Molecular characterization of a large MYBPC3 rearrangement in a cohort of 100 unrelated patients with hypertrophic cardiomyopathy.

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%).

[Fabry disease among hypertrophic cardiomyopathy of genetic origin]. / Place de la maladie de Fabry au sein des cardiomyopathies hypertrophiques d'origine génétique.

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.

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.

[Cardiac troponin I and C: analytical comparison and clinical-biological interpretation of three troponins assays]. / Troponines I et T cardiaques: comparaison analytique et étude clinicobiologique de trois trousses de troponine.

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.

[HDL-cholesterol: role of its dosage in the assessment of cardiovascular risk]. / HDL-cholestérol: place de son dosage dans l'évaluation d'un risque cardiovasculaire.

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.

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.

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.

Non-invasive testing of acquired long QT syndrome: evidence for multiple arrhythmogenic substrates.

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.

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.

Usefulness of procalcitonin for diagnosis of infection in cardiac surgical patients.

OBJECTIVE: To determine the value of procalcitonin (PCT) as a marker of postoperative infection after cardiac surgery. DESIGN: A prospective single institution three phase study. SETTING: University cardiac surgical intensive care unit (31 beds). PATIENTS: Phase 1: To determine the normal perioperative kinetics of PCT, 20 consecutive patients undergoing elective cardiac surgery with cardiopulmonary bypass were included. Phase 2: To determine whether PCT may be useful for diagnosis of postoperative infection, 97 consecutive patients with suspected infection were included. Phase 3: To determine the ability of PCT to differentiate patients with septic shock from those with cardiogenic shock, 26 patients with postoperative circulatory failure were compared. MEASUREMENTS AND MAIN RESULTS: Phase 1: Serum samples were drawn for PCT determination after induction of anesthesia (baseline), at the end of surgery, and daily until postoperative day (POD) 8. Baseline serum PCT concentration was 0.17 +/- 0.08 ng/mL (mean +/- SD). Serum PCT increased after cardiac surgery with a peak on POD 1 (1.08 +/- 1.36). Serum PCT returned to normal range on POD 3 and remained stable thereafter. Phase 2: In patients with suspected infection, serum PCT was measured at the same time of C-reactive protein (CRP) and bacteriologic samples. Among the 97 included patients, 54 were infected with pneumonia (n = 17), bacteremia (n = 16), mediastinitis (n = 9), or septic shock (n = 12). In the 43 remaining patients, infection was excluded by microbiological examinations. In noninfected patients, serum PCT concentration was 0.41 +/- 0.36 ng/mL (range, 0.08-1.67 ng/mL). Serum PCT concentration was markedly higher in patients with septic shock (96.98 +/- 119.61 ng/mL). Moderate increase in serum PCT concentration occurred during pneumonia (4.85 +/-3.31 ng/mL) and bacteremia (3.57 +/- 2.98 ng/mL). Serum PCT concentration remained low during mediastinitis (0.80 +/- 0.58 ng/mL). Five patients with mediastinitis, two patients with bacteremia, and one patient with pneumonia had serum PCT concentrations of <1 ng/mL. These eight patients were administered antibiotics previously and serum PCT was measured during a therapeutic antibiotic window. For prediction of infection by PCT, the best cutoff value was 1 ng/mL, with sensitivity 85%, specificity 95%, positive predictive value 96%, and negative predictive value 84%. Serum CRP was high in all patients without intergroup difference. For prediction of infection by CRP, a value of 50 mg/L was sensitive (84%) but poorly specific (40%). Comparing the area under the receiver operating characteristic curves, PCT was better than CRP for diagnosis of postoperative sepsis (0.82 for PCT vs. 0.68 for CRP). Phase 3: Serum PCT concentration was significantly higher in patients with septic shock than in those with cardiogenic shock (96.98 +/- 119.61 ng/mL vs. 11.30 +/- 12.3 ng/mL). For discrimination between septic and cardiogenic shock, the best cutoff value was 10 ng/mL, with sensitivity of 100% and specificity of 62%. CONCLUSION: Cardiac surgery with cardiopulmonary bypass influences serum PCT concentration with a peak on POD 1. In the presence of fever, PCT is a reliable marker for diagnosis of infection after cardiac surgery, except in patients who previously received antibiotics. PCT was more relevant than CRP for diagnosis of postoperative infection. During a postoperative circulatory failure, a serum PCT concentration >10 ng/mL is highly indicative of a septic shock.

Effect of cardiopulmonary bypass on serum procalcitonin and C-reactive protein concentrations.

We have measured serum procalcitonin (PCT) concentrations after cardiac surgery in 36 patients allocated to one of three groups: group 1, coronary artery bypass grafting (CABG) with cardiopulmonary bypass (CPB) (n = 12); group 2, CABG without CPB (n = 12); and group 3, valvular surgery with CPB (n = 12). Serum PCT and C-reactive protein (CRP) concentrations were measured before operation, at the end of surgery and daily until postoperative day 8. Serum PCT concentrations increased, irrespective of the type of cardiac surgery, with maximum concentrations on day 1: mean 1.3 (SD 1.8), 1.1 (1.2) and 1.4 (1.2) ng ml-1 in groups 1, 2 and 3, respectively (ns). Serum PCT concentrations remained less than 5 ng ml-1 in all patients. Concentrations returned to normal by day 5 in all groups. To determine the effect of the systemic inflammatory response (SIRS) on serum PCT concentrations, patients were divided post hoc, without considering the type of cardiac surgery, into patients with SIRS (n = 19) and those without SIRS (n = 17). The increase in serum PCT was significantly greater in SIRS (peak PCT 1.79 (1.64) ng ml-1 vs 0.34 (0.32) ng ml-1 in patients without SIRS) (P = 0.005). Samples for PCT and CRP measurements were obtained from 10 other patients with postoperative complications (circulatory failure n = 7; active endocarditis n = 2; septic shock n = 1). In these patients, serum PCT concentrations ranged from 6.2 to 230 ng ml-1. Serum CRP concentrations increased in all patients, with no differences between groups. The postoperative increase in CRP lasted longer than that of PCT. We conclude that SIRS induced by cardiac surgery, with and without CPB, influenced serum PCT concentrations with a moderate and transient postoperative peak on the first day after operation. A postoperative serum PCT concentration of more than 5 ng ml-1 is highly suggestive of a postoperative complication.

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.

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.

Deletion of arginine (608) in acid sphingomyelinase is the prevalent mutation among Niemann-Pick disease type B patients from northern Africa.

There is a high incidence of Niemann-Pick type B disease in the Maghreb region of North Africa, which includes Morocco, Algeria and Tunisia. A hypothesis that there may well be a common, predominant mutant acid sphingomyelinase allele responsible for the type B phenotype in this population has been tested. A deletion of an arginine codon at amino acid residue 608 was found in one patient. The same mutation was also observed in another of our cases. An original screening procedure using 3'-end digoxigenin-labeled allele-specific oligonucleotides and chemiluminescent detection was developed and used parallel to the conventional assay with 5'-end radiolabeled oligonucleotides. Of the 15 non-related, non-Jewish North African type B patients studied, 12 were homozygous and two compound heterozygous for this deletion (26 delta R608 alleles/30 mutant alleles). Among type B patients from other geographic regions (France, UK, Italy, Czechoslovakia), this mutation was observed in only one of the 16 alleles studied. Our results indicate that deletion of arginine 608 in the acid sphingomyelinase gene is the highly prevalent mutation underlying Niemann-Pick type B disease in the population of Maghreb. A varying severity of the clinical and enzymatic expression within the non-neuronopathic phenotype has however been observed in patients homozygous for the mutation.

Use of Percoll gradients for isolation of human placenta mitochondria suitable for investigating outer membrane proteins.

Human mitochondria were isolated from placenta by a combination of differential and Percoll gradient centrifugation, resulting in a highly pure and intact preparation as assessed by marker enzyme analysis and electron microscopy. The advantages over previous methods are the rapidity of the procedure and the excellent resolution of mitochondria and lysosomes. Moreover, the high extent of intactness of the mitochondria so obtained made them particularly well suited for investigating outer membrane proteins. Taking advantage of this method, we have purified human mitochondrial porin. The purified protein consists of a single unglycosylated polypeptide of molecular mass 33 kDa.

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.

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.

Mitochondrial dolichyl-phosphate mannose synthase. Purification and immunogold localization by electron microscopy.

Mitochondrial dolichyl-phosphate mannose synthase has been purified to homogeneity using an original procedure, reconstitution into specific phospholipid vesicles and sedimentation on a sucrose gradient as final step. The enzyme has an apparent molecular mass of 30 kDa on an SDS/polyacrylamide gel. Increased enzyme activity could be correlated with this polypeptide band. A specific antibody was raised in rabbits against this transferase. Specific IgG obtained from the immune serum removed enzymatic activity from a detergent extract of mitochondrial outer membrane and reacted specifically with the 30-kDa band on immunoblots. Furthermore, an immunocytochemical experiment proved the localization of dolichyl-phosphate mannose synthase on the cytosolic face of the outer membrane of mitochondria.

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.