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.

Homozygous MFN2 variants causing severe antenatal encephalopathy with clumped mitochondria.

Pathogenic variants in the MFN2 gene are commonly associated with autosomal dominant (CMT2A2A) or recessive (CMT2A2B) Charcot-Marie-Tooth disease, with possible involvement of the CNS. Here, we present a case of severe antenatal encephalopathy with lissencephaly, polymicrogyria and cerebellar atrophy. Whole genome analysis revealed a homozygous deletion c.1717-274_1734 del (NM_014874.4) in the MFN2 gene, leading to exon 16 skipping and in-frame loss of 50 amino acids (p.Gln574_Val624del), removing the proline-rich domain and the transmembrane domain 1 (TM1). MFN2 is a transmembrane GTPase located on the mitochondrial outer membrane that contributes to mitochondrial fusion, shaping large mitochondrial networks within cells. In silico modelling showed that the loss of the TM1 domain resulted in a drastically altered topological insertion of the protein in the mitochondrial outer membrane. Fetus fibroblasts, investigated by fluorescent cell imaging, electron microscopy and time-lapse recording, showed a sharp alteration of the mitochondrial network, with clumped mitochondria and clusters of tethered mitochondria unable to fuse. Multiple deficiencies of respiratory chain complexes with severe impairment of complex I were also evidenced in patient fibroblasts, without involvement of mitochondrial DNA instability. This is the first reported case of a severe developmental defect due to MFN2 deficiency with clumped mitochondria.

New description of an MRPS2 homozygous patient: Further features to help expend the phenotype.

Mutated mito-ribosomal protein S2 (MRPS2) was already described in only three subjects, two with sensorineural hearing impairment, mild developmental delay, hypoglycemia, lactic acidemia and combined oxidative phosphorylation system deficiency and another, recently, presenting with a less severe phenotype. In order to expand the phenotype, we describe a new MRPS2 homozygous subject who shows particular features which have not yet been reported: initial microcephaly, joint hypermobility and autistic features.

Targeting mitochondrial function in macrophages: A novel treatment strategy for atherosclerotic cardiovascular disease?

Atherosclerotic cardiovascular disease is a major cause of morbidity and mortality due to chronic arterial injury caused by hyperlipidemia, hypertension, inflammation and oxidative stress. Recent studies have shown that the progression of this disease is associated with mitochondrial dysfunction and with the accumulation of mitochondrial alterations within macrophages of atherosclerotic plaques. These alterations contribute to processes of inflammation and oxidative stress. Among the many players involved, macrophages play a pivotal role in atherogenesis as they can exert both beneficial and deleterious effects due to their anti- and pro-inflammatory properties. Their atheroprotective functions, such as cholesterol efflux and efferocytosis, as well as the maintenance of their polarization towards an anti-inflammatory state, are particularly dependent on mitochondrial metabolism. Moreover, in vitro studies have demonstrated deleterious effects of oxidized LDL on macrophage mitochondrial function, resulting in a switch to a pro-inflammatory state and to a potential loss of atheroprotective capacity. Therefore, preservation of mitochondrial function is now considered a legitimate therapeutic strategy. This review focuses on the potential therapeutic strategies that could improve the mitochondrial function of macrophages, enabling them to maintain their atheroprotective capacity. These emerging therapies could play a valuable role in counteracting the progression of atherosclerotic lesions and possibly inducing their regression.

UQCRC2-related mitochondrial complex III deficiency, about 7 patients.

Isolated complex III defect is a relatively rare cause of mitochondrial disorder. New genes involved were identified in the last two decades, with only a few cases described for each deficiency. UQCRC2, which encodes ubiquinol-cytochrome c reductase core protein 2, is one of the eleven structural subunits of complex III. We report seven French patients with UQCRC2 deficiency to complete the phenotype reported so far. We highlight the similarities with neoglucogenesis defect during decompensations - hypoglycaemias, liver failure and lactic acidosis - and point out the rapid improvement with glucose fluid infusion, which is a remarkable feature for a mitochondrial disorder. Finally, we discuss the relevance of coenzyme Q10 supplementation in this defect.

MPC2 variants disrupt mitochondrial pyruvate metabolism and cause an early-onset mitochondriopathy.

Pyruvate is an essential metabolite produced by glycolysis in the cytosol and must be transported across the inner mitochondrial membrane into the mitochondrial matrix, where it is oxidized to fuel mitochondrial respiration. Pyruvate import is performed by the mitochondrial pyruvate carrier (MPC), a hetero-oligomeric complex composed by interdependent subunits MPC1 and MPC2. Pathogenic variants in the MPC1 gene disrupt mitochondrial pyruvate uptake and oxidation and cause autosomal-recessive early-onset neurological dysfunction in humans. The present work describes the first pathogenic variants in MPC2 associated with human disease in four patients from two unrelated families. In the first family, patients presented with antenatal developmental abnormalities and harboured a homozygous c.148T>C (p.Trp50Arg) variant. In the second family, patients that presented with infantile encephalopathy carried a missense c.2T>G (p.Met1?) variant disrupting the initiation codon. Patient-derived skin fibroblasts exhibit decreased pyruvate-driven oxygen consumption rates with normal activities of the pyruvate dehydrogenase complex and mitochondrial respiratory chain and no defects in mitochondrial content or morphology. Re-expression of wild-type MPC2 restored pyruvate-dependent respiration rates in patient-derived fibroblasts. The discovery of pathogenic variants in MPC2 therefore broadens the clinical and genetic landscape associated with inborn errors in pyruvate metabolism.

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.

Cholesterol accumulation induced by acetylated LDL exposure modifies the enzymatic activities of the TCA cycle without impairing the respiratory chain functionality in macrophages.

The unregulated uptake of modified low-density lipoproteins (LDL) by macrophages leads to foam cell formation, promoting atherosclerotic plaque progression. The cholesterol efflux capacity of macrophages by the ATP-Binding Cassette transporters depends on the ATP mitochondrial production. Therefore, the mitochondrial function maintenance is crucial in limiting foam cell formation. Thus, we aimed to investigate the mechanisms involved in the mitochondrial dysfunction that may occur in cholesterol-laden macrophages. We incubated THP-1 macrophages with acetylated LDL (acLDL) to obtain cholesterol-laden cells or with mildly oxidized LDL (oxLDL) to generate cholesterol- and oxidized lipids-laden cells. Cellular cholesterol content was measured in each condition. Mitochondrial function was evaluated by measurement of several markers of energetic metabolism, oxidative phosphorylation, oxidative stress, mitochondrial biogenesis and dynamics. OxLDL-exposed macrophages exhibited a significantly reduced mitochondrial respiration and complexes I and III activities, associated to an oxidative stress state and a reduced mitochondrial DNA copy number. Meanwhile, acLDL-exposed macrophages featured an efficient oxidative phosphorylation despite the decreased activities of aconitase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase. Our study revealed that mitochondrial function was differently impacted according to the nature of modified LDL. Exposure to cholesterol and oxidized lipids carried by oxLDL leads to a mitochondrial dysfunction in macrophages, affecting the mitochondrial respiratory chain functional capacity, whereas the cellular cholesterol enrichment induced by acLDL exposure results in a tricarboxylic acid cycle shunt while maintaining mitochondrial energetic production, reflecting a metabolic adaptation to cholesterol intake. These new mechanistic insights are of direct relevance to the understanding of the mitochondrial dysfunction in foam cells.

FDX2 and ISCU Gene Variations Lead to Rhabdomyolysis With Distinct Severity and Iron Regulation.

BACKGROUND AND OBJECTIVES: To determine common clinical and biological traits in 2 individuals with variants in ISCU and FDX2, displaying severe and recurrent rhabdomyolyses and lactic acidosis. METHODS: We performed a clinical characterization of 2 distinct individuals with biallelic ISCU or FDX2 variants from 2 separate families and a biological characterization with muscle and cells from those patients. RESULTS: The individual with FDX2 variants was clinically more affected than the individual with ISCU variants. Affected FDX2 individual fibroblasts and myoblasts showed reduced oxygen consumption rates and mitochondrial complex I and PDHc activities, associated with high levels of blood FGF21. ISCU individual fibroblasts showed no oxidative phosphorylation deficiency and moderate increase of blood FGF21 levels relative to controls. The severity of the FDX2 individual was not due to dysfunctional autophagy. Iron was excessively accumulated in ISCU-deficient skeletal muscle, which was accompanied by a downregulation of IRP1 and mitoferrin2 genes and an upregulation of frataxin (FXN) gene expression. This excessive iron accumulation was absent from FDX2 affected muscle and could not be correlated with variable gene expression in muscle cells. DISCUSSION: We conclude that FDX2 and ISCU variants result in a similar muscle phenotype, that differ in severity and skeletal muscle iron accumulation. ISCU and FDX2 are not involved in mitochondrial iron influx contrary to frataxin.

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.

Galaxy Is a Suitable Bioinformatics Platform for the Molecular Diagnosis of Human Genetic Disorders Using High-Throughput Sequencing Data Analysis: Five Years of Experience in a Clinical Laboratory.

BACKGROUND: To date, the usage of Galaxy, an open-source bioinformatics platform, has been reported primarily in research. We report 5 years' experience (2015 to 2020) with Galaxy in our hospital, as part of the "Assistance Publique-Hôpitaux de Paris" (AP-HP), to demonstrate its suitability for high-throughput sequencing (HTS) data analysis in a clinical laboratory setting. METHODS: Our Galaxy instance has been running since July 2015 and is used daily to study inherited diseases, cancer, and microbiology. For the molecular diagnosis of hereditary diseases, 6970 patients were analyzed with Galaxy (corresponding to a total of 7029 analyses). RESULTS: Using Galaxy, the time to process a batch of 23 samples-equivalent to a targeted DNA sequencing MiSeq run-from raw data to an annotated variant call file was generally less than 2 h for panels between 1 and 500 kb. Over 5 years, we only restarted the server twice for hardware maintenance and did not experience any significant troubles, demonstrating the robustness of our Galaxy installation in conjunction with HTCondor as a job scheduler and a PostgreSQL database. The quality of our targeted exome sequencing method was externally evaluated annually by the European Molecular Genetics Quality Network (EMQN). Sensitivity was mean (SD)% 99 (2)% for single nucleotide variants and 93 (9)% for small insertion-deletions. CONCLUSION: Our experience with Galaxy demonstrates it to be a suitable platform for HTS data analysis with vast potential to benefit patient care in a clinical laboratory setting.

Improved detection of mitochondrial DNA instability in mitochondrial genome maintenance disorders.

PURPOSE: Diseases caused by defects in mitochondrial DNA (mtDNA) maintenance machinery, leading to mtDNA deletions, form a specific group of disorders. However, mtDNA deletions also appear during aging, interfering with those resulting from mitochondrial disorders. METHODS: Here, using next-generation sequencing (NGS) data processed by eKLIPse and data mining, we established criteria distinguishing age-related mtDNA rearrangements from those due to mtDNA maintenance defects. MtDNA deletion profiles from muscle and urine patient samples carrying pathogenic variants in nuclear genes involved in mtDNA maintenance (n = 40) were compared with age-matched controls (n = 90). Seventeen additional patient samples were used to validate the data mining model. RESULTS: Overall, deletion number, heteroplasmy level, deletion locations, and the presence of repeats at deletion breakpoints were significantly different between patients and controls, especially in muscle samples. The deletion number was significantly relevant in adults, while breakpoint repeat lengths surrounding deletions were discriminant in young subjects. CONCLUSION: Altogether, eKLIPse analysis is a powerful tool for measuring the accumulation of mtDNA deletions between patients of different ages, as well as in prioritizing novel variants in genes involved in mtDNA stability.

Response to systemic therapy in fumarate hydratase-deficient renal cell carcinoma.

PURPOSE: Fumarate hydratase-deficient (FHdef) renal cell carcinoma (RCC) is a rare entity associated with the hereditary leiomyomatosis and RCC syndrome with no standard therapy approved. The aim of this retrospective study was to evaluate the efficacy of different systemic treatments in this population. METHODS: We performed a multicentre retrospective analysis of Fhdef RCC patients to determine the response to systemic treatments. The endpoints were objective response rate (ORR), time-to-treatment failure (TTF), and overall survival (OS). The two latter were estimated using the Kaplan-Meier method. RESULTS: Twenty-four Fhdef RCC patients were identified, and 21 under systemic therapy were included in the analysis: ten received cabozantinib, 14 received sunitinib, nine received "other antiangiogenics" (sorafenib, pazopanib, and axitinib), three received erlotinib-bevacizumab (E-B), three received mTOR inhibitors, and 11 received immune checkpoint blockers (ICBs). ORR for treatments were 50% for cabozantinib, 43% for sunitinib, 63% for "other antiangiogenics," and 30% for E-B, whereas ORR was 0% for mTOR inhibitors and 18% for ICBs. The median TTF (mTTF) was significantly higher with antiangiogenics (11.6 months) than with mTOR inhibitors (4.4 months) or ICBs (2.7 months). In the first-line setting, antiangiogenics presented a higher ORR compared with nivolumab-ipilimumab (64% versus 25%) and a significantly superior mTTF (11.0 months vs 2.5 months; p = 0.0027). The median OS from the start of the first systemic treatment was 44.0 months (95% confidence interval: 13.0-95.0). CONCLUSIONS: We report the first European retrospective study of Fhdef RCC patients treated with systemic therapy with a remarkably long median OS of 44.0 months. Our results suggest that antiangiogenics may be superior to ICB/mTOR inhibitors in this population.

Enteral tube feeding in patients receiving dietary treatment for metabolic diseases: A retrospective analysis in a large French cohort.

CONTEXT: A strictly controlled diet (often involving enteral tube feeding (ETF)) is part of the treatment of many inherited metabolic diseases (IMDs). OBJECTIVE: To describe the use of ETF in a large cohort of patients with IMDs. DESIGN: A retrospective analysis of ETF in patients with urea cycle disorders (UCDs), organic aciduria (OA), maple syrup disease (MSUD), glycogen storage diseases (GSDs) or fatty acid oxidation disorders (FAODs) diagnosed before the age of 12 months. SETTING: The reference center for IMDs at Necker Hospital (Paris, France). RESULTS: 190 patients born between January 1991 and August 2017 were being treated for OA (n = 60), UCDs (n = 55), MSUD (n = 32), GSDs (n = 26) or FAODs (n = 17). Ninety-eight of these patients (52%) received ETF (OA subgroup: n = 40 (67%); UCDs: n = 12 (22%); MSUD: n = 9 (28%); GSDs: n = 23 (88%); FAODs: n = 14 (82%)). Indications for ETF were feeding difficulties in 64 (65%) patients, cessation of fasting in 39 (40%), and recurrent metabolic decompensation in 14 (14%). Complications of ETF were recorded in 48% of cases, more frequently with nasogastric tube (NGT) than with gastrostomy. Among patients in whom ETF was withdrawn, the mean duration of ETF was 5.9 (SD: 4.8) years (range: 0.6-19.8 years). The duration of ETF was found to vary from one disease subgroup to another (p = 0.051). While the longest median duration was found in the GSD subgroup (6.8 years), the shortest one was found in the UCD subgroup (0.9 years). CONCLUSION: ETF is an integral part of the dietary management of IMDs. The long duration of ETF and the specific risks of NGT highlights the potential value of gastrostomy.In this study at a French tertiary hospital, we documented the indications, modalities, duration and complications of enteral tube feeding in a cohort of patients with inherited metabolic diseases.

Additive protective effects of sacubitril/valsartan and bosentan on vascular remodelling in experimental pulmonary hypertension.

AIMS: Although right ventricular (RV) function is an important determinant of morbidity and mortality in patients with pulmonary arterial hypertension (PAH), there is no treatment targeting directly the RV. We evaluate the efficacy of sacubitril/valsartan (LCZ 696) as add-on therapy to bosentan in rats with severe pulmonary hypertension (PH). METHODS AND RESULTS: Combination therapy of LCZ 696 and bosentan has additive vascular protective effects against the pulmonary vascular remodelling and PH in two preclinical models of severe PH. Compared with monotherapy, co-treatment of LCZ 696 (30 or 68 mg/kg/day for 2 weeks, per os) and bosentan (100 mg/kg/day for 2 weeks, per os) started 7 days after monocrotaline (MCT) injection substantially reduces pulmonary pressures, vascular remodelling, and RV hypertrophy and fibrosis in rats. Consistent with these observations, co-treatment of rats with established PH induced by sugen/hypoxia (SuHx) with LCZ 696 (30 mg/kg/day for 3 weeks, per os) and bosentan (100 mg/kg/day for 3 weeks, per os) started 5 weeks after Sugen injection partially attenuate total pulmonary vascular resistance and cardiovascular structures. We also obtained evidence showing that LCZ 696 has anti-proliferative effect on cultured human pulmonary artery smooth muscle cells derived from patients with idiopathic PAH, an effect that is more pronounced in presence of bosentan. Finally, we found that the plasma levels of atrial natriuretic peptide (ANP) and cyclic guanosine monophosphate (cGMP) are higher in rats co-treated with LCZ 696 (30 mg/kg/day) and bosentan (100 mg/kg/day) than in MCT and SuHx rats treated with vehicle. CONCLUSION: Dual therapy with LCZ 696 plus bosentan proved significantly superior beneficial effect to LCZ 696 or bosentan alone on vascular remodelling and severity of experimental PH.

Adenosine kinase deficiency: Three new cases and diagnostic value of hypermethioninemia.

Adenosine kinase (ADK) deficiency is characterized by liver disease, dysmorphic features, epilepsy and developmental delay. This defect disrupts the adenosine/AMP futile cycle and interferes with the upstream methionine cycle. We report the clinical, histological and biochemical courses of three ADK children carrying two new mutations and presenting with neonatal cholestasis and neurological disorders. One of them died of liver failure whereas the other two recovered from their liver damage. As the phenotype was consistent with a mitochondrial disorder, we studied liver mitochondrial respiratory chain activities in two patients and revealed a combined defect of several complexes. In addition, we retrospectively analyzed methionine plasma concentration, a hallmark of ADK deficiency, in a cohort of children and showed that methionine level in patients with ADK deficiency was strongly increased compared with patients with other liver diseases. ADK deficiency is a cause of neonatal or early infantile liver disease that may mimic primary mitochondrial disorders. In this context, an elevation of methionine plasma levels over twice the upper limit should not be considered as a nonspecific finding. ADK deficiency induced-liver dysfunction is most often transient, but could be life-threatening.

Mitochondrial dysfunction caused by novel ATAD3A mutations.

Mitochondrial respiratory chain integrity depends on a number of proteins encoded by nuclear and mitochondrial genomes. Mutations of such factors can result in isolated or combined respiratory chain deficits, some of which can induce abnormal morphology of the mitochondrial network or accumulation of intermediary metabolites. Consequently, affected patients are clinically heterogeneous, presenting with central nervous system, muscular, or neurodegenerative disorders. ATAD3A is a nuclear-encoded ATPase protein of the AAA+ family and has been localized to the inner mitochondrial membrane. Recently reported mutations or large deletions in the ATDA3A gene in patients have been shown to induce altered mitochondrial structure and function and abnormal cholesterol metabolism in a recessive or dominant manner. Here, we report two siblings presenting axonal sensory-motor neuropathy associated with neonatal cataract. Genetic analyses identified two novel mutations in ATAD3A; a point mutation and an intronic 15 bp deletion affecting splicing and leading to exon skipping. Biochemical analysis in patient cells and tissues showed abnormal function of the mitochondrial respiratory chain in muscle and abnormal mitochondrial cristae structure. These new cases underline the large spectrum of biochemical and clinical presentations of ATAD3A deficiency and the different modes of inheritance, making it an atypical mitochondrial disorder.