Novel NDUFA12 variants are associated with isolated complex I defect and variable clinical manifestation.

Isolated biochemical deficiency of mitochondrial complex I is the most frequent signature among mitochondrial diseases and is associated with a wide variety of clinical symptoms. Leigh syndrome represents the most frequent neuroradiological finding in patients with complex I defect and more than 80 monogenic causes have been involved in the disease. In this report, we describe seven patients from four unrelated families harboring novel NDUFA12 variants, with six of them presenting with Leigh syndrome. Molecular genetic characterization was performed using next-generation sequencing combined with the Sanger method. Biochemical and protein studies were achieved by enzymatic activities, blue native gel electrophoresis, and western blot analysis. All patients displayed novel homozygous mutations in the NDUFA12 gene, leading to the virtual absence of the corresponding protein. Surprisingly, despite the fact that in none of the analyzed patients, NDUFA12 protein was detected, they present a different onset and clinical course of the disease. Our report expands the array of genetic alterations in NDUFA12 and underlines phenotype variability associated with NDUFA12 defect.

NDUFB8 Mutations Cause Mitochondrial Complex I Deficiency in Individuals with Leigh-like Encephalomyopathy.

Respiratory chain complex I deficiency is the most frequently identified biochemical defect in childhood mitochondrial diseases. Clinical symptoms range from fatal infantile lactic acidosis to Leigh syndrome and other encephalomyopathies or cardiomyopathies. To date, disease-causing variants in genes coding for 27 complex I subunits, including 7 mitochondrial DNA genes, and in 11 genes encoding complex I assembly factors have been reported. Here, we describe rare biallelic variants in NDUFB8 encoding a complex I accessory subunit revealed by whole-exome sequencing in two individuals from two families. Both presented with a progressive course of disease with encephalo(cardio)myopathic features including muscular hypotonia, cardiac hypertrophy, respiratory failure, failure to thrive, and developmental delay. Blood lactate was elevated. Neuroimaging disclosed progressive changes in the basal ganglia and either brain stem or internal capsule. Biochemical analyses showed an isolated decrease in complex I enzymatic activity in muscle and fibroblasts. Complementation studies by expression of wild-type NDUFB8 in cells from affected individuals restored mitochondrial function, confirming NDUFB8 variants as the cause of complex I deficiency. Hereby we establish NDUFB8 as a relevant gene in childhood-onset mitochondrial disease.

Bi-allelic Mutations in the Mitochondrial Ribosomal Protein MRPS2 Cause Sensorineural Hearing Loss, Hypoglycemia, and Multiple OXPHOS Complex Deficiencies.

Biogenesis of the mitochondrial oxidative phosphorylation system, which produces the bulk of ATP for almost all eukaryotic cells, depends on the translation of 13 mtDNA-encoded polypeptides by mitochondria-specific ribosomes in the mitochondrial matrix. These mitoribosomes are dual-origin ribonucleoprotein complexes, which contain mtDNA-encoded rRNAs and tRNAs and ∼80 nucleus-encoded proteins. An increasing number of gene mutations that impair mitoribosomal function and result in multiple OXPHOS deficiencies are being linked to human mitochondrial diseases. Using exome sequencing in two unrelated subjects presenting with sensorineural hearing impairment, mild developmental delay, hypoglycemia, and a combined OXPHOS deficiency, we identified mutations in the gene encoding the mitochondrial ribosomal protein S2, which has not previously been implicated in disease. Characterization of subjects' fibroblasts revealed a decrease in the steady-state amounts of mutant MRPS2, and this decrease was shown by complexome profiling to prevent the assembly of the small mitoribosomal subunit. In turn, mitochondrial translation was inhibited, resulting in a combined OXPHOS deficiency detectable in subjects' muscle and liver biopsies as well as in cultured skin fibroblasts. Reintroduction of wild-type MRPS2 restored mitochondrial translation and OXPHOS assembly. The combination of lactic acidemia, hypoglycemia, and sensorineural hearing loss, especially in the presence of a combined OXPHOS deficiency, should raise suspicion for a ribosomal-subunit-related mitochondrial defect, and clinical recognition could allow for a targeted diagnostic approach. The identification of MRPS2 as an additional gene related to mitochondrial disease further expands the genetic and phenotypic spectra of OXPHOS deficiencies caused by impaired mitochondrial translation.

A retrospective study on the efficacy of prenatal diagnosis for pregnancies at risk of mitochondrial DNA disorders.

PURPOSE: Prenatal diagnosis of mitochondrial DNA (mtDNA) disorders is challenging due to potential instability of fetal mutant loads and paucity of data connecting prenatal mutant loads to postnatal observations. Retrospective study of our prenatal cohort aims to examine the efficacy of prenatal diagnosis to improve counseling and reproductive options for those with pregnancies at risk of mtDNA disorders. METHODS: We report on a retrospective review of 20 years of prenatal diagnosis of pathogenic mtDNA variants in 80 pregnant women and 120 fetuses. RESULTS: Patients with undetectable pathogenic variants (n = 29) consistently had fetuses free of variants, while heteroplasmic women (n = 51) were very likely to transmit their variant (57/78 fetuses, 73%). In the latter case, 26 pregnancies were terminated because fetal mutant loads were >40%. Of the 84 children born, 27 were heteroplasmic (mutant load <65%). To date, no medical problems related to mitochondrial dysfunction have been reported. CONCLUSION: Placental heterogeneity of mutant loads questioned the reliability of chorionic villous testing. Fetal mutant load stability, however, suggests the reliability of a single analysis of amniotic fluid at any stage of pregnancy for prenatal diagnosis of mtDNA disorders. Mutant loads under 40% reliably predict lack of symptoms in the progeny of heteroplasmic women.

Biallelic IARS2 mutations presenting as sideroblastic anemia.

Not available.

Clinical, neuroimaging and biochemical findings in patients and patient fibroblasts expressing ten novel GFM1 mutations.

Pathogenic GFM1 variants have been linked to neurological phenotypes with or without liver involvement, but only a few cases have been reported in the literature. Here, we report clinical, biochemical, and neuroimaging findings from nine unrelated children carrying GFM1 variants, 10 of which were not previously reported. All patients presented with neurological involvement-mainly axial hypotonia and dystonia during the neonatal period-with five diagnosed with West syndrome; two children had liver involvement with cytolysis episodes or hepatic failure. While two patients died in infancy, six exhibited a stable clinical course. Brain magnetic resonance imaging showed the involvement of basal ganglia, brainstem, and periventricular white matter. Mutant EFG1 and OXPHOS proteins were decreased in patient's fibroblasts consistent with impaired mitochondrial translation. Thus, we expand the genetic spectrum of GFM1-linked disease and provide detailed clinical profiles of the patients that will improve the diagnostic success for other patients carrying GFM1 mutations.

Biallelic Mutations in MRPS34 Lead to Instability of the Small Mitoribosomal Subunit and Leigh Syndrome.

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 natural history of infantile mitochondrial DNA depletion syndrome due to RRM2B deficiency.

PURPOSE: Mitochondrial DNA (mtDNA) depletion syndrome (MDDS) encompasses a group of genetic disorders of mtDNA maintenance. Mutation of RRM2B is an uncommon cause of infantile-onset encephalomyopathic MDDS. Here we describe the natural history of this disease. METHODS: Multinational series of new genetically confirmed cases from six pediatric centers. RESULTS: Nine new cases of infantile-onset RRM2B deficiency, and 22 previously published cases comprised a total cohort of 31 patients. Infants presented at a mean of 1.95 months with truncal hypotonia, generalized weakness, and faltering growth. Seizures evolved in 39% at a mean of 3.1 months. Non-neurological manifestations included respiratory distress/failure (58%), renal tubulopathy (55%), sensorineural hearing loss (36%), gastrointestinal disturbance (32%), eye abnormalities (13%), and anemia (13%). Laboratory features included elevated lactate (blood, cerebrospinal fluid (CSF), urine, magnetic resonance (MR), spectroscopy), ragged-red and cytochrome c oxidase-deficient fibers, lipid myopathy, and multiple oxidative phosphorylation enzyme deficiencies in skeletal muscle. Eight new RRM2B variants were identified. Patients with biallelic truncating variants had the worst survival. Overall survival was 29% at 6 months and 16% at 1 year. CONCLUSIONS: Infantile-onset MDDS due to RRM2B deficiency is a severe disorder with characteristic clinical features and extremely poor prognosis. Presently management is supportive as there is no effective treatment. Novel treatments are urgently needed.

Mutations in Complex I Assembly Factor TMEM126B Result in Muscle Weakness and Isolated Complex I Deficiency.

Mitochondrial complex I deficiency results in a plethora of often severe clinical phenotypes manifesting in early childhood. Here, we report on three complex-I-deficient adult subjects with relatively mild clinical symptoms, including isolated, progressive exercise-induced myalgia and exercise intolerance but with normal later development. Exome sequencing and targeted exome sequencing revealed compound-heterozygous mutations in TMEM126B, encoding a complex I assembly factor. Further biochemical analysis of subject fibroblasts revealed a severe complex I deficiency caused by defective assembly. Lentiviral complementation with the wild-type cDNA restored the complex I deficiency, demonstrating the pathogenic nature of these mutations. Further complexome analysis of one subject indicated that the complex I assembly defect occurred during assembly of its membrane module. Our results show that TMEM126B defects can lead to complex I deficiencies and, interestingly, that symptoms can occur only after exercise.

High predictive value of brain MRI imaging in primary mitochondrial respiratory chain deficiency.

BACKGROUND: Because the mitochondrial respiratory chain (RC) is ubiquitous, its deficiency can theoretically give rise to any symptom in any organ or tissue at any age with any mode of inheritance, owing to the twofold genetic origin of respiratory enzyme machinery, that is, nuclear and mitochondrial. Not all respiratory enzyme deficiencies are primary and secondary or artefactual deficiency is frequently observed, leading to a number of misleading conclusions and inappropriate investigations in clinical practice. This study is aimed at investigating the potential role of brain MRI in distinguishing primary RC deficiency from phenocopies and other aetiologies. METHODS: Starting from a large series of 189 patients (median age: 3.5 years (8 days-56 years), 58% males) showing signs of RC enzyme deficiency, for whom both brain MRIs and disease-causing mutations were available, we retrospectively studied the positive predictive value (PPV) and the positive likelihood ratio (LR+) of brain MRI imaging and its ability to discriminate between two groups: primary deficiency of the mitochondrial RC machinery and phenocopies. RESULTS: Detection of (1) brainstem hyperintensity with basal ganglia involvement (P≤0.001) and (2) lactate peak with either brainstem or basal ganglia hyperintensity was highly suggestive of primary RC deficiency (P≤0.01). Fourteen items had a PPV>95% and LR+ was greater than 9 for seven signs. Biallelic SLC19A3 mutations represented the main differential diagnosis. Non-significant differences between the two groups were found for cortical/subcortical atrophy, leucoencephalopathy and involvement of caudate nuclei, spinothalamic tract and corpus callosum. CONCLUSION: Based on these results and owing to invasiveness of skeletal muscle biopsies and cost of high-throughput DNA sequencing, we suggest giving consideration to brain MRI imaging as a diagnostic marker and an informative investigation to be performed in patients showing signs of RC enzyme deficiency.

Inhibition of mitochondrial translation in fibroblasts from a patient expressing the KARS p.(Pro228Leu) variant and presenting with sensorineural deafness, developmental delay, and lactic acidosis.

Aminoacyl-tRNA synthetases are ubiquitous enzymes, which universally charge tRNAs with their cognate amino acids for use in cytosolic or organellar translation. In humans, mutations in mitochondrial tRNA synthetases have been linked to different tissue-specific pathologies. Mutations in the KARS gene, which encodes both the cytosolic and mitochondrial isoform of lysyl-tRNA synthetase, cause predominantly neurological diseases that often involve deafness, but have also been linked to cardiomyopathy, developmental delay, and lactic acidosis. Using whole exome sequencing, we identified two compound heterozygous mutations, NM_001130089.1:c.683C>T p.(Pro228Leu) and NM_001130089.1:c.1438del p.(Leu480TrpfsX3), in a patient presenting with sensorineural deafness, developmental delay, hypotonia, and lactic acidosis. Nonsense-mediated mRNA decay eliminated the truncated mRNA transcript, rendering the patient hemizygous for the missense mutation. The c.683C>T mutation was previously described, but its pathogenicity remained unexamined. Molecular characterization of patient fibroblasts revealed a multiple oxidative phosphorylation deficiency due to impaired mitochondrial translation, but no evidence of inhibition of cytosolic translation. Reintroduction of wild-type mitochondrial KARS, but not the cytosolic isoform, rescued this phenotype confirming the disease-causing nature of p.(Pro228Leu) exchange and demonstrating the mitochondrial etiology of the disease. We propose that mitochondrial translation deficiency is the probable disease culprit in this and possibly other patients with mutations in KARS.

Mutations in GTPBP3 cause a mitochondrial translation defect associated with hypertrophic cardiomyopathy, lactic acidosis, and encephalopathy.

Respiratory chain deficiencies exhibit a wide variety of clinical phenotypes resulting from defective mitochondrial energy production through oxidative phosphorylation. These defects can be caused by either mutations in the mtDNA or mutations in nuclear genes coding for mitochondrial proteins. The underlying pathomechanisms can affect numerous pathways involved in mitochondrial physiology. By whole-exome and candidate gene sequencing, we identified 11 individuals from 9 families carrying compound heterozygous or homozygous mutations in GTPBP3, encoding the mitochondrial GTP-binding protein 3. Affected individuals from eight out of nine families presented with combined respiratory chain complex deficiencies in skeletal muscle. Mutations in GTPBP3 are associated with a severe mitochondrial translation defect, consistent with the predicted function of the protein in catalyzing the formation of 5-taurinomethyluridine (τm(5)U) in the anticodon wobble position of five mitochondrial tRNAs. All case subjects presented with lactic acidosis and nine developed hypertrophic cardiomyopathy. In contrast to individuals with mutations in MTO1, the protein product of which is predicted to participate in the generation of the same modification, most individuals with GTPBP3 mutations developed neurological symptoms and MRI involvement of thalamus, putamen, and brainstem resembling Leigh syndrome. Our study of a mitochondrial translation disorder points toward the importance of posttranscriptional modification of mitochondrial tRNAs for proper mitochondrial function.

Distinct Clinical Courses and Shortened Lifespans in Childhood-Onset DNA Polymerase Gamma Deficiency.

Background and Objectives: DNA polymerase subunit gamma (POLG) deficiency is likely the most frequent cause of nuclear-encoded mitochondrial disorders. POLG-related disorders reportedly constitute a spectrum of overlapping phenotypes from infancy to late adulthood. We retrospectively reviewed natural histories for 40 children carrying biallelic pathogenic POLG variants. Methods: The patients were identified by the French coordinating center for mitochondrial disorders (CARAMMEL), making this a large monocentric series on childhood-onset POLG deficiency. Results: Three patterns of clinical course and survival were observed, distinguished by main category of symptoms: neurologic, hepatic, and gastrointestinal. A total of 24 patients needed urgent neurointensive care for tonic-clonic seizures, myoclonic epilepsy, and status epilepticus, occasionally precipitated by valproate administration. Other neurologic symptoms included dystonia, cerebellar ataxia, and peripheral neuropathy. We report 6 POLG-deficient patients with polyradiculoneuropathy mimicking subacute Guillain-Barré syndrome and provide postgadolinium MRI evidence of diffuse cranial nerve root and cauda equina enhancement, suggesting these disorders have an inflammatory component. Children presenting with enteral nervous system involvement had vomiting, gastroparesis, and chronic intestinal pseudo-obstruction. They had later ages of onset and lived much longer. Primarily, hepatic presentations had the earliest onset and shortest survivals. Secondary hepatic failure was frequently precipitated by valproate administration given before diagnosis to patients with focal impaired awareness seizures or absence of seizures. These POLG deficiencies were often fatal, with age at death ranging from 3 months to 10 years, with a significant difference in survival between the 3 clinical forms; 6 of the 40 children did survive. No genotype-phenotype correlations were found for the 3 clinical course types. Discussion: The study demonstrates the prevalence of neurologic presentation and the extent of central, peripheral, and autonomous nervous system involvement in 60% of patients. Most of the patients with early onset and rapidly fatal hepatic failure did not live long enough to develop neurologic symptoms. The study revealed a new clinical form of POLG deficiency presenting with neurodigestive symptoms with longer lifespan. We also propose that POLG deficiency should be considered in children presenting with unexplained polyradiculoneuropathy, demyelinating neuropathy, and elevated CSF protein. Finally, valproate administration remains a notable cause of avoidable death in POLG-deficient patients.

Novel ELAC2 Mutations in Individuals Presenting with Variably Severe Neurological Disease in the Presence or Absence of Cardiomyopathy.

Transcription of mitochondrial DNA generates long polycistronic precursors whose nucleolytic cleavage yields the individual mtDNA-encoded transcripts. In most cases, this cleavage occurs at the 5'- and 3'-ends of tRNA sequences by the concerted action of RNAseP and RNaseZ/ELAC2 endonucleases, respectively. Variants in the ELAC2 gene have been predominantly linked to severe to mild cardiomyopathy that, in its milder forms, is accompanied by variably severe neurological presentations. Here, we report five patients from three unrelated families. Four of the patients presented mild to moderate cardiomyopathy and one died at 1 year of age, one patient had no evidence of cardiomyopathy. The patients had variable neurological presentations that included intellectual disability, ataxia, refractory epilepsy, neuropathy and deafness. All patients carried previously unreported missense and nonsense variants. Enzymatic analyses showed multiple OXPHOS deficiencies in biopsies from two patients, whereas immunoblot analyses revealed a decreased abundance of ELAC2 in fibroblasts from three patients. Northern blot analysis revealed an accumulation of unprocessed mt-tRNAVal-precursor consistent with the role of ELAC2 in transcript processing. Our study expands the genetic spectrum of ELAC2-linked disease and suggests that cardiomyopathy is not an invariably present clinical hallmark of this pathology.

Maternal uniparental disomy of chromosome 2 in a patient with a DGUOK mutation associated with hepatocerebral mitochondrial DNA depletion syndrome.

We report maternal uniparental disomy of chromosome 2 (matUPD2) in a 9-month-old girl presenting with hepatocerebral mitochondrial DNA depletion syndrome. This patient was homozygous for the c.352C>T (p.Arg118Cys) mutation in DGUOK gene. The proband's mother was heterozygous for the mutation was absent in DNA of the father. For proband, the absence of paternal contribution at the DGUOK locus prompted us to exclude intragenic DGUOK deletion of the paternal allele with Multiplex ligation-dependent probe amplification (MLPA) analysis. We also excluded non-paternity by studying various markers at different loci. Then we performed an analysis of copy number variations and absence of heterozygosity (AOH) on the proband DNA using high resolution oligonucleotides microarray. Several large regions of AOH with no copy number change were detected on chromosome 2 and one of these AOH regions encompassed DGUOK gene. These results were confirmed with haplotype analysis using polymorphic markers. Informative SNPs and microsatellites markers spanning the whole chromosome 2 showed a matUPD2 with heterodisomy and isodisomy regions, the absence of paternal allele and presence of two maternal alleles, with only one maternal allele on the region of DGUOK locus in 2p13.1. This is the first demonstration of matUPD2 with segmental isodisomy at 2p13.1 locus in hepatocerebral mitochondrial DNA depletion syndrome. The identification of UPD2 will impact genetic counseling for the proband's parents. Because the recurrence risk for UPD2 is very low, the risk for disease in further offspring for this couple is negligible.

Exome sequencing identifies MRPL3 mutation in mitochondrial cardiomyopathy.

By combining exome sequencing in conjunction with genetic mapping, we have identified the first mutation in large mitochondrial ribosomal protein MRPL3 in a family of four sibs with hypertrophic cardiomyopathy, psychomotor retardation, and multiple respiratory chain deficiency. Affected sibs were compound heterozygotes for a missense MRPL3 mutation (P317R) and a large-scale deletion, inherited from the mother and the father, respectively. These mutations were shown to alter ribosome assembly and cause a mitochondrial translation deficiency in cultured skin fibroblasts resulting in an abnormal assembly of several complexes of the respiratory chain. This observation gives support to the view that exome sequencing combined with genetic mapping is a powerful approach for the identification of new genes of mitochondrial disorders.

Novel FARS2 variants in patients with early onset encephalopathy with or without epilepsy associated with long survival.

Mitochondrial translation is essential for the biogenesis of the mitochondrial oxidative phosphorylation system (OXPHOS) that synthesizes the bulk of ATP for the cell. Hypomorphic and loss-of-function variants in either mitochondrial DNA or in nuclear genes that encode mitochondrial translation factors can result in impaired OXPHOS biogenesis and mitochondrial diseases with variable clinical presentations. Compound heterozygous or homozygous missense and frameshift variants in the FARS2 gene, that encodes the mitochondrial phenylalanyl-tRNA synthetase, are commonly linked to either early-onset epileptic mitochondrial encephalopathy or spastic paraplegia. Here, we expand the genetic spectrum of FARS2-linked disease with three patients carrying novel compound heterozygous variants in the FARS2 gene and presenting with spastic tetraparesis, axial hypotonia and myoclonic epilepsy in two cases.

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.