Recessive RYR1 mutations cause unusual congenital myopathy with prominent nuclear internalization and large areas of myofibrillar disorganization.

AIMS: To report the clinical, pathological and genetic findings in a group of patients with a previously not described phenotype of congenital myopathy due to recessive mutations in the gene encoding the type 1 muscle ryanodine receptor channel (RYR1). METHODS: Seven unrelated patients shared a predominant axial and proximal weakness of varying severity, with onset during the neonatal period, associated with bilateral ptosis and ophthalmoparesis, and unusual muscle biopsy features at light and electron microscopic levels. RESULTS: Muscle biopsy histochemistry revealed a peculiar morphological pattern characterized by numerous internalized myonuclei in up to 51% of fibres and large areas of myofibrillar disorganization with undefined borders. Ultrastructurally, such areas frequently occupied the whole myofibre cross section and extended to a moderate number of sarcomeres in length. Molecular genetic investigations identified recessive mutations in the ryanodine receptor (RYR1) gene in six compound heterozygous patients and one homozygous patient. Nine mutations are novel and four have already been reported either as pathogenic recessive mutations or as changes affecting a residue associated with dominant malignant hyperthermia susceptibility. Only two mutations were located in the C-terminal transmembrane domain whereas the others were distributed throughout the cytoplasmic region of RyR1. CONCLUSION: Our data enlarge the spectrum of RYR1 mutations and highlight their clinical and morphological heterogeneity. A congenital myopathy featuring ptosis and external ophthalmoplegia, concomitant with the novel histopathological phenotype showing fibres with large, poorly delimited areas of myofibrillar disorganization and internal nuclei, is highly suggestive of an RYR1-related congenital myopathy.

Mutational spectrum and phenotypes in Danish families with hereditary angioedema because of C1 inhibitor deficiency.

BACKGROUND: Hereditary angioedema (HAE), type I and II, is an autosomal dominant disease with deficiency of functional C1 inhibitor protein causing episodic swellings of skin, mucosa and viscera. HAE is a genetically heterogeneous disease with more than 200 different mutations in the SERPING1 gene. A genotype-phenotype relationship does not seem to exist in HAE, although the polymorphism c.-21T>C of exon 2 has been reported to be associated with a more severe phenotype. We aimed to establish the mutational spectrum of C1 inhibitor deficiency in Denmark and investigate the possible disease-aggravating effect of the c.-21T>C polymorphism. METHODS: Hereditary angioedema was diagnosed based on clinical features and C1 inhibitor deficiency. A general severity score ranging from 0 to 10 was developed based on age at disease onset, clinical manifestations and treatment experiences. SERPING1 gene investigation was performed by exon sequencing followed by multiplex ligation-dependent probe amplification genomic rearrangement analysis in all known Danish HAE families. RESULTS: Fifty-nine patients with HAE from 26 families were included in this study. The mean disease severity score was 7.12 [1-10], and the mean C1 inhibitor function was 26% [20-46%]. The sensitivity of the mutational screening was 96%, and 13 new mutations were found in this Danish patient cohort. Nine patients (15%) carried the c.-21T>C polymorphism, but they didn't have a more severe phenotype. CONCLUSION: Thirteen new mutations were identified in the Danish HAE population. No correlation between the c.-21T>C polymorphism, the biochemical values of C1 inhibitor function and the clinical severity score was found.

Type III hereditary angio-oedema: clinical and biological features in a French cohort.

BACKGROUND: Hereditary angio-oedema (HAE) has been associated with C1inhibitor deficiency. The first cases of type III HAE were described in patients with normal C1Inh antigenic protein level and function and normal C4 levels in 2000. This finding has been reported mostly in women with a family history and may be influenced by exogenous oestrogen exposure. OBJECTIVES: The purpose of this article is to describe the clinical, biological and genetic characteristics of a French population suffering from type III HAE. PATIENTS AND METHODS: We conducted a retrospective analysis of angio-oedema (AE) cases seen in the National Reference Centre of AE between 2000 and 2009. RESULTS: We found 26 patients (from 15 unrelated families) with type III HAE. All but four were women and presented with typical AE attacks, exacerbated by pregnancy or oral contraceptives containing oestrogens (OC). We also found that 54.5% of women were worsened with oestrogen and 23% were oestrogen dependent. All patients improved on long-term prophylactic tranexamic acid treatment; some acute attacks improved with C1Inh concentrate infusion. All of the patients had normal C1Inh and C4 levels. C1Inh function was also normal, except in women receiving OC or during a pregnancy: transient, moderately low levels (32-74% of the normal range) were found in 18 patients tested (67%). No SERPING1 gene mutation was found. Six patients from three unrelated families were heterozygous for an F12 gene variant. CONCLUSION: Diagnosis of type III HAE should be based on clinical (typical attacks, often hormonally influenced), laboratory (normal C1Inh antigenic protein) and genetic (F12 gene mutation) evidence.

Early onset of hypokalaemic periodic paralysis caused by a novel mutation of the CACNA1S gene.

We report a precocious and atypical form of hypokalaemic periodic paralysis, with clinical manifestations at birth and first episodes of paralysis occurring as early as 1 year of age, although onset of this disease usually occurs between 5-35 years. Extensive molecular analysis showed that the disease was caused by a novel de novo p.Arg897Ser mutation in the CACNA1S gene. The mutation mapped to a new region of the protein, the S4 voltage sensing segment of domain III, at odds with previously reported mutations that exclusively affected domains II and IV.

[Function of aurora kinase C (AURKC) in human reproduction]. / Rôle d'aurora kinase C (AURKC) dans la reproduction humaine.

Infertility concerns at least 70 million couples worldwide. An important proportion of cases is believed to have a genetic component, yet few causal genes have been identified so far. Hundreds of genes are probably involved in spermatogenesis and oogenesis and this genetic heterogeneity has so far hindered the identification of genes causing infertility in the human. Careful morphological examination of spermatozoa can provide cues to identify homogeneous cohorts of patients likely to have the same genetic defect. We studied a cohort of North-Africans patients with a rare phenotype of large-headed spermatozoa. Using a homozygosity mapping strategy, we could map the morbid gene and we identified the same homozygous mutation (c.144delC) in the aurora kinase C gene (AURKC) of all patients studied initially. We then genotyped a total of 62 patients. All who had a typical phenotype with close to 100% large-headed spermatozoa were homozygously mutated (n=34), whereas no AURKC mutations were detected in the others. A carrier frequency of 1/50 was established from individuals from the Maghrebian population, indicating that 1 in 10,000 men from North-African can be expected to present this form of infertility, a frequency comparable to that of Y-microdeletions, thus far the only known recurrent genetic event altering spermatogenesis. Then we demonstrated by flow cytometry that all spermatozoa have in fact a homogeneous 4C. We recommend the realisation of a molecular diagnosis to all patients with large-headed spermatozoa. ICSI is formally contraindicated for all homozygous patients who can have recourse to donor sperm or adoption. One cannot be as categorical for the patients not harbouring an AURKC mutation.

Nuclear DNA origin of mitochondrial complex I deficiency in fatal infantile lactic acidosis evidenced by transnuclear complementation of cultured fibroblasts.

We have studied complex I (NADH-ubiquinone reductase) defects of the mitochondrial respiratory chain in 2 infants who died in the neonatal period from 2 different neurological forms of severe neonatal lactic acidosis. Specific and marked decrease in complex I activity was documented in muscle, liver, and cultured skin fibroblasts. Biochemical characterization and study of the genetic origin of this defect were performed using cultured fibroblasts. Immunodetection of 6 nuclear DNA-encoded (20, 23, 24, 30, 49, and 51 kDa) and 1 mitochondrial DNA-encoded (ND1) complex I subunits in fibroblast mitochondria revealed 2 distinct patterns. In 1 patient, complex I contained reduced amounts of the 24- and 51-kDa subunits and normal amounts of all the other investigated subunits. In the second patient, amounts of all the investigated subunits were severely decreased. The data suggest partial or extensive impairment of complex I assembly in both patients. Cell fusion experiments between 143B206 rho degrees cells, fully depleted of mitochondrial DNA, and fibroblasts from both patients led to phenotypic complementation of the complex I defects in mitochondria of the resulting cybrid cells. These results indicate that the complex I defects in the 2 reported cases are due to nuclear gene mutations.

[Oculo-cerebro-renal Lowe syndrome: clinical, biochemical and molecular studies in a Moroccan patient]. / Syndrome oculo-cérébro-rénal de Lowe: étude clinique, biochimique et moléculaire chez un patient marocain.

The oculo-cerebro-renal syndrome of Lowe is a rare X-linked disorder, caused by the inositol biphosphate 5-phosphatase deficiency, localized to the Golgi complex. Several mutations were reported in patient's OCRL gene leading to enzyme deficiency. We report a Moroccan case of OCRL syndrome of Lowe with a neo mutation in exon 10. The patient aged of 19 months was referred to our medical centre because of a psychomotor retardation. He had a medical history of eye abnormalities including cataract and bilateral glaucoma, diagnosed when he was 5 weeks old. Cataract has been treated after chirurgical therapy but ocular hypertonia persisted. Physical examination revealed an axial hypotonia and walking difficulties. Laboratory tests revealed a moderate acidosis (20 mmol/L), a slight decrease of serum phosphate level (24 mg/L) and an increased serum phosphatase activity. Further studies showed mild proteinuria, urinary bicarbonates loosing and generalised hyperaminoaciduria. Based on both clinical and biological data, Lowe syndrome has been suggested. In this context, molecular investigation has been performed using dHPLC/sequencing techniques which allow identifying an original mutation c.776T>C (p.Phe259Ser), localized on the exon 10 of the OCRL gene. The mutation was not found in the probant's mother suggesting a neo mutation. Lowe syndrome is a rare hereditary X-linked disorder resulting from a variety of heterogeneous mutations of OCRL gene. Indeed, numerous mutations have been reported, variations were noted concerning their localization as well as their type. To our knowledge, this is the first report of the neo mutation c.776T>C of OCRL gene and the first published case report of the Lowe syndrome in a Moroccan patient.

[Molecular genetics of cardiac arrhythmias]. / Génétique moléculaire des arythmies cardiaques.

INTRODUCTION: Recent progress in molecular biology led to the identification of the genes involved in various cardiac arrhythmias causing syncope and sudden death in young people. EXEGESIS: This article briefly describes the clinical features and the genes associated with the congenital long QT syndrome, the short QT syndrome, Brugada's syndrome, catecholaminergic polymorphic ventricular tachycardias and arrhythmogenic right ventricular dysplasia. CONCLUSION: Identification of genetic variations that cause cardiac tachyarrhythmias can help to identify at risk-patients and to propose clinical follow-up and preventive therapy. Further studies are needed to discover other cardiac genetic disorders and to understand cellular mechanisms involved in these diseases.

Evidence for a quinone binding site close to the interface between NUOD and NUOB subunits of Complex I.

Piericidin, rotenone and pyridaben are specific inhibitors of the NADH-ubiquinone oxidoreductase (Complex I) that bind to its ubiquinone binding site(s). Using site directed mutagenesis, we demonstrate that residues G409, D412, R413 and V407 of the C-terminus of Complex I NUOD subunit are directly involved in the binding of these inhibitors. We propose that the corresponding inhibitor/quinone binding site would be located close to NUOD-NUOB interface.

Studies of the nucleotide-binding sites on the mitochondrial F1-ATPase through the use of a photoactivable derivative of adenylyl imidodiphosphate.

(1)N-4-Azido-2-nitrophenyl-gamma-[3H]aminobutyryl-AdoPP[NH] P(NAP4-AdoPP[NH]P) a photoactivable derivative of 5-adenylyl imidodiphosphate (AdoPP[NH]P), was synthesized. (2) Binding of [3H]NAP4-AdoPP[NH]P to soluble ATPase from beef heart mitochondria (F1) was studied in the absence of photoirradiation, and compared to that of [3H]AdoPP[NH]P. The photoactivable derivative of AdoPP[NH]P was found to bind to F1 with high affinity, like AdoPP[NH]P. Once [3H]NAP4-AdoPP[NH]P had bound to F1 in the dark, it could be released by AdoPP[NH]P, ADP and ATP, but not at all by NAP4 or AMP. Furthermore, preincubation of F1 with unlabeled AdoPP[NH]P, ADP, or ATP prevented the covalent labeling of the enzyme by [3H]NAP4-AdoPP[NH]P upon photoirradiation. (3) Photoirradiation of F1 by [3H]NAP4-AdoPP[NH]P resulted in covalent photolabeling and concomitant inactivation of the enzyme. Full inactivation corresponded to the binding of about 2 mol [3H]NAP4-AdoPP[NH]P/mol F1. Photolabeling by NAP4-AdoPP[NH]P was much more efficient in the presence than in the absence of MgCl2. (4) Bound [3H]NAP4-AdoPP[NH]P was localized on the alpha- and beta- subunits of F1. At low concentrations (less than 10 microM), bound [3H]NAP4-AdoPP[NH]P was predominantly localized on the alpha-subunit; at concentrations equal to, or greater than 75 microM, both alpha- and beta-subunits were equally labeled. (5) The extent of inactivation was independent of the nature of the photolabeled subunit (alpha or beta), suggesting that each of the two subunits, alpha and beta, is required for the activity of F1. (6) The covalently photolabeled F1 was able to form a complex with aurovertin, as does native F1. The ADP-induced fluorescence enhancement was more severely inhibited than the fluorescence quenching caused by ATP. The precentage of inactivation of F1 was virtually the same as the percentage of inhibition of the ATP-induced fluorescence quenching, suggestion that fluorescence quenching is related to the binding of ATP to the catalytic site of F1.

Effect of the natural ATPase inhibitor on the binding of adenine nucleotides and inorganic phosphate to mitochondrial F1-ATPase.

(1) Incubation of the beef heart mitochondrial ATPase, F1 with Mg-ATP was required for the binding of the natural inhibitor, IF1, to F1 to form the inactive F1-IF1 complex. When F1 was incubated in the presence of [14C]ATP and MgCl2, about 2 mol 14C-labeled adenine nucleotides were found to bind per mol of F1; the bound 14C-labeled nucleotides consisted of [14C]ADP arising from [14C]ATP hydrolysis and [14C]ATP. The 14C- labeled nucleotide binding was not prevented by IF1. These data are in agreement with the idea that the formation of the F1-IF1 complex requires an appropriate conformation of F1. (2) The 14C-labeled adenine nucleotides bound to F1 following preincubation of F1 with Mg-[14C] ATP could be exchanged with added [3H]ADP or [3H]ATP. No exchange occurred between added [3H]ADP or [3H]ATP and the 14 C-labeled adenine nucleotides bound to the F1-IF1 complex. These data suggest that the conformation of F1 in the isolated F1-IF1 complex is further modified in such a way that the bound 14C-labeled nucleotides are no longer available for exchange. (3) 32Pi was able to bind to isolated F1 with a stoichiometry of about 1 mol of Pi per mol of F1 (Penefsky, H.S. (1977) J. Biol. Chem. 252, 2891-2899). There was no binding of 32Pi to the F1-IF1 complex. Thus, not only the nucleotides sites, but also the Pi site, are masked from interaction with external ligands in the isolated F1-IF1 complex.

The nuoM arg368his mutation in NADH:ubiquinone oxidoreductase from Rhodobacter capsulatus: a model for the human nd4-11778 mtDNA mutation associated with Leber's hereditary optic neuropathy.

Mutation at position 11778 in the nd4 gene of the human mitochondrial complex I is associated with Leber's hereditary optic neuropathy. Type I NADH:ubiquinone oxidoreductase of Rhodobacter capsulatus displays similar properties to complex I of the mitochondrial respiratory chain. The NUOM subunit of the bacterial enzyme is homologous to the ND4 subunit. Disruption of the nuoM gene led to a bacterial mutant exhibiting a defect in complex I activity and assembly. A nuoM-1103 point mutant reproducing the nd4-11778 mutation has been introduced in the R. capsulatus genome. This mutant showed a reduced ability to grow in a medium containing malate instead of lactate which indicated a clear impairment in oxidative phosphorylation capacity. NADH supported respiration of porous bacterial cells was significantly decreased in the nuoM-1103 mutant while no significant reduction could be observed in isolated bacterial membranes. As it has been observed in the case of the nd4-11778 mitochondrial mutation, proton-pump activity of the bacterial enzyme was not affected by the nuoM-1103 mutation. All these data which reproduce most of the biochemical features observed in patient mitochondria harboring the nd4-11778 mutation show that the R. capsulatus complex I might be used as a useful model to investigate mutations of the mitochondrial DNA which are associated with complex I deficiencies in human pathologies.

The complex I from Rhodobacter capsulatus.

The NADH-ubiquinone oxidoreductase (type I NDH) of Rhodobacter capsulatus is a multisubunit enzyme encoded by the 14 genes of the nuo operon. This bacterial enzyme constitutes a valuable model for the characterization of the mitochondrial Complex I structure and enzymatic mechanism for the following reasons. (i) The mitochondria-encoded ND subunits are not readily accessible to genetic manipulation. In contrast, the equivalents of the mitochondrial ND1, ND2, ND4, ND4L, ND5 and ND6 genes can be easily mutated in R. capsulatus by homologous recombination. (ii) As illustrated in the case of ND1 gene, point mutations associated with human cytopathies can be reproduced and studied in this model system. (iii) The R. capsulatus model also allows the recombinant manipulations of iron-sulfur (Fe-S) subunits and the assignment of Fe-S clusters as illustrated in the case of the NUOI subunit (the equivalent of the mitochondrial TYKY subunit). (iv) Finally, like mitochondrial Complex I, the NADH-ubiquinone oxidoreductase of R. capsulatus is highly sensitive to the inhibitor piericidin-A which is considered to bind to or close to the quinone binding site(s) of Complex I. Therefore, isolation of R. capsulatus mutants resistant to piericidin-A represents a straightforward way to map the inhibitor binding sites and to try and define the location of quinone binding site(s) in the enzyme. These illustrations that describe the interest in the R. capsulatus NADH-ubiquinone oxidoreductase model for the general study of Complex I will be critically developed in the present review.

cDNA sequence and chromosomal localization of the NDUFS8 human gene coding for the 23 kDa subunit of the mitochondrial complex I.

We have sequenced the cDNA for the 23 kDa subunit of the human mitochondrial respiratory complex I. The deduced protein consists of 210 amino acids (Mr = 23705 Da) with a 34 amino acid N terminus presumably acting as a presequence for mitochondrial import. The predicted mature protein (Mr = 20290 Da) is 92% identical to the bovine mitochondrial subunit and 72% to the Rhodobacter capsulatus NUOI counterpart. Two clusters of four cysteine residues are conserved among these proteins. The gene (NDUFS8) coding for the human subunit has been mapped to chromosome 11q13.

5-lipoxygenase expression and activity in aorta from streptozotocin-induced diabetic rats.

We previously reported an activation of the 5-lipoxygenase pathway in aorta from streptozotocin-induced diabetic rats. The aim of this study was to investigate whether this activation was associated with an increased expression of 5-lipoxygenase, an increased cysteinyl leukotriene (CysLT) production in response to arachidonic acid or calcium ionophore A23187 and/or a hypersensitivity of the aorta to CysLTs in streptozotocin-induced diabetic rats. In aorta from diabetic and control rats, reverse transcriptase-PCR and western blot analysis with a specific 5-lipoxygenase antibody provided evidence for the presence of 5-lipoxygenase in aorta. However, the expression of 5-lipoxygenase was not significantly different between diabetic and control rats. Challenge by A23187 (10 microM) and arachidonic acid (10 microM and 0.1 mM) with or without A23187 (10 micromol/l) induced a significant increase of CysLT release (measured by enzyme immunoassay) that was in the same range in aorta from control and diabetic rats. In contrast, aortas from diabetic rats showed a greater sensitivity to LTC4 and LTD4 contractile effects. These data suggested that the activation of the 5-lipoxygenase pathway previously reported in streptozotocin-induced diabetic rats could be explained by an augmented sensitivity to CysLTs of the diabetic aorta.

Clinical and histopathological aspects of central core disease associated and non-associated with RYR1 locus.

We analysed the clinical, histochemical, ultrastructural and genetic data of patients affected by central core disease (CCD) studied during the last 20 years. From a total series of 86 CCD-families, we have identified 46 CCD families with RYR1 mutations (16 autosomal dominant, 8 autosomal recessive, 17 sporadic cases and 5 de novo mutations). Out of the other 40 CCD families, the RyR1 gene was entirely excluded in 7 families, by cDNA sequencing or linkage analysis, indicating a genetic heterogeneity of CCD.

Genomic structure of the human NDUFS8 gene coding for the iron-sulfur TYKY subunit of the mitochondrial NADH:ubiquinone oxidoreductase.

The structural organization of the NDUFS8 gene coding for the TYKY subunit of the human mitochondrial NADH:ubiquinone oxidoreductase (Complex I) has been determined by sequencing of a genomic fragment cloned from a cosmid library. The NDUFS8 gene is located on chromosome 11q13 immediately downstream of the ALDH7 isoform gene. It spans about 6kb and contains seven exons ranging in size from 51 to 186bp. Three CCAAT box sequence motifs are present upstream of the transcription start. Sp1 and NRF1 binding site motifs are present in the first intron. Expression of the gene is ubiquitous but predominant in heart and skeletal muscle. Immunodetection of the TYKY subunit in placental mitochondria after two-dimensional gel electrophoresis revealed that the mature protein has a molecular mass of 22kDa and a pI in the range of 4.9-5.0.

Identification of five Rhodobacter capsulatus genes encoding the equivalent of ND subunits of the mitochondrial NADH-ubiquinone oxidoreductase.

We previously reported the sequencing of two genes (ndhA and ndhI) encoding two of the subunits of the type-I NADH-ubiquinone oxidoreductase from Rhodobacter capsulatus (Rc). The present paper deals with the cloning and characterization of a chromosomal fragment clustering five new Rc genes which encode subunits of this enzyme. This gene cluster is located immediately downstream from ndhA and ndhI, and also contains two unidentified open reading frames (urf2, urf3). The five genes, nuoJ, nuoK, nuoL, nuoM and nuoN, encode proteins related, respectively, to mitochondrial (mt) subunits ND6, ND4L, ND5, ND4 and ND2. The overall organization of the nuo genes identified in Rc shows similarity to that of the Paracoccus denitrificans (Pd) nqo gene cluster.

The 49-kDa subunit of NADH-ubiquinone oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone-related inhibitors.

Piericidin is a potent inhibitor of the mitochondrial and bacterial type I NADH-ubiquinone oxidoreductases (Complex I) and is considered to bind at or close to the ubiquinone binding site(s) of the enzyme. Piericidin-resistant mutants of the bacterium Rhodobacter capsulatus have been isolated and the present work demonstrates that a single missense mutation at the level of the gene encoding the peripheral 49-kDa/NUOD subunit of Complex I is definitely associated with this resistance. Based on this original observation, we propose a model locating the binding site for piericidin (and quinone) at the interface between the hydrophilic and hydrophobic domains of Complex I.