOS complexes were shown to be decreased in patient fibroblasts by quantitative proteomics and western blotting. In contrast, small mitoribosome subunit proteins were unchanged. Conclusion(s): This study identified biallelic loss-of-function mutations in MRPL39 as a novel cause of mitochondrial disease via mitoribosomal large subunit instability. It highlights the utility of WGS, RNAseq and quantitative proteomics for gene prioritization plus discovery and validation of pathogenic variants beyond the exome.

Background: Mitochondrial ribosomes (mitoribosomes) synthesize 13 mitochondrial DNA encoded proteins essential for cellular energy generation via oxidative phosphorylation (OXPHOS). Mitoribosomes are composed of ~80 proteins, with biallelic mutations in 11 of these thus far demonstrated to cause severe recessive multisystemic mitochondrial disorders. We have identified MRPL39, encoding a protein component of the mitoribosomal large subunit, as a novel disease gene associated with Leigh and Leigh-like syndrome. Methods and Results: Two unrelated patients with Leigh or Leigh-like syndrome each had single rare heterozygous variants (c.912+5G>A and c.526delT) identified by exome sequencing in MRPL39, predicted to result in premature stop codons and nonsense mediated decay (NMD). Subsequent whole genome sequencing (WGS) uncovered a rare heterozygous deep intronic variant (c.589-924G>A) present in both patients, which created a cryptic exon and also underwent NMD. The variants were confirmed to be biallelic by segregation analysis. RNA sequencing (RNAseq) and full length cDNA analysis of patient fibroblasts established aberrant splicing and aberrant expression corresponding to each of the mutations identified. Steady state levels of large mitoribosome subunit proteins (including MRPL39) and OXPHOS complexes were shown to be decreased in patient fibroblasts by quantitative proteomics and western blotting. In contrast, small mitoribosome subunit proteins were unchanged. Conclusion(s): This study identified biallelic loss-of-function mutations in MRPL39 as a novel cause of mitochondrial disease via mitoribosomal large subunit instability. It highlights the utility of WGS, RNAseq and quantitative proteomics for gene prioritization plus discovery and validation of pathogenic variants beyond the exome.