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.

An early onset benign myopathy with glycogen storage caused by a de novo 1.4 Mb-deletion of chromosome 14.

Early onset myopathies are a clinically and histologically heterogeneous monogenic diseases linked to approximately 90 genes. Molecular diagnosis is challenging, especially in patients with a mild phenotype. We describe a 26-year-old man with neonatal hypotonia, motor delay and seizures during infancy, and non-progressive, mild muscular weakness in adulthood. Serum Creatine kinase level was normal. Whole-body muscle MRI showed thin muscles, and brain MRI was unremarkable. A deltoid muscle biopsy showed glycogen storage. WGS revealed a de novo 1.4 Mb-deletion of chromosome 14, confirmed by Array-CGH. This microdeletion causes the loss of ten genes including RALGAPA1, encoding for RalA, a regulator of glucose transporter 4 (GLUT4) expression at the membrane of myofibers. GLUT4 was overexpressed in patient's muscle. Here we highlight the importance to search for chromosomal alterations in the diagnostic workup of early onset myopathies.

Genotypic and phenotypic spectrum of infantile liver failure due to pathogenic TRMU variants.

PURPOSE: This study aimed to define the genotypic and phenotypic spectrum of reversible acute liver failure (ALF) of infancy resulting from biallelic pathogenic TRMU variants and determine the role of cysteine supplementation in its treatment. METHODS: Individuals with biallelic (likely) pathogenic variants in TRMU were studied within an international retrospective collection of de-identified patient data. RESULTS: In 62 individuals, including 30 previously unreported cases, we described 47 (likely) pathogenic TRMU variants, of which 17 were novel, and 1 intragenic deletion. Of these 62 individuals, 42 were alive at a median age of 6.8 (0.6-22) years after a median follow-up of 3.6 (0.1-22) years. The most frequent finding, occurring in all but 2 individuals, was liver involvement. ALF occurred only in the first year of life and was reported in 43 of 62 individuals; 11 of whom received liver transplantation. Loss-of-function TRMU variants were associated with poor survival. Supplementation with at least 1 cysteine source, typically N-acetylcysteine, improved survival significantly. Neurodevelopmental delay was observed in 11 individuals and persisted in 4 of the survivors, but we were unable to determine whether this was a primary or a secondary consequence of TRMU deficiency. CONCLUSION: In most patients, TRMU-associated ALF was a transient, reversible disease and cysteine supplementation improved survival.

Very long-term outcomes in 23 patients with cblA type methylmalonic acidemia.

OBJECTIVES: To present the very long-term follow up of patients with cobalamin A (cblA) deficiency. METHODS: A retrospective case series of adult (>16 years) patients with molecular or enzymatic diagnosis of cblA deficiency. RESULTS: We included 23 patients (mean age: 27 ± 7.6 years; mean follow-up: 24.9 ± 7.6 years). Disease onset was mostly pediatric (78% < 1 year, median = 4 months) with acute neurologic deterioration (65%). Eight patients presented with chronic symptoms, and one had an adult-onset mild cblA deficiency. Most of the patients (61%) were initially classified as vitamin B12-unresponsive methylmalonic aciduria (MMA); in vitro B12 responsiveness was subsequently found in all the tested patients (n = 13). Initial management consisted of protein restriction (57%), B12 (17%), or both (26%). The main long-term problems were intellectual disability (39%) and renal failure (30%). However, 56.5% of the patients were living independently. Intellectual disability was equally distributed among the initial treatment groups, while renal failure (moderate and beginning at the age of 38 years) was present in only one out of seven patients initially treated with B12. CONCLUSIONS: We provide a detailed picture of the long-term outcome of a series of adult cblA patients, mostly diagnosed before the enzymatic and molecular era. We confirm that about 35% of the patients do not present acutely, underlining the importance of measuring MMA in any case of unexplained chronic renal failure, intellectual disability, or growth delay. In addition, we describe a patient with a milder adult-onset form. Early B12 supplementation seems to protect from severe renal insufficiency.

Heterozygous PNPT1 Variants Cause Spinocerebellar Ataxia Type 25.

OBJECTIVE: Dominant spinocerebellar ataxias (SCA) are characterized by genetic heterogeneity. Some mapped and named loci remain without a causal gene identified. Here we applied next generation sequencing (NGS) to uncover the genetic etiology of the SCA25 locus. METHODS: Whole-exome and whole-genome sequencing were performed in families linked to SCA25, including the French family in which the SCA25 locus was originally mapped. Whole exome sequence data were interrogated in a cohort of 796 ataxia patients of unknown etiology. RESULTS: The SCA25 phenotype spans a slowly evolving sensory and cerebellar ataxia, in most cases attributed to ganglionopathy. A pathogenic variant causing exon skipping was identified in the gene encoding Polyribonucleotide Nucleotidyltransferase PNPase 1 (PNPT1) located in the SCA25 linkage interval. A second splice variant in PNPT1 was detected in a large Australian family with a dominant ataxia also mapping to SCA25. An additional nonsense variant was detected in an unrelated individual with ataxia. Both nonsense and splice heterozygous variants result in premature stop codons, all located in the S1-domain of PNPase. In addition, an elevated type I interferon response was observed in blood from all affected heterozygous carriers tested. PNPase notably prevents the abnormal accumulation of double-stranded mtRNAs in the mitochondria and leakage into the cytoplasm, associated with triggering a type I interferon response. INTERPRETATION: This study identifies PNPT1 as a new SCA gene, responsible for SCA25, and highlights biological links between alterations of mtRNA trafficking, interferonopathies and ataxia. ANN NEUROL 2022;92:122-137.

Heterogeneity of PNPT1 neuroimaging: mitochondriopathy, interferonopathy or both?

BACKGROUND: Biallelic variants in PNPT1 cause a mitochondrial disease of variable severity. PNPT1 (polynucleotide phosphorylase) is a mitochondrial protein involved in RNA processing where it has a dual role in the import of small RNAs into mitochondria and in preventing the formation and release of mitochondrial double-stranded RNA into the cytoplasm. This, in turn, prevents the activation of type I interferon response. Detailed neuroimaging findings in PNPT1-related disease are lacking with only a few patients reported with basal ganglia lesions (Leigh syndrome) or non-specific signs. OBJECTIVE AND METHODS: To document neuroimaging data in six patients with PNPT1 highlighting novel findings. RESULTS: Two patients exhibited striatal lesions compatible with Leigh syndrome; one patient exhibited leukoencephalopathy and one patient had a normal brain MRI. Interestingly, two unrelated patients exhibited cystic leukoencephalopathy resembling RNASET2-deficient patients, patients with Aicardi-Goutières syndrome (AGS) or congenital CMV infection. CONCLUSION: We suggest that similar to RNASET2, PNPT1 be searched for in the setting of cystic leukoencephalopathy. These findings are in line with activation of type I interferon response observed in AGS, PNPT1 and RNASET2 deficiencies, suggesting a common pathophysiological pathway and linking mitochondrial diseases, interferonopathies and immune dysregulations.

Expanding the phenotypic spectrum of BCS1L-related mitochondrial disease.

OBJECTIVE: To delineate the full phenotypic spectrum of BCS1L-related disease, provide better understanding of the genotype-phenotype correlations and identify reliable prognostic disease markers. METHODS: We performed a retrospective multinational cohort study of previously unpublished patients followed in 15 centres from 10 countries. Patients with confirmed biallelic pathogenic BCS1L variants were considered eligible. Clinical, laboratory, neuroimaging and genetic data were analysed. Patients were stratified into different groups based on the age of disease onset, whether homozygous or compound heterozygous for the c.232A>G (p.Ser78Gly) variant, and those with other pathogenic BCS1L variants. RESULTS: Thirty-three patients were included. We found that growth failure, lactic acidosis, tubulopathy, hepatopathy and early death were more frequent in those with disease onset within the first month of life. In those with onset after 1 month, neurological features including movement disorders and seizures were more frequent. Novel phenotypes, particularly involving movement disorder, were identified in this group. The presence of the c.232A>G (p.Ser78Gly) variant was associated with significantly worse survival and exclusively found in those with disease onset within the first month of life, whilst other pathogenic BCS1L variants were more frequent in those with later symptom onset. INTERPRETATION: The phenotypic spectrum of BCS1L-related disease comprises a continuum of clinical features rather than a set of separate syndromic clinical identities. Age of onset defines BCS1L-related disease clinically and early presentation is associated with poor prognosis. Genotype correlates with phenotype in the presence of the c.232A>G (p.Ser78Gly) variant.

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.

Cerebral blood flow and acute episodes of Leigh syndrome in neurometabolic disorders.

AIM: To investigate cerebral blood flow (CBF) in acute episodes of Leigh syndrome compared with basal state in patients carrying pathogenic mitochondrial disease gene variants responsible for neurometabolic disorders. METHOD: Arterial spin labelling (ASL) magnetic resonance imaging (MRI) sequences were used to measure CBF in 27 patients with mitochondrial respiratory chain enzyme deficiencies, ascribed to pathogenic variants of reported disease genes who were undergoing either urgent neuroimaging for acute episodes of Leigh syndrome (Group I: 15 MRI, seven females, eight males; mean age 7y; range 7mo-14y) or routine brain MRI (Group II: 15 MRI, eight females, seven males; mean age 5y 2mo; range 2mo-12y). RESULTS: Patients displayed markedly increased CBF in the striatum (2.8-fold greater, p<0.001 [1.05-2.53]) during acute episodes of Leigh syndrome compared to basal conditions. Detection of elevated CBF preceded identification of structural MRI lesions in four out of 15 cases. INTERPRETATION: Our results suggest that increased CBF is an overt hallmark of Leigh syndrome episodes and ASL MRI sequences should facilitate early diagnosis of acute episodes of Leigh syndrome, especially during the first attack in young children, when structural MRI is insufficiently informative.

Biallelic IARS2 mutations presenting as sideroblastic anemia.

Not available.

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.

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.

Nocturnal enteral nutrition is therapeutic for growth failure in Fanconi-Bickel syndrome.

Fanconi-Bickel syndrome (FBS) is a rare autosomal recessive disorder characterised by impaired glucose liver homeostasis and proximal renal tubular dysfunction. It is caused by pathogenic variants in SLC2A2 coding for the glucose transporter GLUT2. Main clinical features include hepatomegaly, fasting hypoglycaemia, postprandial hyperglycaemia, Fanconi-type tubulopathy occasionally with rickets, and a severe growth disorder. While treatment for renal tubular dysfunction is well established, data regarding optimal nutritional therapy are scarce. Similarly, detailed clinical evaluation of treated FBS patients is lacking. These unmet needs were an incentive to conduct the present pilot study. We present clinical findings, laboratory parameters and molecular genetic data on 11 FBS patients with emphasis on clinical outcome under various nutritional interventions. At diagnosis, the patients' phenotypic severity could be classified into two categories: a first group with severe growth failure and rickets, and a second group with milder signs and symptoms. Three patients were diagnosed early and treated because of family history. All patients exhibited massive glucosuria at diagnosis and some in both groups had fasting hypoglycaemic episodes. Growth retardation improved drastically in all five patients treated by intensive nutritional intervention (nocturnal enteral nutrition) and uncooked cornstarch with final growth parameters in the normal range. The four severely affected patients who were treated with uncooked cornstarch alone did not catch up growth. All patients received electrolytes and l-carnitine supplementation to compensate for the tubulopathy. This is one of the largest series of FBS on therapeutic management with evidence that nocturnal enteral nutrition rescues growth failure.

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.