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.

Biallelic Mutations in LIPT2 Cause a Mitochondrial Lipoylation Defect Associated with Severe Neonatal Encephalopathy.

Lipoate serves as a cofactor for the glycine cleavage system (GCS) and four 2-oxoacid dehydrogenases functioning in energy metabolism (α-oxoglutarate dehydrogenase [α-KGDHc] and pyruvate dehydrogenase [PDHc]), or amino acid metabolism (branched-chain oxoacid dehydrogenase, 2-oxoadipate dehydrogenase). Mitochondrial lipoate synthesis involves three enzymatic steps catalyzed sequentially by lipoyl(octanoyl) transferase 2 (LIPT2), lipoic acid synthetase (LIAS), and lipoyltransferase 1 (LIPT1). Mutations in LIAS have been associated with nonketotic hyperglycinemia-like early-onset convulsions and encephalopathy combined with a defect in mitochondrial energy metabolism. LIPT1 deficiency spares GCS deficiency and has been associated with a biochemical signature of combined 2-oxoacid dehydrogenase deficiency leading to early death or Leigh-like encephalopathy. We report on the identification of biallelic LIPT2 mutations in three affected individuals from two families with severe neonatal encephalopathy. Brain MRI showed major cortical atrophy with white matter abnormalities and cysts. Plasma glycine was mildly increased. Affected individuals' fibroblasts showed reduced oxygen consumption rates, PDHc, α-KGDHc activities, leucine catabolic flux, and decreased protein lipoylation. A normalization of lipoylation was observed after expression of wild-type LIPT2, arguing for LIPT2 requirement in intramitochondrial lipoate synthesis. Lipoic acid supplementation did not improve clinical condition nor activities of PDHc, α-KGDHc, or leucine metabolism in fibroblasts and was ineffective in yeast deleted for the orthologous LIP2.

E4F1 controls a transcriptional program essential for pyruvate dehydrogenase activity.

The mitochondrial pyruvate dehydrogenase (PDH) complex (PDC) acts as a central metabolic node that mediates pyruvate oxidation and fuels the tricarboxylic acid cycle to meet energy demand. Here, we reveal another level of regulation of the pyruvate oxidation pathway in mammals implicating the E4 transcription factor 1 (E4F1). E4F1 controls a set of four genes [dihydrolipoamide acetlytransferase (Dlat), dihydrolipoyl dehydrogenase (Dld), mitochondrial pyruvate carrier 1 (Mpc1), and solute carrier family 25 member 19 (Slc25a19)] involved in pyruvate oxidation and reported to be individually mutated in human metabolic syndromes. E4F1 dysfunction results in 80% decrease of PDH activity and alterations of pyruvate metabolism. Genetic inactivation of murine E4f1 in striated muscles results in viable animals that show low muscle PDH activity, severe endurance defects, and chronic lactic acidemia, recapitulating some clinical symptoms described in PDC-deficient patients. These phenotypes were attenuated by pharmacological stimulation of PDH or by a ketogenic diet, two treatments used for PDH deficiencies. Taken together, these data identify E4F1 as a master regulator of the PDC.

Fluxomic assay-assisted diagnosis orientation in a cohort of 11 patients with myopathic form of CPT2 deficiency.

Carnitine palmitoyltransferase type 2 (CPT2) deficiency, a mitochondrial fatty acid oxidation disorder (MFAOD), is a cause of myopathy in its late clinical presentation. As for other MFAODs, its diagnosis may be evocated when blood acylcarnitine profile is abnormal. However, a lack of abnormalities or specificity in this profile is not exclusive of CPT2 deficiency. Our retrospective study reports clinical and biological data in a cohort of 11 patients with circulating acylcarnitine profile unconclusive enough for a specific diagnosis orientation. In these patients, CPT2 gene studies was prompted by prior fluxomic explorations of mitochondrial ß-oxidation on intact whole blood cells incubated with pentadeuterated ([16-2H3, 15-2H2])-palmitate. Clinical indication for fluxomic explorations was at least one acute rhabdomyolysis episode complicated, in 5 of 11 patients, by acute renal failure. Major trigger of rhabdomyolysis was febrile infection. In all patients, fluxomic data indicated deficient CPT2 function showing normal deuterated palmitoylcarnitine (C16-Cn) formation rates associated with increased ratios between generated C16-Cn and downstream deuterated metabolites (Σ deuterated C2-Cn to C14-Cn). Subsequent gene studies showed in all patients pathogenic gene variants in either homozygous or compound heterozygous forms. Consistent with literature data, allelic frequency of the c.338C > T[p.Ser113Leu] mutation amounted to 68.2% in our cohort. Other missense mutations included c.149C > A[p.Pro50His] (9%), c.200C > G[p.Ala200Gly] (4.5%) and previously unreported c.1171A > G[p.ser391Gly] (4.5%) and c.1420G > C[p.Ala474Pro] (4.5%) mutations. Frameshift c.1666-1667delTT[p.Leu556val*16] mutation (9%) was observed in two patients unknown to be related.

Prenatal sonographic description of fetuses affected by pyruvate dehydrogenase or pyruvate carboxylase deficiency.

OBJECTIVE: Pyruvate dehydrogenase deficiency (PDHD) and pyruvate carboxylase deficiency (PCD) are diseases with severe neonatal forms, and their low prevalence makes them difficult to diagnose during pregnancy. Our objective was to describe prenatal ultrasound features that may be suggestive of these diagnoses. METHODS: We analyzed 3 cases from our institution and reviewed 12 published cases of PDHD and 6 cases of PCD, recording all of the ultrasound signs, as well as magnetic resonance findings when available. Because of the small number of cases of PCD, we also included postnatal signs that could have been observed during imaging during pregnancy, for a total of 11 cases of PCD. RESULTS: We conclude that PDHD can be suggested in the presence of ventriculomegaly or paraventricular cysts, associated with an abnormality of the cerebral parenchyma such as abnormal gyration or involvement of the corpus callosum. Pyruvate carboxylase deficiency can be suggested in the presence of ventriculomegaly, frontal horn impairment associated with subependymal, and paraventricular cysts. CONCLUSION: When confronted to the ultrasound abnormalities we described, and after eliminating the most frequent etiologies, a metabolic deficiency should be considered. Furthermore, the hereditary character of these diseases makes that it is important to send the family with genetic advice in particular in case of history of a fetal death in utero or a death neonatal unexplained.

Assembly defects of multiple respiratory chain complexes in a child with cardiac hypertrophy associated with a novel ACAD9 mutation.

Patients carrying Acyl-CoA dehydrogenase 9 (ACAD9) mutations reported to date mainly present with severe hypertrophic cardiomyopathy and isolated complex I (CI) dysfunction. Here we report a novel ACAD9 mutation in a young girl presenting with severe hypertrophic cardiomyopathy, isolated CI deficiency and interestingly multiple respiratory chain complexes assembly defects. We show that ACAD9 analysis has to be performed in first intention in patients presenting with cardiac hypertrophy even in the presence of multiple assembly defects.

UQCRC2 mutation in a patient with mitochondrial complex III deficiency causing recurrent liver failure, lactic acidosis and hypoglycemia.

An isolated mitochondrial complex III (CIII) defect constitutes a rare cause of mitochondrial disorder. Here we present the second case involving UQCRC2 gene, which encodes core protein 2, one of the 11 structural subunits of CIII. The patient has the same mutation (c.547C>T; p.Arg183Trp) as the first case and presented with neonatal lactic acidosis, hypoglycemia and severe episodes of liver failure. Our study expands the few reported cases of CIII deficiency of nuclear origin.

Severe defect in mitochondrial complex I assembly with mitochondrial DNA deletions in ACAD9-deficient mild myopathy.

INTRODUCTION: Acyl-coenzyme A dehydrogenase 9 (ACAD9) has a role in mitochondrial complex I (CI) assembly. Only a few patients who carry ACAD9 mutations have been reported. They mainly present with severe hypertrophic cardiomyopathy, although a minority have only mild isolated myopathy. Although the secondary factors influencing disease severity have not been elucidated, conservation of CI assembly and residual enzymatic activity have been suggested as explanations for the mild phenotypes associated with ACAD9 mutations. METHODS: We report a novel homozygous ACAD9 mutation (c.1240C>T; p.Arg414Cys) in a 34-year-old woman who presented with non-progressive myopathy. RESULTS: We show that this ACAD9 mutation led to a severe defect in CI assembly in the patient's muscle. Furthermore, the impact of CI deficiency is confirmed by accumulation of mitochondrial DNA deletions. CONCLUSION: Our data suggest that a major defect of CI assembly is not responsible for a severe phenotype. Muscle Nerve 55: 919-922, 2017.

Mitochondrial trifunctional protein deficiency in human cultured fibroblasts: effects of bezafibrate.

Mitochondrial trifunctional protein (MTP) deficiency caused by HADHA or HADHB gene mutations exhibits substantial molecular, biochemical, and clinical heterogeneity and ranks among the more severe fatty acid oxidation (FAO) disorders, without pharmacological treatment. Since bezafibrate has been shown to potentially correct other FAO disorders in patient cells, we analyzed its effects in 26 MTP-deficient patient fibroblasts representing 16 genotypes. Overall, the patient cell lines exhibited variable, complex, biochemical profiles and pharmacological responses. HADHA-deficient fibroblasts showed markedly reduced alpha subunit protein levels together with decreased beta-subunit abundance, exhibited a -86 to -96% defect in LCHAD activity, and produced large amounts of C14 and C16 hydroxyacylcarnitines. In control fibroblasts, exposure to bezafibrate (400 µM for 48 h) increased the abundance of HADHA and HADHB mRNAs, immune-detectable alpha and beta subunit proteins, activities of LCHAD and LCKAT, and stimulated FAO capacities, clearly indicating that MTP is pharmacologically up-regulated by bezafibrate in human fibroblasts. In MTP-deficient patient fibroblasts, which were found markedly FAO-deficient, bezafibrate improved FAO capacities in six of 26 (23%) cases, including three cell lines heterozygous for the common c1528G > C mutation. Altogether, our results strongly suggest that, due to variable effects of HADHA and HADHB mutations on MTP abundance and residual activity, improvement of MTP deficiency in response to bezafibrate was achieved in a subset of responsive genotypes.

Fructose 1,6-bisphosphatase deficiency: clinical, biochemical and genetic features in French patients.

Fructose-1,6-bisphosphatase (FBPase) deficiency is a very rare autosomal recessive disorder caused by a mutation of the fructose-1,6-bisphosphatase gene(FBP1). Disease is mainly revealed by hypoglycemia and lactic acidosis, both symptoms being characteristic for an enzymatic block in the last steps of the gluconeogenesis. Twelve patients with FBPase deficiency were diagnosed in France in the 2001-2013 period, using a diagnostic system based on a single blood sample which allows simultaneous enzyme activity measurement on mononuclear white blood cells and molecular analysis. Sequencing of exons and intron-exon junctions of FBP1 gene was completed in unsolved cases by a gene dosage assay developed for each exon. For most patients, first metabolic decompensation occurred before two years of age with a similar sequence: the triggering factors were fever, fasting, or decrease of food intake. However, diagnosis was made late at a mean age of 3 years, as mitochondrial defects or glycogen storage diseases were firstly suspected. Enzyme activity in leukocytes was dramatically decreased (<10%). Twelve different mutations were identified in 22 alleles among them seven were novels: one missense mutation c.472C > T, one point deletion c.48del, one point duplication c.865dupA, one deletion-insertion, and two splice mutations (c.427-1del and c.825 + 1G > A). We described the first intragenic deletion in FBP1 (g.97,364,754_97,382,011del) in homozygous state. Our report also confirms that this very rare disease is misdiagnosed, as other energetic defects are firstly suspected.

Dihydrolipoamide dehydrogenase deficiency: a still overlooked cause of recurrent acute liver failure and Reye-like syndrome.

The causes of Reye-like syndrome are not completely understood. Dihydrolipoamide dehydrogenase (DLD or E3) deficiency is a rare metabolic disorder causing neurological or liver impairment. Specific changes in the levels of urinary and plasma metabolites are the hallmark of the classical form of the disease. Here, we report a consanguineous family of Algerian origin with DLD deficiency presenting without suggestive clinical laboratory and anatomopathological findings. Two children died at birth from hepatic failure and three currently adult siblings had recurrent episodes of hepatic cytolysis associated with liver failure or Reye-like syndrome from infancy. Biochemical investigation (lactate, pyruvate, aminoacids in plasma, organic acids in urine) was normal. Histologic examination of liver and muscle showed mild lipid inclusions that were only visible by electron microscopy. The diagnosis of DLD deficiency was possible only after genome-wide linkage analysis, confirmed by a homozygous mutation (p.G229C) in the DLD gene, previously reported in patients with the same geographic origin. DLD and pyruvate dehydrogenase activities were respectively reduced to 25% and 70% in skin fibroblasts of patients and were unresponsive to riboflavin supplementation. In conclusion, this observation clearly supports the view that DLD deficiency should be considered in patients with Reye-like syndrome or liver failure even in the absence of suggestive biochemical findings, with the p.G229C mutation screening as a valuable test in the Arab patients because of its high frequency. It also highlights the usefulness of genome-wide linkage analysis for decisive diagnosis advance in inherited metabolic disorders.

Mutation spectrum in the French cohort of galactosemic patients and structural simulation of 27 novel missense variations.

BACKGROUND: Classic galactosemia refers to galactose-1-phosphate uridyltransferase (GALT) deficiency and is characterized by long-term complications of unknown mechanism and high allelic heterogeneity of GALT gene. AIM: To report molecular characterization of GALT variations in 210 French families, to analyze the structural effects of novel missense variations and to assess informativity of structural data in predicting outcome. METHODS: Sequencing of exons and intron-exon junctions of GALT gene was completed in unsolved cases by analysis of a long range PCR product. Structural consequences of novel missense variations were predicted using a homology model of GALT protein and a semi-automated analysis which integrates simulation of variations, structural analyses and two web servers dedicated to identify mutation-induced change of protein stability. RESULTS: Forty four novel variations were identified, among them 27 nucleotide substitutions. In silico modeling of these missense variations showed that 12 variations are predicted to impair subunit interactions and/or active site conformation and that 23 variations modify H-bond or salt-bridge networks. Twenty variations decrease the global stability of the protein. Five variations had apparently no structural effect. CONCLUSION: Our results expand the mutation spectrum in GALT gene and the list of GALT variations analyzed at the structural level, providing new data to assess the pathophysiology of galactosemia.

Two human patient mitochondrial pyruvate carrier mutations reveal distinct molecular mechanisms of dysfunction.

The Mitochondrial Pyruvate Carrier (MPC) occupies a central metabolic node by transporting cytosolic pyruvate into the mitochondrial matrix and linking glycolysis with mitochondrial metabolism. Two reported human MPC1 mutations cause developmental abnormalities, neurological problems, metabolic deficits, and for one patient, early death. We aimed to understand biochemical mechanisms by which the human patient C289T and T236A MPC1 alleles disrupt MPC function. MPC1 C289T encodes two protein variants, a mis-spliced, truncation mutant (A58G) and a full length point mutant (R97W). MPC1 T236A encodes a full length point mutant (L79H). Using human patient fibroblasts and complementation of CRISPR-deleted, MPC1 null mouse C2C12 cells, we investigated how MPC1 mutations cause MPC deficiency. Truncated MPC1 A58G protein was intrinsically unstable and failed to form MPC complexes. The MPC1 R97W protein was less stable but when overexpressed formed complexes with MPC2 that retained pyruvate transport activity. Conversely, MPC1 L79H protein formed stable complexes with MPC2, but these complexes failed to transport pyruvate. These findings inform MPC structure-function relationships and delineate three distinct biochemical pathologies resulting from two human patient MPC1 mutations. They also illustrate an efficient gene pass-through system for mechanistically investigating human inborn errors in pyruvate metabolism.

Carnitine palmitoyltransferase type 2 deficiency: novel mutation in a Native South American family with whole-body muscle magnetic resonance imaging findings: two case reports.

BACKGROUND: The myopathic form of carnitine palmitoyltransferase type II deficiency is an inherited autosomal recessive metabolic myopathy usually starting in childhood. Most reports have been on European and Japanese populations, and no Native South American patients have been reported to date. The p.Ser113Leu mutation is the most frequent in the European population. Only lower-leg magnetic resonance imaging findings have been reported, with gluteus maximus involvement in one case and normal imaging in other patients. CASE PRESENTATION: Two Native South American siblings, a boy and a girl, presented to our neuromuscular clinic with recurrent rhabdomyolysis associated with transient muscle weakness after prolonged exercise. During episodes, their creatine kinase concentrations were markedly increased, up to 148,000 (1.48 × 105) IU/L in the boy and 18,000 (1.8 × 104) IU/L in the girl. The results of electroneuromyography and histopathology suggested a nonspecific myopathy. CPT2 gene sequencing showed two heterozygous mutations: the p.Ser113Leu variant and a novel one (predicted to be deleterious by in silico analysis), the p.Ser373Pro variant. The patients' parents were asymptomatic carriers. Whole-body magnetic resonance imaging showed mild selective involvement in the thoracic extensors and pelvic girdle in both siblings, and in the thighs and lower legs in one of them. Dietary and bezafibrate treatment was started, and symptomatic relief was observed. CONCLUSIONS: To the best of our knowledge, this is the first reported Native South American family with a CPT2 deficiency carrying a novel mutation and particular features visualized by whole-body magnetic resonance imaging.

Phenotypic and Neuropathological Characterization of Fetal Pyruvate Dehydrogenase Deficiency.

To distinguish pyruvate dehydrogenase deficiency (PDH) from other antenatal neurometabolic disorders thereby improving prenatal diagnosis, we describe imaging findings, clinical phenotype, and brain lesions in fetuses from 3 families with molecular characterization of this condition. Neuropathological analysis was performed in 4 autopsy cases from 3 unrelated families with subsequent biochemical and molecular confirmation of PDH complex deficiency. In 2 families there were mutations in the PDHA1 gene; in the third family there was a mutation in the PDHB gene. All fetuses displayed characteristic craniofacial dysmorphism of varying severity, absence of visceral lesions, and associated encephaloclastic and developmental supra- and infratentorial lesions. Neurodevelopmental abnormalities included microcephaly, migration abnormalities (pachygyria, polymicrogyria, periventricular nodular heterotopias), and cerebellar and brainstem hypoplasia with hypoplastic dentate nuclei and pyramidal tracts. Associated clastic lesions included asymmetric leukomalacia, reactive gliosis, large pseudocysts of germinolysis, and basal ganglia calcifications. The diagnosis of PDH deficiency should be suspected antenatally with the presence of clastic and neurodevelopmental lesions and a relatively characteristic craniofacial dysmorphism. Postmortem examination is essential for excluding other closely related entities, thereby allowing for biochemical and molecular confirmation.

AIP mutations impair AhR signaling in pituitary adenoma patients fibroblasts and in GH3 cells.

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene predispose humans to pituitary adenomas through unknown molecular mechanisms. The best-known interacting partner of AIP is the aryl hydrocarbon receptor (AhR), a transcription factor that mediates the effects of xenobiotics implicated in carcinogenesis. As 75% of AIP mutations disrupt the physical and/or functional interaction with AhR, we postulated that the tumorigenic potential of AIP mutations might result from altered AhR signaling. We evaluated the impact of AIP mutations on the AhR signaling pathway, first in fibroblasts from AIP-mutated patients with pituitary adenomas, by comparison with fibroblasts from healthy subjects, then in transfected pituitary GH3 cells. The AIP protein level in mutated fibroblasts was about half of that in cells from healthy subjects, but AhR expression was unaffected. Gene expression analyses showed significant modifications in the expression of the AhR target genes CYP1B1 and AHRR in AIP-mutated fibroblasts, both before and after stimulation with the endogenous AhR ligand kynurenine. Kynurenine increased Cyp1b1 expression to a greater extent in GH3 cells overexpressing wild type compared with cells expressing mutant AIP Knockdown of endogenous Aip in these cells attenuated Cyp1b1 induction by the AhR ligand. Both mutant AIP expression and knockdown of endogenous Aip affected the kynurenine-dependent GH secretion of GH3 cells. This study of human fibroblasts bearing endogenous heterozygous AIP mutations and transfected pituitary GH3 cells shows that AIP mutations affect the AIP protein level and alter AhR transcriptional activity in a gene- and tissue-dependent manner.

New spastic paraplegia phenotype associated to mutation of NFU1.

Recently an early onset lethal encephalopathy has been described in relation to mutations of NFU1, one of the genes involved in iron-sulfur cluster metabolism. We report a new NFU1 mutated patient presenting with a milder phenotype characterized by a later onset, a slowly progressive spastic paraparesis with relapsing-remitting episodes, mild cognitive impairment and a long survival. The early white matter abnormalities observed on MRI was combined with a mixed sensory-motor neuropathy in the third decade. Our case clearly suggests the importance of considering NFU1 mutation in slowly evolving leukoencephalopathy with high glycine concentration.

The deleterious G15498A mutation in mitochondrial DNA-encoded cytochrome b may remain clinically silent in homoplasmic carriers.

We report on a patient with severe growth retardation and IgF1 deficiency, in which a mitochondrial abnormality was suspected. An isolated mitochondrial respiratory chain complex III deficiency was found in blood lymphocytes and skin fibroblasts. Sequence analysis of the cytochrome b, which is the only mitochondrial DNA-encoded subunit of complex III, revealed a homoplasmic G15498A mutation, resulting in the substitution of a highly conserved amino acid (glycine 251 into an aspartic acid). The mutation was found to be homoplasmic in all tissues examined from the mother and her brother (lymphocytes, fibroblasts, hair roots and buccal cells). Complex III deficiency was also demonstrated in these cells. Nevertheless, the mother and the brother were asymptomatic. This mutation had been considered as a cardiomyopathy-generating mutation in a previously reported case, and its pathogenicity has been demonstrated recently in yeast. However, it seems not to fulfil the classical criteria for pathogenicity of a mitochondrial DNA mutation, especially the heteroplasmic status, and to be clinically silent, albeit present, in nonaffected relatives. We suggest that other factors are contributing to the clinical variability expression of the G15498A mtDNA mutation.