Primary mitochondrial disorders and mimics: Insights from a large French cohort.

OBJECTIVE: The objective of this study was to evaluate the implementation of NGS within the French mitochondrial network, MitoDiag, from targeted gene panels to whole exome sequencing (WES) or whole genome sequencing (WGS) focusing on mitochondrial nuclear-encoded genes. METHODS: Over 2000 patients suspected of Primary Mitochondrial Diseases (PMD) were sequenced by either targeted gene panels, WES or WGS within MitoDiag. We described the clinical, biochemical, and molecular data of 397 genetically confirmed patients, comprising 294 children and 103 adults, carrying pathogenic or likely pathogenic variants in nuclear-encoded genes. RESULTS: The cohort exhibited a large genetic heterogeneity, with the identification of 172 distinct genes and 253 novel variants. Among children, a notable prevalence of pathogenic variants in genes associated with oxidative phosphorylation (OXPHOS) functions and mitochondrial translation was observed. In adults, pathogenic variants were primarily identified in genes linked to mtDNA maintenance. Additionally, a substantial proportion of patients (54% (42/78) and 48% (13/27) in children and adults, respectively), undergoing WES or WGS testing displayed PMD mimics, representing pathologies that clinically resemble mitochondrial diseases. INTERPRETATION: We reported the largest French cohort of patients suspected of PMD with pathogenic variants in nuclear genes. We have emphasized the clinical complexity of PMD and the challenges associated with recognizing and distinguishing them from other pathologies, particularly neuromuscular disorders. We confirmed that WES/WGS, instead of panel approach, was more valuable to identify the genetic basis in patients with "possible" PMD and we provided a genetic testing flowchart to guide physicians in their diagnostic strategy.

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.

Infantile onset carnitine palmitoyltransferase 2 deficiency: Cortical polymicrogyria, schizencephaly, and gray matter heterotopias in an adolescent with normal development.

OBJECTIVE: To report an adolescent with infantile-onset carnitine palmitoyltransferase 2 (CPT2) deficiency and cerebral malformations and to review the occurrence of brain malformations in CPT2 deficiency. The patient presented clinically at age 5 months with dehydration and hepatomegaly. He also has an unrelated condition, X-linked nephrogenic diabetes insipidus. He had recurrent rhabdomyolysis but normal psychomotor development. At age 17 years, he developed spontaneous focal seizures. Cerebral magnetic resonance imaging revealed extensive left temporo-parieto-occipital polymicrogyria, white matter heterotopias, and schizencephaly. Neuronal migration defects were previously reported in lethal neonatal CPT2 deficiency but not in later-onset forms. DESIGN AND METHODS: We searched PubMed, Google Scholar, and the bibliographies of the articles found by these searches, for cerebral malformations in CPT2 deficiency. All antenatal, neonatal, infantile, and adult-onset cases were included. Exclusion criteria included insufficient information about age of clinical onset and lack of confirmation of CPT2 deficiency by enzymatic assay or genetic testing. For each report, we noted the presence of cerebral malformations on brain imaging or pathological examination. RESULTS: Of 26 neonatal-onset CPT2-deficient patients who met the inclusion criteria, brain malformations were reported in 16 (61.5%). In 19 infantile-onset cases, brain malformations were not reported, but only 3 of the 19 reports (15.8%) include brain imaging or neuropathology data. In 276 adult-onset cases, no brain malformations were reported. CONCLUSION: To the best of our knowledge, this is the first report of cerebral malformations in an infantile onset CPT2-deficient patient. Brain imaging should be considered in patients with CPTII deficiency and neurological manifestations, even in those with later clinical onset.

Altered expression of fragile X mental retardation-1 (FMR1) in the thymus in autoimmune myasthenia gravis.

Predisposition to autoimmunity and inflammatory disorders is observed in patients with fragile X-associated syndromes. These patients have increased numbers of CGG triplets in the 5' UTR region of FMR1 (Fragile X Mental Retardation 1) gene, that affects its expression. FMR1 is decreased in the thymus of myasthenia gravis (MG) patients, a prototypical autoimmune disease. We thus analyzed the number of CGG triplets in FMR1 in MG, and explored the regulatory mechanisms affecting thymic FMR1 expression. We measured the number of CGGs using thymic DNA from MG and controls, but no abnormalities in CGGs were found in MG that could explain thymic decrease of FMR1. We next analyzed by RT-PCR the expression of FMR1 and its transcription factors in thymic samples, and in thymic epithelial cell cultures in response to inflammatory stimuli. In control thymuses, FMR1 expression was higher in males than females, and correlated with CTCF (CCCTC-binding factor) expression. In MG thymuses, decreased expression of FMR1 was correlated with both CTCF and MAX (Myc-associated factor X) expression. Changes in FMR1 expression were supported by western blot analyses for FMRP. In addition, we demonstrated that FMR1, CTCF and MAX expression in thymic epithelial cells was also sensitive to inflammatory signals. Our results suggest that FMR1 could play a central role in the thymus and autoimmunity. First, in relation with the higher susceptibility of females to autoimmune diseases. Second, due to the modulation of its expression by inflammatory signals that are known to be altered in MG thymuses.

OTC deficiency in females: Phenotype-genotype correlation based on a 130-family cohort.

OTC deficiency, an inherited urea cycle disorder, is caused by mutations in the X-linked OTC gene. Phenotype-genotype correlations are well understood in males but still poorly known in females. Taking advantage of a cohort of 130 families (289 females), we assessed the relative contribution of OTC enzyme activity, X chromosome inactivation, and OTC gene sequencing to genetic counseling in heterozygous females. Twenty two percent of the heterozygous females were clinically affected, with episodic (11%), chronic (7.5%), or neonatal forms of the disease (3.5%). Overall mortality rate was 4%. OTC activity, ranging from 0% to 60%, did not correlate with phenotype at the individual level. Analysis of multiple samples from 4 mutant livers showed intra-hepatic variability of OTC activity and X inactivation profile (range of variability: 30% and 20%, respectively) without correlation between both parameters for 3 of the 4 livers. Ninety disease-causing variants were found, 27 of which were novel. Mutations were classified as "mild" or "severe," based on male phenotypes and/or in silico prediction. In our cohort, a serious disease occurred in 32% of females with a severe mutation, compared to 4% in females with a mild mutation (odds ratio = 1.365; P = 1.6e-06). These data should help prenatal diagnosis for heterozygous females and genetic counseling after fortuitous findings of OTC variants in pangenomic sequencing.

Mitochondrial DNA mutations do not impact early human embryonic development.

Mitochondrial DNA (mtDNA) mutations cause severe maternally inherited disorders, although mechanisms regulating mother-to-offspring transmission have not yet been elucidated. To investigate if mtDNA mutations affect embryonic development, we compared morphology, viability and mtDNA content in control (n = 165) and mitochondrial (n = 16) human embryos at the cleavage-stage. mtDNA copy number (CN) was assessed in one or two embryonic cells, by real-time PCR. The presence of a maternal or embryonic mtDNA mutation did not impact on either embryonic quality or viability. mtDNA CN was not altered by mtDNA mutations, suggesting that mtDNA defects do not modify mtDNA metabolism at this early stage.

Biallelic IARS2 mutations presenting as sideroblastic anemia.

Not available.

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.

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.

Improving post-natal detection of mitochondrial DNA mutations.

INTRODUCTION: Currently, genetic testing of mitochondrial DNA mutations includes screening for single-nucleotide variants, several base pair insertions or deletions, large-scale deletions, or relative depletion of total mitochondrial DNA content. Within the last decade, next-generation sequencing (NGS) has resulted in remarkable advances in the field of mitochondrial diseases (MD) and has become a routine step of the diagnostic workup. AREAS COVERED: We aimed to present an overview of current technologies employed in molecular diagnosis of mitochondrial DNA diseases. We report on the recent contributions of NGS testing to the diagnosis and understanding of MD. EXPERT OPINION: The progress of NGS technologies allows the simultaneous detection of mutations and quantification of the heteroplasmy level, ensuring sensitivity and specificity requested for the detection of mitochondrial DNA point mutations. NGS protocols enabling the simultaneous analysis of mitochondrial and nuclear DNA are now efficient and cost-saving approaches, and have become the gold-standard technique in diagnostic laboratories.

PRPS1 loss-of-function variants, from isolated hearing loss to severe congenital encephalopathy: New cases and literature review.

We describe two sporadic and two familial cases with loss-of-function variants in PRPS1, which is located on the X chromosome and encodes phosphoribosyl pyrophosphate synthetase 1 (PRS-1). We illustrate the clinical variability associated with decreased PRS-1 activity, ranging from mild isolated hearing loss to severe encephalopathy. One of the variants we identified has already been reported with a phenotype similar to our patient's, whereas the other three were unknown. The clinical and biochemical information we provide will hopefully contribute to gain insight into the correlation between genotype and phenotype of this rare condition, both in females and in males. Moreover, our observation of a new family in which hemizygous males display hearing loss without any neurological or ophthalmological symptoms prompts us to suggest analysing PRPS1 in cases of isolated hearing loss. Eventually, PRPS1 variants should be considered as a differential diagnosis of mitochondrial disorders.

Evidence of diaphragmatic dysfunction with severe alveolar hypoventilation syndrome in mitochondrial respiratory chain deficiency.

Diaphragmatic dysfunction has been reported in congenital myopathies, muscular dystrophies, and occasionally, mitochondrial respiratory chain deficiency. Using a minimally invasive procedure in 3 young girls, 1 with a heteroplasmic MT-CYB mutation and 2 with biallelic pathogenic TK2 variants, we provided functional evidence of diaphragmatic dysfunction with global respiratory muscle weakness in mitochondrial respiratory chain deficiency. Analysis of respiratory muscle performance using esogastric pressures revealed paradoxical breathing and severe global inspiratory and expiratory muscle weakness with a sniff esophageal inspiratory pressure and a gastric pressure during cough averaging 50% and 40% of predicted values, respectively. This diaphragmatic dysfunction was responsible for severe undiagnosed nocturnal hypoventilation, requiring noninvasive ventilation. Our results underline the interest of this minimally invasive procedure for the evaluation of respiratory muscle performance and its potential value for the monitoring of future clinical trials in respiratory chain deficiency.

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.

Epilepsy with migrating focal seizures: KCNT1 mutation hotspots and phenotype variability.

OBJECTIVE: To report new sporadic cases and 1 family with epilepsy of infancy with migrating focal seizures (EIMFSs) due to KCNT1 gain-of-function and to assess therapies' efficacy including quinidine. METHODS: We reviewed the clinical, EEG, and molecular data of 17 new patients with EIMFS and KCNT1 mutations, in collaboration with the network of the French reference center for rare epilepsies. RESULTS: The mean seizure onset age was 1 month (range: 1 hour to 4 months), and all children had focal motor seizures with autonomic signs and migrating ictal pattern on EEG. Three children also had infantile spasms and hypsarrhythmia. The identified KCNT1 variants clustered as "hot spots" on the C-terminal domain, and all mutations occurred de novo except the p.R398Q mutation inherited from the father with nocturnal frontal lobe epilepsy, present in 2 paternal uncles, one being asymptomatic and the other with single tonic-clonic seizure. In 1 patient with EIMFS, we identified the p.R1106Q mutation associated with Brugada syndrome and saw no abnormality in cardiac rhythm. Quinidine was well tolerated when administered to 2 and 4-year-old patients but did not reduce seizure frequency. CONCLUSIONS: The majority of the KCNT1 mutations appear to cluster in hot spots essential for the channel activity. A same mutation can be linked to a spectrum of conditions ranging from EMFSI to asymptomatic carrier, even in the same family. None of the antiepileptic therapies displayed clinical efficacy, including quinidine in 2 patients.

Expanding the clinical spectrum of MTTF mutations.

We report on a de novo m.586G > A MTTF mutation in a 14 yrs old boy with non-progressive muscle weakness, myalgia, normal brain MRI, normal schooling and absent central nervous system involvement. The same m.586G > A MTTF mutation has been previously reported in a 57 yrs-old woman with a progressive neurodegenerative disorder, akinesia-rigidity, abnormal movements, dementia, and psychiatric disorder. Those two strikingly different clinical presentations emphasize the impact of either mitochondrial factors (heteroplasmy, mitotic segregation) or hitherto unknown nuclear factors on the clinical expression of genetically homogeneous mtDNA mutations.

Risk estimation of uniparental disomy of chromosome 14 or 15 in a fetus with a parent carrying a non-homologous Robertsonian translocation. Should we still perform prenatal diagnosis?

OBJECTIVE: Uniparental disomy (UPD) testing is currently recommended during pregnancy in fetuses carrying a balanced Robertsonian translocation (ROB) involving chromosome 14 or 15, both chromosomes containing imprinted genes. The overall risk that such a fetus presents a UPD has been previously estimated to be around ~0.6-0.8%. However, because UPD are rare events and this estimate has been calculated from a number of studies of limited size, we have reevaluated the risk of UPD in fetuses for whom one of the parents was known to carry a nonhomologous ROB (NHROB). METHOD: We focused our multicentric study on NHROB involving chromosome 14 and/or 15. A total of 1747 UPD testing were performed in fetuses during pregnancy for the presence of UPD(14) and/or UPD(15). RESULT: All fetuses were negative except one with a UPD(14) associated with a maternally inherited rob(13;14). CONCLUSION: Considering these data, the risk of UPD following prenatal diagnosis of an inherited ROB involving chromosome 14 and/or 15 could be estimated to be around 0.06%, far less than the previous estimation. Importantly, the risk of miscarriage following an invasive prenatal sampling is higher than the risk of UPD. Therefore, we do not recommend prenatal testing for UPD for these pregnancies and parents should be reassured.