RESUMO
Human placental sphingomyelinase was highly purified through an original six-step scheme in order to raise a specific rabbit anti-sphingomyelinase antibody. Pure enzyme preparations showed specific activities ranging between 100 and 150 mumol/h per mg protein and gave two constant silver-stained bands (Mr 70,000 and 57,000) on acrylamide after electrophoresis under denaturing conditions. These two bands were the sole areas detected by the described antibody on Western blots from normal placental preparations at various stages of purification. A similar procedure was applied to the separate study of placental sphingomyelinase from two prenatally diagnosed foetuses with confirmed Niemann-Pick disease type A. During purification, the mutant enzyme could be followed owing to its minute but measurable level of catalytic activity, and behaved normally at the various chromatographic steps. In the purified preparations, specific activities of 0.18 and 0.49 mumol/h per mg protein, respectively, were reached. No alteration of the Km value (19 mumol/l) was observed, while the Vmax was 0.5-1% of normal. With immunostaining of Western blots obtained as above, results similar to those described for normal tissue were found, leading to the conclusion that immunoreactive sphingomyelinase is present in Niemann-Pick disease type A.
Assuntos
Doenças de Niemann-Pick/enzimologia , Diester Fosfórico Hidrolases/isolamento & purificação , Placenta/enzimologia , Esfingomielina Fosfodiesterase/isolamento & purificação , Animais , Eletroforese em Gel de Poliacrilamida , Feminino , Humanos , Imunoquímica , Gravidez , Coelhos , gama-GlobulinasRESUMO
The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.
Assuntos
Metabolismo dos Carboidratos , Glicosídeo Hidrolases/isolamento & purificação , Sítios de Ligação , Fibroblastos/enzimologia , Galactosidases/deficiência , Galactosidases/isolamento & purificação , Gangliosidoses/enzimologia , Doença de Gaucher/enzimologia , Glucosidases/isolamento & purificação , Humanos , Fígado/enzimologia , Baço/enzimologia , Especificidade por SubstratoRESUMO
Addition of low-density lipoprotein (LDL) to cholesterol-deprived human skin fibroblast cultures treated by imipramine at a 20 microM concentration induced a significant intracellular accumulation of unesterified cholesterol. Intracytoplasmic inclusions were already visible by histochemical filipin staining after 2 h of LDL uptake and were progressively mobilized towards the perinuclear region within 24 h. At this concentration of the drug, the rate of proteolytic 125I-LDL hydrolysis was similar in treated and untreated cells. Treated cells maintained in lipoprotein-deficient medium showed no abnormality, indicating the exogenous origin of the accumulated sterol. Further, the drug induced a drastic dose-dependent impairment of LDL-stimulated cholesterol esterification, not related to an inhibition of acyl CoA:cholesterol acyltransferase, and a significant delay in down-regulation of de novo cholesterol synthesis. However, imipramine did not affect 25-hydroxycholesterol-mediated regulation of the two latter processes. These results resemble those observed in Niemann-Pick type C disease and suggest an impaired mobilization of LDL-derived cholesterol in imipramine-treated cells.
Assuntos
LDL-Colesterol/metabolismo , Fibroblastos/metabolismo , Imipramina/farmacologia , Doenças de Niemann-Pick/metabolismo , Acetatos/metabolismo , Células Cultivadas , Ésteres do Colesterol/biossíntese , Regulação para Baixo , Fibroblastos/efeitos dos fármacos , Fibroblastos/ultraestrutura , Humanos , Hidroxicolesteróis/metabolismo , Cinética , Doenças de Niemann-Pick/induzido quimicamente , Doenças de Niemann-Pick/patologiaRESUMO
The 20-fold increase of free sphingoid bases found in liver from a murine model of Niemann-Pick type C (NPC) combined to the NPC-like phenotype induced by addition of sphinganine to normal fibroblast cultures prompted us to investigate the potential involvement of these compounds in the human disease. The contents of sphingosine and sphinganine were measured in liver, spleen, brain and skin fibroblast cultures by a sensitive HPLC method. In liver and spleen from NPC patients, a 6- to 24-fold elevation of sphingosine and sphinganine already prominent at the fetal stage of the disease was observed, while no clear increase could be evidenced in brain tissue. A significant increase, not modulated by the intralysosomal content of free cholesterol, also occurred in skin fibroblast cultures. To investigate the specificity of these findings, other lysosomal storage disorders were studied. A striking accumulation was found in liver and spleen (24- to 36-fold) from patients with Niemann-Pick disease type A and B (sphingomyelinase-deficient forms), and in cerebral cortex of type A Niemann-Pick disease. A significant storage also occurred in Sandhoff disease, while several other sphingolipidoses showed a moderate elevation. In all cases but Sandhoff disease brain, the sphingosine/sphinganine ratio remained unchanged, suggesting that the accumulated free sphingoid bases derived from sphingolipid catabolism. Formation of complexes between sphingosine and the lipid material accumulated in lysosomes might be a general mechanism in lysosomal lipidoses. In NPC, however, an increase of free sphingoid bases disproportionate to the degree of lysosomal storage and a specific involvement of cultured fibroblasts suggested a more complex or combined mechanism.
Assuntos
Doenças por Armazenamento dos Lisossomos/metabolismo , Doenças de Niemann-Pick/metabolismo , Esfingosina/análogos & derivados , Química Encefálica , Feto/química , Humanos , Fígado/química , Pele/química , Esfingosina/análise , Baço/químicaRESUMO
To investigate biochemical heterogeneity within Niemann-Pick type C disease (NPC), the two most characteristic abnormalities, namely (1) kinetics of LDL-stimulated cholesteryl ester formation and (2) intravesicular accumulation of LDL-derived unesterified cholesterol, evaluated by histochemical filipin staining, were studied in cultured skin fibroblasts from a population of 125 NPC patients. Profound alterations (esterification rates less than 10% of normal, very numerous and intensely fluorescent cholesterol-filipin granules) were demonstrated in 86% of the cases, depicting the 'classical' NPC phenotype. The remaining cell lines showed a graded less severe impairment and more transient delay in the induction of LDL-mediated cholesteryl esterification, along with an attenuated accumulation of unesterified cholesterol. In particular, cells from a small group (7%) of patients, which have been individualized as representative of a 'variant' phenotype, showed only slight alterations of esterification, restricted to the early phase of LDL uptake and undistinguishable from those in heterozygotes. In these cells, an abnormal cytochemical distribution of LDL-derived cholesterol, although moderate, was still evident provided rigorous experimental conditions were followed. A third, less clearly individualized group (7%), differing from the classical phenotype mostly by higher rates of cholesteryl ester formation, has been designated as an 'intermediary' phenotype to reflect a more difficult diagnosis of such patients. These findings have an important bearing with regard to diagnosis and genetic counselling, although the significance of such a phenotypic variation in terms of genetic heterogeneity has still to be demonstrated. A given biochemical phenotype was however a constant observation within a family (14 pairs of siblings tested so far). The unique feature of LDL-cholesterol processing alterations in NPC has been further established from comparative studies in Wolman disease and I-cell disease, showing normal or different intracellular distribution of unesterified LDL-derived cholesterol in the latter disorders. Correlation between biochemical and clinical NPC phenotypes was only partial, but a correlation between the severity of alterations in cholesterol processing and sphingomyelin catabolism could be established.
Assuntos
LDL-Colesterol/metabolismo , Doenças de Niemann-Pick/metabolismo , Adolescente , Adulto , Células Cultivadas , Criança , Pré-Escolar , Ésteres do Colesterol/metabolismo , Esterificação , Histocitoquímica , Homeostase , Humanos , Cinética , Doenças de Niemann-Pick/genética , Fenótipo , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Many assays 1(st), 2(nd) even 3(rd) generation are at present available to determine the concentration of cardiac troponin I and T. With the redefinition of upper reference value in the acute coronary syndromes, the aim of this study was to evaluate the clinical and analytical performance of 2 troponins assays: Troponin Ic 2(nd) generation (AccuTnI) on Access 2 of Beckman Coulter and Troponin Tc 3(rd)generation (Troponin T STAT) on Elecsys 2010 of Roche Diagnostics. The analytical performance observed with these 2 assays are accurate (analytical and functional sensitivity, repetability and reproductibility). Comparing each method with Dade Behring assay (Flex Troponine-I Cardiaque, TROP) on Dimension RxL, the correlation observed with AccuTnI kit on Access 2 can be put into the equation: AccuTnI = 1.08 (TnIc TROP) - 0.34, r = 0.99. On the contrary, it's more difficult to compare cTnI and cTnT. The study of decisonnal values indicated by Beckman Coulter for cTnI (0.04 microg/L at the 99 degrees percentil, 0.06 microg/L for a CV < or =10%) show a better specificity (76%) and predictive positive value (89%) with a sensitivity at 100% at 0.1 microg/L, fixed and used in the laboratory for its better agreement between sensibility / specificity and its imprecision below 10 %. For the cTnT values published by Roche Diagnostics (0.01 microg/L), at the 99 degrees percentil and 0.03 microg/L for a CV < or = 10%, the specificity is lower, so the decisionnal value 0.1 microg/L seems to be more suitable. During this study, few false positive and negative cTnT values have been observed, in patients with complex pathologies; this eventuality must be taken in consideration if clinical findings are not in good accordance with laboratory results.
Assuntos
Angina Instável/sangue , Angina Instável/diagnóstico , Infarto do Miocárdio/sangue , Infarto do Miocárdio/diagnóstico , Troponina C/sangue , Troponina I/sangue , Diagnóstico Diferencial , Eletrocardiografia , Cardiopatias/sangue , Cardiopatias/diagnóstico , Humanos , Técnicas Imunoenzimáticas , Sensibilidade e Especificidade , SíndromeRESUMO
BACKGROUND: Although well-defined clinically and electrocardiographically, Acquired Long QT Syndrome (LQTS) remains elusive from a pathophysiologic point of view. An increasingly accepted hypothesis is that it represents an attenuated form of Congenital Long QT Syndrome. To test this hypothesis further, we investigated patients with Acquired LQTS, using various investigations that are known to give information in patients with Congenital LQTS. METHODS: All the investigations were performed in patients with a history of Acquired Long QT Syndrome, defined by marked transient QT lengthening (QT>600 ms) and/or torsades de pointes. Measurement of the QT interval dispersion, the interlead difference for the QT interval on a 12-lead ECG, was performed in 18 patients and compared with 18 controls, matched for age and sex. To assess sympathetic myocardial innervation, I-123 Meta-iodobenzylguanidine (I-123-MIBG) scintigraphy was performed in 12 patients, together with Thallium scintigraphy, to rule out abnormal myocardial perfusion. Time-frequency analysis of a high-resolution ECG using a wavelet technique, was made for nine patients and compared with 38 healthy controls. Finally, genetic studies were performed prospectively in 16 consecutive patients, to look for HERG, KCNE1, KCNE2 and KCNQ1 mutations. The functional profile of a mutated HERG protein was performed using the patch-clamp technique. RESULTS: Compared with the control group, a significant increase in QT dispersion was observed in the patients with a history of Acquired LQTS (55+/-15 vs. 33+/-9 ms, P<0.001). In another group of patients with Acquired LQTS, 123 I-MIBG tomoscintigraphy demonstrated a decrease in the sympathetic myocardial innervation. Time--frequency analysis using wavelet transform, demonstrated an abnormal frequency content within the QRS complexes, in the patients with Acquired LQTS, similar to that found in Congenital LQTS patients. Molecular screening in 16 consecutive patients, identified one patient with a missense mutation on HERG, one of the LQTS genes. Expression of the mutated HERG protein led to altered K(+) channel function. CONCLUSION: Our results suggest that Acquired and Congenital Long QT Syndromes have some common features. They allow the mechanism of the clinical heterogeneity, found in both syndromes, to be understood. Further multi-facet approaches are needed to decipher the complex interplay between the main determinants of these arrhythmogenic diseases.
Assuntos
Proteínas de Transporte de Cátions , Proteínas de Ligação a DNA , Síndrome do QT Longo/fisiopatologia , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Transativadores , Idoso , Canal de Potássio ERG1 , Eletrocardiografia , Canais de Potássio Éter-A-Go-Go , Feminino , Coração/inervação , Humanos , Síndrome do QT Longo/induzido quimicamente , Síndrome do QT Longo/congênito , Síndrome do QT Longo/genética , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Canais de Potássio/genética , Estudos Prospectivos , Sistema Nervoso Simpático/fisiopatologia , Tomografia Computadorizada de Emissão , Regulador Transcricional ERGRESUMO
To establish the profile of the multiple forms of placental lysosomal sphingomyelinase, we have developed in a preliminary step, a new sequence of purification. This protocol has a good yield such that the final preparation is representative of the whole enzyme. Using chromatofocusing, purified sphingomyelinase is resolved into 4 major forms whose pI's are 7.2, 6.5, 5.9 and 5.2. Chromatofocusing is preferable to isoelectric focusing because it is more rapid, more reproducible and above all it provides a constant profile of activity which is not influenced by experimental conditions, in particular, detergent concentration.
Assuntos
Diester Fosfórico Hidrolases/isolamento & purificação , Placenta/enzimologia , Esfingomielina Fosfodiesterase/isolamento & purificação , Cromatografia/métodos , Feminino , Humanos , Focalização Isoelétrica , GravidezRESUMO
An anti-acid sphingomyelinase monoclonal antibody has been prepared using an in vitro booster technique. The antigen, acid sphingomyelinase, was purified from human placentas by sequential chromatographic steps in the presence of the non-ionic detergent Nonidet P40. This monoclonal antibody (MAB 236) precipitates specifically the enzyme activity by immunoadsorption techniques and presents the same specificity to normal and mutated sphingomyelinase in Niemann-Pick type A patients. MAB 236 is the first antibody able to precipitate the protein in the presence of detergent thereby permitting the quantitative determination of normal and mutated sphingomyelinase in tissue and cell extracts. Polypeptide analysis and quantitative determination experiments using this monoclonal antibody showed no difference between patients and normal controls.
Assuntos
Anticorpos Monoclonais , Doenças de Niemann-Pick/diagnóstico , Esfingomielina Fosfodiesterase/imunologia , Humanos , Lisossomos/enzimologia , Peso Molecular , Placenta/enzimologia , Esfingomielina Fosfodiesterase/químicaRESUMO
Sphingomyelinase activity of cultivated skin fibroblast extracts from normal individuals was resolved by chromatofocusing in the pH range 8-5 into three major components with pI's of 7.3, 6.3 and 5.9, respectively. Chromatofocusing proved a more efficient and reproducible separation technique than preparative flat-bed isoelectric focusing and it gave a constant profile even when detergent concentration varied. In skin fibroblasts from five patients with Niemann-Pick disease type C, a varying degree of reduction in the proportion of the 7.3 peak was observed. In a patient with clinical features of Niemann-Pick disease type C, the finding of such a profile would thus be a good argument for the diagnosis, but it is not pathognomonic as we found similar changes in two cases with GM1-gangliosidosis, while some cases of Niemann-Pick disease type C have borderline normal profiles. These results challenge the concept of a specific sphingomyelinase isoenzyme deficiency as the basic defect in Niemann-Pick disease type C.
Assuntos
Fibroblastos/enzimologia , Gangliosidoses/enzimologia , Focalização Isoelétrica , Doenças de Niemann-Pick/enzimologia , Diester Fosfórico Hidrolases/isolamento & purificação , Esfingomielina Fosfodiesterase/isolamento & purificação , Gangliosídeo G(M1) , Humanos , Concentração de Íons de Hidrogênio , Ponto Isoelétrico , Pele/enzimologiaRESUMO
Glycoconjugates are directly involved in major skeletal muscle functions. As little is known about glycosylation processes in muscle, we investigated glycoconjugate synthesis in subcellular fractions from human skeletal muscle tissue. Mitochondria and microsomal membranes were prepared from muscle biopsies by thorough mechanical disruption and differential centrifugations. This procedure resulted in the isolation of intact mitochondria (1 mg protein/g muscle) and of a microsomal fraction (1.5 mg protein/g muscle). Glycosyltransferases were studied in both subcellular fractions using either dolichylmonophosphate as a polyprenic acceptor or chemically modified fetuin as a glycoprotein substrate. Our results provide evidence for high rates of glycosylation in muscle. The highest activities were obtained with GDP-mannose: dilichylmonophosphate mannosyltransferase, a key enzyme in glycosylation process (220 pmol/mg per h in mitochondria and 1,550 pmol/mg per h in microsomal membranes). Substantial individual variations were observed for dolichol pathway glycosyltransferases but low individual variations were found for glycosyltransferases involved in maturation of glycoproteins. The role which glycosylation defects may play in muscle dysfunction has yet to be defined.
Assuntos
Glucose/metabolismo , Microssomos/metabolismo , Mitocôndrias Musculares/metabolismo , Músculos/metabolismo , Adolescente , Adulto , Dolicóis/metabolismo , Feminino , Galactose/metabolismo , Glucosiltransferases/metabolismo , Humanos , Masculino , Membranas/metabolismo , Microssomos/enzimologia , Pessoa de Meia-Idade , Mitocôndrias Musculares/enzimologia , Proteínas Musculares/metabolismo , Músculos/enzimologia , Músculos/ultraestrutura , Doenças Musculares/metabolismo , alfa-Fetoproteínas/metabolismoRESUMO
PURPOSE: To clarify the molecular mechanisms leading to radiation-induced apoptosis or resistance, the kinetics (1-48 h) and sequence of events triggered in response to 10 Gy irradiation were investigated in three cell lines displaying a gradient of sensitivity to 7-rays. MATERIALS AND METHODS: Ceramide levels were measured by high performance liquid chromatography (HPLC). Mitochondrial function was evaluated in terms of transmembrane potential (delta(psi)m), reactive oxygen species (ROS) and glutathione levels analysed by flow cytometry or HPLC. Caspase activation was assessed by immunoblotting, and apoptosis by flow cytometry. RESULTS: In Jurkat radiosensitive cells and SCC61 adherent cells with intermediate radiosensitivity, the degree of delayed ceramide release was directly related to their propensity to undergo apoptosis. Transduction of the death signal was mediated by a drop in delta(psi)m and glutathione levels, ROS accumulation and activation of effector caspases. Experiments conducted with caspase inhibitors, bongkrekic acid, or DL-PDMP indicated that ceramide triggers mitochondrial collapse, followed by the activation of caspases-9, -8 and -3, and poly(ADP-ribose)polymerase cleavage. In SQ20B radioresistant cells, gamma-radiation did not induce ceramide generation or subsequent activation of the mitochondrial/caspase apoptotic pathway. CONCLUSIONS: Ceramide appears to be a determining factor in the commitment phase of radiation-induced apoptosis. When ceramide is not generated, the whole pathway is ineffective and resistance to apoptosis may result.
Assuntos
Caspases/metabolismo , Ceramidas/metabolismo , Mitocôndrias/metabolismo , Mitocôndrias/efeitos da radiação , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Ácido Bongcréquico/farmacologia , Inibidores de Caspase , Linhagem Celular , Ativação Enzimática/efeitos da radiação , Raios gama , Glutationa/metabolismo , Humanos , Células Jurkat , Cinética , Potenciais da Membrana/efeitos da radiação , Mitocôndrias/efeitos dos fármacos , Morfolinas/farmacologia , Tolerância a Radiação , Espécies Reativas de Oxigênio/metabolismoRESUMO
Our objective was to compare different methods for studying programmed cell death in adherent H460 non-small lung cancer cells of moderate clonogenic radiosensitivity. The major effect of gamma-radiation was found to be the release of cells from the substratum. The different methods gave complementary and unexpected information: a) with the TUNEL method, a few non-apoptotic cells were found in the culture medium; b) with the flow cytometry after propidium iodide labeling, some hypodiploid cells which remained attached to the substratum were apoptotic, as demonstrated by the effect of a caspase inhibitor; c) with the annexin V labeling, the detached cells were demonstrated either necrotic or very late apoptotic; d) the mitochondria transmembrane potential (deltapsim), measurements demonstrated that the mitochondria were implicated in cell death induced by gamma-radiation. These data illustrate the need to use several complementary methods in the study of apoptosis in adherent cells exposed to gamma-radiation.
Assuntos
Raios gama , Anexina A5/farmacologia , Adesão Celular , Ciclo Celular/efeitos da radiação , Morte Celular , Diploide , Relação Dose-Resposta à Radiação , Inibidores Enzimáticos/farmacologia , Citometria de Fluxo , Imuno-Histoquímica , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana , Mitocôndrias/metabolismo , Fosfatidilserinas/metabolismo , Propídio/farmacologia , Células Tumorais CultivadasRESUMO
In an increasing number of biochemical variants of the sphingolipidoses, demonstration of the enzyme defect cannot be achieved by the (briefly summarized) conventional techniques. Alternative methods allowing diagnosis of such patients - in particular metabolic studies in living cell cultures - are discussed. The current knowledge and practical impact of molecular biology in the sphingolipidoses field is shortly reviewed. The authors finally discuss the recent findings highlighting in Niemann-Pick disease type C metabolic abnormalities in the cellular processing of cholesterol and their consequences on diagnosis and pathogenesis of this disorder.
Assuntos
Esfingolipidoses/diagnóstico , Células Cultivadas , Humanos , Métodos , Doenças de Niemann-Pick/diagnóstico , Doenças de Niemann-Pick/etiologia , Esfingolipidoses/etiologiaRESUMO
The interest of HDL-cholesterol (HDLC) to evaluate a cardiovascular risk has been studied since many years. According to Framingham Heart studies, this factor is inversely correlated to a future ischaemic heart disease. At high level, HDLC is considered as a cardiovascular protecting factor, and is known since few years as "good cholesterol". In the year 2000, the ANAES (Agence nationale de l'accréditation et évaluation en santé) has redefined the role of HDLC in the exploration of dyslipidaemia. In the case of a cardiovascular-risk history, HDLC, with total cholesterol, triglycerides, and LDL-cholesterol (by Friedewald method) will be analyzed. Usually, HDLC is not very accessible to conventional treatments. So, according to ANAES, the treatment of dyslipidaemia will be based on LDL-cholesterol levels only. Nevertheless, HDLC is a major lipid factor to evaluate a cardiovascular risk. The object of this review is, on one hand, to situate HDLC in the evaluation of cardiovascular risk, by showing its key role in lipid metabolism, and, on the other hand, to report the main direct assays of this parameter.
Assuntos
Doenças Cardiovasculares/sangue , Doenças Cardiovasculares/epidemiologia , HDL-Colesterol/sangue , Anticolesterolemiantes/uso terapêutico , Análise Química do Sangue/métodos , Doenças Cardiovasculares/etiologia , Doenças Cardiovasculares/fisiopatologia , Doenças Cardiovasculares/prevenção & controle , Humanos , Obesidade/complicações , Fatores de RiscoRESUMO
Hypertrophic cardiomyopathy (HCM), a common and clinically heterogeneous disease characterized by unexplained ventricular myocardial hypertrophy and a high risk of sudden cardiac death, is mostly caused by mutations in MYH7 and MYBPC3 genes. As 70% of MYBPC3 mutations introduce a premature termination codon, the purpose of the current study was to report the prevalence of large MYBPC3 rearrangements. A large French cohort of 100 HCM patients, for whom no putatively causative point mutations were identified previously in the most prevalent HCM-causing genes, was investigated using an MLPA methodology. One HCM patient was identified to carry a large MYBPC3 rearrangement (<1%). This patient presents a 3505-bp deletion, which begins in the intron 27 and ends 485 bp after the MYBPC3 stop codon (g.47309385_47312889del). It was originated by recombination of a 296 bp AluSz sequence located in intron 27 and a 300 bp AluSx sequence located immediately downstream of exon 35. This study allowed the characterization of the first large MYBPC3 deletion reported in the literature. However, it appears that MLPA strategy, that moderates the identification of large MYBPC3 rearrangements, might confirm a clinical diagnosis only in a small number of patients (<1%).
Assuntos
Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Deleção de Genes , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-IdadeRESUMO
Primary hypertrophic cardiomyopathy is a relatively frequent disease (1/500) which results from a mutation in a gene encoding a sarcomeric protein. In a series of 184 cases, nearly half (46 %) were secondary to a mutation in one of the 4 following genes : MYBPC3, MYH7, TNNI3, TNNT2. In Fabry disease, an exclusive or nearly exclusive cardiac expression is possible and referred to as "cardiac variant". The hypertrophic cardiomyopathy of Fabry disease is usually unspecific. Two series reported a prevalence of Fabry disease of about 6% among male cases. An Italian series of 34 female cases with hypertrophic cardiomyopathy demonstrated that it was feasible to diagnose Fabry disease in females by screening for specific lesions in myocardial biopsies. We detected a patient who initially presented with a common hypertrophic cardiomyopathy except that his ECG showed depression of ST segment and inversion of T wave in leads D1, VL and in precordial leads. The family history revealed several affected relatives and female carriers. In conclusion, an isolated common hypertrophic cardiomyopathy may be secondary to Fabry disease. Male patients should be screened systemically for enzyme defect except in cases of father-to-son transmission. In females, an affected male relative should be searched for screening or the GLA gene should be sequenced. It is important to think about a putative Fabry disease in cases with hypertrophic cardiomyopathy not associated with any obvious cause.
Assuntos
Miosinas Cardíacas/genética , Cardiomiopatia Hipertrófica/genética , Proteínas de Transporte/genética , Doença de Fabry/genética , Cadeias Pesadas de Miosina/genética , Troponina I/genética , Troponina T/genética , Cardiomiopatia Hipertrófica/patologia , Diagnóstico Diferencial , Doença de Fabry/patologia , Feminino , Marcadores Genéticos/genética , Genótipo , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Linhagem , Fenótipo , Sarcômeros/genéticaRESUMO
Long QT syndrome (LQTS) is a rare and clinically heterogeneous inherited disorder characterized by a long QT interval on the electrocardiogram, increased risk of syncope and sudden death caused by arrhythmias. This syndrome is mostly caused by mutations in genes encoding various cardiac ion channels. The clinical heterogeneity is usually attributed to variable penetrance. One of the reasons for this variability in expression could be the coexistence of common single nucleotide polymorphisms (SNPs) on LQTS-causing genes and/or unknown genes. Some synonymous and nonsynonymous exonic SNPs identified in LQTS-causing genes may have an effect on the cardiac repolarization process and modulate the clinical expression of a latent LQTS pathogenic mutation. We report the molecular pattern of 44 unrelated patients with LQTS using denaturing high-performance liquid chromatography analysis of the KCNQ1, KCNH2, SCN5A, KCNE1 and KCNE2 genes. Forty-five disease-causing mutations (including 24 novel ones) were identified in this cohort. Most of our patients (84%) showed complex molecular pattern with one mutation (and even two for four patients) associated with several SNPs located in several LQTS genes.
Assuntos
Síndrome do QT Longo/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Sódio/genética , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Substituição de Aminoácidos , Criança , Estudos de Coortes , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Mutação , Canal de Sódio Disparado por Voltagem NAV1.5 , Polimorfismo de Nucleotídeo Único , Canais de Potássio de Abertura Dependente da Tensão da Membrana/química , Canais de Sódio/químicaRESUMO
The French experience in the long term follow-up of 105 cases of organic aciduria (45 maple syrup urine disease, 12 isovaleric acidaemia, 19 propionic acidaemia, 24 methylmalonic aciduria and some rare allied disorders) is reported. Main conclusions drawn from this survey are the poor overall prognosis and the slow improvement in the outcome of such disorders over the last 15 years. In MSUD, while early diagnosis and early management remain a basic requirement, intellectual development did not improve as much as expected. In propionic and methylmalonic acidaemia modern treatment does not prevent a fatal outcome in the classical neonatal forms. It should be also emphasized that in the rare cases where a coenzyme deficiency has been demonstrated, vitamin therapy is very often ineffective in vivo.