Expanding the phenotype of mitochondrial disease: Novel pathogenic variant in ISCA1 leading to instability of the iron-sulfur cluster in the protein.

We report a patient carrying a novel pathogenic variant p.(Tyr101Cys) in ISCA1 leading to MMDS type 5. He initially presented a psychomotor regression with loss of gait and language skills and a tetrapyramidal spastic syndrome. Biochemical analysis of patient fibroblasts revealed impaired lipoic acid synthesis and decreased activities of complex I and II of respiratory chain. While ISCA1 is involved in the mitochondrial machinery for iron-sulfur cluster biogenesis, these dysfunctions are secondary to impaired maturation of mitochondrial proteins containing the [4Fe-4S] clusters. Expression and purification of the human ISCA1 showed a decreased stability of the [2Fe-2S] cluster in the mutated protein.

Ketone Bodies as a Possible Adjuvant to Ketogenic Diet in PDHc Deficiency but Not in GLUT1 Deficiency.

OBJECTIVE: Ketogenic diet is the first line therapy for neurological symptoms associated with pyruvate dehydrogenase deficiency (PDHD) and intractable seizures in a number of disorders, including GLUT1 deficiency syndrome (GLUT1-DS). Because high-fat diet raises serious compliance issues, we investigated if oral L,D-3-hydroxybutyrate administration could be as effective as ketogenic diet in PDHD and GLUT1-DS. METHODS: We designed a partial or total progressive substitution of KD with L,D-3-hydroxybutyrate in three GLUT1-DS and two PDHD patients. RESULTS: In GLUT1-DS patients, we observed clinical deterioration including increased frequency of seizures and myoclonus. In parallel, ketone bodies in CSF decreased after introducing 3-hydroxybutyrate. By contrast, two patients with PDHD showed clinical improvement as dystonic crises and fatigability decreased under basal metabolic conditions. In one of the two PDHD children, 3-hydroxybutyrate has largely replaced the ketogenic diet, with the latter that is mostly resumed only during febrile illness. Positive direct effects on energy metabolism in PDHD patients were suggested by negative correlation between ketonemia and lactatemia (r 2 = 0.59). Moreover, in cultured PDHc-deficient fibroblasts, the increase of CO2 production after 14C-labeled 3-hydroxybutyrate supplementation was consistent with improved Krebs cycle activity. However, except in one patient, ketonemia tended to be lower with 3-hydroxybutyrate administration compared to ketogenic diet. CONCLUSION: 3-hydroxybutyrate may be an adjuvant treatment to ketogenic diet in PDHD but not in GLUT1-DS under basal metabolic conditions. Nevertheless, ketogenic diet is still necessary in PDHD patients during febrile illness.

Impact of mutations within the [Fe-S] cluster or the lipoic acid biosynthesis pathways on mitochondrial protein expression profiles in fibroblasts from patients.

Lipoic acid (LA) is the cofactor of the E2 subunit of mitochondrial ketoacid dehydrogenases and plays a major role in oxidative decarboxylation. De novo LA biosynthesis is dependent on LIAS activity together with LIPT1 and LIPT2. LIAS is an iron­sulfur (Fe-S) cluster-containing mitochondrial protein, like mitochondrial aconitase (mt-aco) and some subunits of respiratory chain (RC) complexes I, II and III. All of them harbor at least one [Fe-S] cluster and their activity is dependent on the mitochondrial [Fe-S] cluster (ISC) assembly machinery. Disorders in the ISC machinery affect numerous Fe-S proteins and lead to a heterogeneous group of diseases with a wide variety of clinical symptoms and combined enzymatic defects. Here, we present the biochemical profiles of several key mitochondrial [Fe-S]-containing proteins in fibroblasts from 13 patients carrying mutations in genes encoding proteins involved in either the lipoic acid (LIPT1 and LIPT2) or mitochondrial ISC biogenesis (FDX1L, ISCA2, IBA57, NFU1, BOLA3) pathway. Ten of them are new patients described for the first time. We confirm that the fibroblast is a good cellular model to study these deficiencies, except for patients presenting mutations in FDX1L and a muscular clinical phenotype. We find that oxidative phosphorylation can be affected by LA defects in LIPT1 and LIPT2 patients due to excessive oxidative stress or to another mechanism connecting LA and respiratory chain activity. We confirm that NFU1, BOLA3, ISCA2 and IBA57 operate in the maturation of [4Fe-4S] clusters and not in [2Fe-2S] protein maturation. Our work suggests a functional difference between IBA57 and other proteins involved in maturation of [Fe-S] proteins. IBA57 seems to require BOLA3, NFU1 and ISCA2 for its stability and NFU1 requires BOLA3. Finally, our study establishes different biochemical profiles for patients according to their mutated protein.

[Molecular diagnosis of respiratory enterovirus infections: Use of PCR and molecular identification for a best approach of the main circulating strains during 2008]. / Diagnostic moléculaire des infections respiratoires à entérovirus : apport de la PCR et du génotypage pour une meilleure approche de la circulation des souches en Basse-Normandie au cours de l'année 2008.

UNLABELLED: The PCR assays are currently used in diagnosis of enterovirus (EV) meningitis. Nevertheless, the use of molecular diagnosis of EV should be investigated in respiratory tract infections (RTI). OBJECTIVES: To perform enterovirus molecular diagnostic tools, PCR and genotyping, in nasal samples for diagnostic and epidemiologic purposes. METHODS: During 2008, 3612 nasal specimen (NS) were studied by IFD and MRC5 culture. Next, we realised successively viral isolation on HuH7 culture (for NS negative by IFD assay) and a duplex PCR enterovirus-rhinovirus for the 816 HuH7 positive supernatants. Furthermore, 327 NS collected from neonates were systematically tested by a real-time RT-PCR. This assay was used in routine for EV diagnosis setting in cerebrospinal fluid. Enterovirus genotyping was then performed for the 68 positive supernatants. RESULTS: Thirty-five NS (0.97%) were positive for EV by culture (MRC5). A combination of both PCR assays, PEVRV and PEV, allowed an additional identification of 41 EV, eight EV-RV and 12 RV, increasing the number of positive to 96 NS (2.6%). Among the neonates, 32 NS (11.3%) were positive for EV by PEV. Of the 98 NS tested by the two PCR assays (PEV and PEVRV), 27 were positive and we detected 10 EV, five EV-RV and 12 RV. From January to December 2008, the circulation of EV showed the usual peak in June-July when a small outbreak of aseptic meningitis occurred and an additional autumnal peak corresponding to respiratory tract infections. Five main serotypes were isolated: 19 EV68 (29.7%), 12 CB3 (18.7%), nine E3 (14,1%), six CA9 (9.4%) and six CB1 (9.4%); the 19 EV68 were isolated in October-November and 17/19 (89.5%) of positive patients were hospitalised for severe respiratory diseases. CONCLUSION: The use of molecular screening techniques (PCR assays and genotyping) on nasal samples collected from patients with respiratory infections allowed a prospective, effective and precise identification of circulating strains.