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C Rodriguez-Lafrasse

Publications and source records attributed to C Rodriguez-Lafrasse.

At least 19 recordsLinked to original sources
PubMed2006

Spectrum of pathogenic mutations and associated polymorphisms in a cohort of 44 unrelated patients with long QT syndrome.

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.

Adolescent↗
PubMed2006

[Long QT syndrome in children: analysis of the Lyon series].

Congenital long QT syndrome is a rare and serious disorder in children. In addition to the clinical and electrocardiographical diagnostic criteria, molecular biochemistry has identified six genes which are implicated in this pathology. Our study involved a retrospective analysis of 23 patients aged less than 21 with congenital long QT syndrome, followed up for an average of two years. Genotypes were obtained for all of the patients. There were unfortunately two deaths, one of which had a mutation in the SCN5A gene. The other patient had a double mutation of the SCN5A and KCNE2 genes. Symptomatic patients had QT and QTc intervals noticeably longer than the asymptomatic patients, although this difference was not shown to be significant. LQT3 patients as well as those with a double mutation were affected more severely because two of the three LQT3 patients and one of the two patients with a double mutation suffered a cardiac arrest. Three patients in our study showed no mutation. Nevertheless, two of them suffered a severe cardiac event. This confirms the limits of genetic diagnosis, which could be envisaged in all cases. All of the clinical and ECG data should be combined with the genetic analysis in order to confirm the diagnosis.

Adolescent↗
PubMed2002

Ceramide induces activation of the mitochondrial/caspases pathway in Jurkat and SCC61 cells sensitive to gamma-radiation but activation of this sequence is defective in radioresistant SQ20B cells.

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.

Apoptosis↗
PubMed2001

Temporal relationships between ceramide production, caspase activation and mitochondrial dysfunction in cell lines with varying sensitivity to anti-Fas-induced apoptosis.

To clarify the chronology of events leading to anti-Fas-induced apoptosis, and the mechanisms of resistance to this death effector, we compared the response kinetics of three tumour cell lines that display varying sensitivity to anti-Fas (based on levels of apoptosis), in terms of ceramide release, mitochondrial function and the caspase-activation pathway. In the highly sensitive Jurkat cell line, early caspase-8 activation, observed from 2 h after treatment, was chronologically associated with an acute depletion of glutathione and the cleavage of caspase-3 and poly-ADP ribosyl polymerase (PARP), followed by a progressive fall in the mitochondrial transmembrane potential (Delta(psi)m), between 4 and 48 h after treatment. Ceramide levels began to increase 2 h after the addition of anti-Fas (with no increase during the first hour), and increased continuously to 640% of control cells at 48 h. In the moderately sensitive SCC61 adherent cells, comparable results were observed, though with lower levels of ceramide and a delay in the response kinetics, with apoptotic cells becoming flotant. Finally, despite early cleavage of caspase-8 at 2 h, and a sustained level of activation until 48 h, no apoptotic response was observed in anti-Fas-resistant SQ20B cells. This was confirmed by a lack of ceramide generation and mitochondrial changes, and by the absence of any detectable cleavage of caspase-3 or PARP. Inhibition of caspase processing, and amplification of endogenous ceramide signalling by pharmacological agents, allowed us to establish the order of cellular events, locating ceramide release after caspase-8 activation and before caspase-3 activation, and demonstrating a direct involvement for ceramide release in mitochondrial dysfunction. Furthermore, these experiments provide strong arguments for the role of endogenous ceramide as a key executor of apoptosis, rather than as a consequence of membrane alterations.

Antibodies, Monoclonal↗
PubMed2001

Comparison of methods used to study cell death in an adherent tumoral cell line with moderate clonogenic radiosensitivity.

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.

Annexin A5↗
PubMed1999

Sphingosylphosphorylcholine in Niemann-Pick disease brain: accumulation in type A but not in type B.

A study of brain lipids in patients with the sphingomyelinase-deficient types of Niemann-Pick disease demonstrated that abnormal accumulation of sphingomyelin occurs only in the brain of neuronopathic type A patients but not in the non-neuronopathic type B. Additional lipid abnormalities were present in the type A brain. In contrast, the brain lipid profile was normal in type B patients. Since lysosphingolipids have been implicated in the biochemical pathogenesis of other genetic lysosomal sphingolipidoses, the occurrence of sphingosylphosphorylcholine (lysosphingomyelin) was specifically investigated in brain and extraneural tissues, using an HPLC method with fluorescent detection of orthophtalaldehyde derivatives. Levels close to or below the limit of detection (10 pmol/mg tissue protein) were observed in normal and pathological controls. A striking accumulation was observed in brain of two Niemann-Pick type A patients (830 and 430 pmol/mg protein in 27-and 16-month-old children with severe and milder neurological course, respectively), which was not present at the fetal stage of the disease. No significant increase was found in brain tissue from a 3.5 year-old type B patient. In liver and spleen, abnormally high sphingosylphosphorylcholine levels were observed in both types of the disease, with indication of a progressive increase during development. This study establishes the integrity of brain tissue in Niemann-Pick disease type B and suggests that the lysocompound sphingosylphosphorylcholine could play a role in the pathophysiology of brain dysfunction in the neuronopathic type A.

Brain↗
PubMed1997

Modulation of protein kinase C by endogenous sphingosine: inhibition of phorbol dibutyrate binding in Niemann-Pick C fibroblasts.

The abnormal and variable increase in levels of free sphingoid bases recently described in fibroblasts from Niemann-Pick C patients allowed us to investigate the modulation of protein kinase C in vivo by endogenous sphingosine. The specific binding of [20-3H]phorbol 12, 13-dibutyrate to the regulatory domain of membrane-bound protein kinase C was significantly decreased in fibroblasts from patients compared with controls. A pronounced difference between the two groups (P<0.0001) was demonstrated in low-density lipoprotein-supplemented medium, i.e. under conditions known to disclose abnormal mobilization of unesterified cholesterol in Niemann-Pick C fibroblasts. Furthermore the degree of impairment of [3H]phorbol 12,13-dibutyrate binding was highly correlated (r=0.95) with the sphingosine levels measured in fibroblasts from those patients. Scatchard analysis of the binding data indicated that Niemann-Pick C and control fibroblasts contained almost the same number of binding sites per cell. A 8-34-fold increase in Kd was measured in Niemann-Pick C fibroblasts with at least a 5-fold increase in sphingosine levels. Removal, by cell fractionation, of membrane-bound protein kinase C from the bulk of sphingosine induced a normalization of Kd values. The overall results suggest that protein kinase C inhibition is directly related to sphingosine accumulation.

Fibroblasts↗
PubMed1996

Genetic heterogeneity in Niemann-Pick C disease: a study using somatic cell hybridization and linkage analysis.

The primary molecular defect underlying Niemann-Pick C disease (NPC) is still unknown. A wide spectrum of clinical and biochemical phenotypes has previously been documented. Indication of genetic heterogeneity has recently been provided for one patient. In the present study, somatic cell hybridization experiments were carried out on skin fibroblast cultures from 32 unrelated NPC patients covering the range of known clinical and biochemical phenotypes. The criterion for complementation was the restoration of a normal intracellular fluorescent pattern in polykaryons stained with filipin to document cholesterol distribution. Crosses between the various cell lines revealed a major complementation group comprising 27 unrelated patients and a second minor group comprising 5 patients. Linkage analysis in one multiplex family belonging to the minor complementation group showed that the mutated gene does not map to the 18q11-12 region assigned to the major gene. Patients in the first group spanned the whole spectrum of clinical and cellular phenotypes. No consistent clinical or biochemical phenotypes was associated with the second complementation group. Three of the five group 2 patients, however, presented with a new rare phenotype associated with severe pulmonary involvement leading to death within the first year of life. No biochemical abnormality specific of either group could be demonstrated with regard to tissue lipid storage pattern, intralysosomal cholesterol storage, and regulation of cholesterol homeostasis. Mutations affecting at least two different genes have thus been shown to underlie NPC. The two gene products may function together or sequentially in a common metabolic pathway affecting intracellular cholesterol transport.

Adolescent↗
PubMed1994

Free sphingoid bases in tissues from patients with type C Niemann-Pick disease and other lysosomal storage disorders.

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.

Brain Chemistry↗
PubMed1993

Preparation of an anti-acid sphingomyelinase monoclonal antibody for the quantitative determination and polypeptide analysis of lysosomal sphingomyelinase in fibroblasts from normal and Niemann-Pick type A patients.

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.

Antibodies, Monoclonal↗
PubMed1992

Type C Niemann-Pick disease: a murine model of the lysosomal cholesterol lipidosis accumulates sphingosine and sphinganine in liver.

We have determined the levels of free sphingoid bases in livers of normal and cholesterol lipidotic Niemann-Pick type C mice. Hepatic sphingosine and sphinganine levels in affected mice (593 pmol/mg protein) were elevated more than 20-fold when compared to levels in age-matched normal mice (26 pmol/mg protein). Upon fractionation of mutant liver homogenates by differential centrifugation, most of the sphingoid bases sedimented with beta-hexosaminidase in the 9000 x g pellet. Co-sedimentation of sphingoid bases with a lighter beta-hexosaminidase peak in Percoll gradients suggests that these bases accumulate in lipid laden lysosomes. A cytosolic sphinganine kinase is the first enzyme in the degradative pathway of sphingoid base metabolism. Activity of this enzyme was partially deficient in crude mutant liver cytosolic extracts due to the presence of an inhibitory substance. Following molecular sieving of mutant cytosolic extracts on Sepharose 4B, sphinganine kinase, with normal levels of activity, was resolved from a complex higher-molecular-weight inhibitor fraction. The Km values for either sphinganine or ATP-Mg substrates with partially purified sphinganine kinase from normal and mutant mouse liver extracts, were similar. These findings indicate that accumulation of free sphingoid bases is not due to a direct inherent deficiency in the catalytic activity of sphinganine kinase. The possible cause and effect relationship between the accumulation of these endogenous hydrophobic amines and the lesion in intracellular cholesterol trafficking in Niemann-Pick type C disease is discussed.

Animals↗
PubMed1992

Prenatal diagnosis of Niemann-Pick type C disease: current strategy from an experience of 37 pregnancies at risk.

Thirty-seven pregnancies at risk for Niemann-Pick type C disease were monitored by study of cultured amniotic fluid cells (8 cases) or chorionic villus cells (29 cases) in 23 couples over the period 1984-91. An early protocol combined determination of sphingomyelinase activity with electron microscopy. The current strategy, based on the demonstration of specific abnormalities in intracellular processing of exogenous cholesterol, combines the study of the early phase (first 6 h) of LDL-induced cholesteryl ester formation and the histochemical evaluation (filipin staining after 24 h of LDL uptake) of the LDL-induced accumulation of unesterified cholesterol. Thirteen fetuses were predicted to be affected. Confirmation of the diagnosis was made by study of cholesterol processing in fetal skin fibroblast cultures and/or by demonstration of a characteristic lipid storage in fetal liver, already present at 14 w gestation. Definition of the biochemical phenotype (classical, variant, or intermediate) of the index case, with regard to cholesterol-processing abnormalities, is an absolute prerequisite to adequate genetic counseling in a given family. Prenatal diagnosis has now proved a safe procedure in the predominant (approximately 85%) group of families with the classical phenotype.

Cells, Cultured↗
PubMed1991

Type C Niemann-Pick disease: spectrum of phenotypic variation in disruption of intracellular LDL-derived cholesterol processing.

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.

Adolescent↗
PubMed1991

Niemann-Pick disease type C: an update.

The concept of Niemann-Pick disease type C as a secondary sphingomyelin storage disorder (in contrast to the sphingomyelinase-deficient types A and B) has become more and more prevalent, in view of the complex lipid storage pattern and variable sphingomyelinase activities. Although the primary lesion is still unknown, studies conducted over the past six years have led to a breakthrough by showing that this disorder is characterized by unique abnormalities of intracellular translocation of exogenous cholesterol. In cultured fibroblasts of patients, this block leads to a delayed induction of the homeostatic responses to exogenous cholesterol, in particular cholesteryl ester formation, and to the accumulation of unesterified cholesterol in a vesicular, essentially lysosomal, compartment. The transport of endogenous cholesterol is apparently unaffected. The spectrum of phenotypic heterogeneity in relation to abnormal LDL-processing has been defined in a large patient population. Clinical presentation of the disease is also reviewed and biochemical correlations are discussed. This discovery has had immediate medical applications, by providing the first strategy for reliable prenatal diagnosis of the disorder and easy diagnosis of patients. To date, the exact implication of the cholesterol transport defect in the pathogenesis of Niemann-Pick type C is not known; recent observations have opened up new possible approaches for the understanding of this lesion. Although final classification of Niemann-Pick disease type C must await elucidation of the primary defect(s), present knowledge already establishes that the disease is a nosological entity distinct from Niemann-Pick disease type A and B, and suggests that it might be the model for a new molecular concept of neurolipidosis--and even of inherited metabolic disease.

Animals↗
PubMed1991

Type C Niemann-Pick disease: biochemical aspects and phenotypic heterogeneity.

Within Niemann-Pick diseases, type C has now been demonstrated to be a nosological entity totally distinct from types A and B, and is best characterized at present by unique abnormalities of intracellular translocation of exogenous cholesterol, which are briefly reviewed. Although the primary defect is still unknown in type C Niemann-Pick disease, this discovery has had immediate medical applications, by providing the first strategy for reliable prenatal detection of the disorder and easy diagnosis of patients. From our personal experience of 134 cases, diagnosis is best reached by the combined demonstration of a deficient induction of esterification and of an intravesicular cholesterol storage by cytochemistry after filipin staining. The prevalence of the various clinical forms observed is given, together with a brief report of 6 adult-onset cases. The spectrum of phenotypic heterogeneity in relation to abnormal LDL processing has been defined, resulting in the delineation of three biochemical groups, classical (86%), variant (7%) and intermediate (7%). Correlations between clinical and biochemical phenotypes have been studied. To get further insight into genetic heterogeneity, complementation studies were performed. Preliminary results have yet given no evidence of several complementation groups within type C Niemann-Pick disease. The recognition of the three biochemical phenotypes is however critical for diagnosis and genetic counselling.

Adolescent↗
PubMed1990

Abnormal cholesterol metabolism in imipramine-treated fibroblast cultures. Similarities with Niemann-Pick type C disease.

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.

Acetates↗
PubMedDate not supplied

[HDL-cholesterol: role of its dosage in the assessment of cardiovascular risk].

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.

Anticholesteremic Agents↗