Plakophilin 2A is the dominant isoform in human heart tissue: consequences for the genetic screening of arrhythmogenic right ventricular cardiomyopathy.

BACKGROUND: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited heart disease in which mutations affecting Plakophilin-2 (PKP2) are the most frequently detected. However, pathogenicity of variants is not always fully determined. PKP2 encodes two isoforms, the longest (PKP2b) includes the alternatively spliced exon 6, which is routinely screened for molecular diagnosis, despite the absence of data on cardiac expression of PKP2 isoforms. OBJECTIVE: To examine the pathogenicity of PKP2 exon 6 mutations by focusing on a missense variant located in this exon. METHODS AND RESULTS: The PKP2 heterozygous p.Arg490Trp variant was identified in two unrelated ARVC probands (absent from 470 controls). In silico analysis suggested that PKP2 exon 6 is an Alu-derived sequence with very low expression level. PKP2a mRNA, which does not include the sequence encoded by exon 6, was the dominant isoform transcribed; at western blot analysis PKP2A was the only clearly detectable isoform in all human heart samples analysed (from six different controls and the proband). Moreover, in the proband's sample, p.Arg490Trp was not associated with aberrant exon 6 splicing or mutant mRNA downregulation. Finally, a heterozygous missense variant (p.Glu2343Lys) in Desmoplakin was identified in this proband and is likely to be the disease-causing mutation. CONCLUSION: PKP2A was shown to be the major isoform expressed in human heart tissue and PKP2B protein was undetectable. The results strongly suggest that p.Arg490Trp and other variants located in PKP2 exon 6 may not be disease causing. Variant splicing also has important consequences for the interpretation of mutation analysis and genetic counselling in ARVC and other hereditary cardiac diseases.

[Genetics of inherited cardiomyopathies]. / Génétique des cardiomyopathies héréditaires.

Hereditary cardiomyopathy is a primitive disorder in which the heart muscle is structurally and functionally abnormal in the absence of any other cause of cardiomyopathy. They are separated into four phenotypic groups, hypertrophic cardiomyopathy, dilated cardiomyopathy, restrictive cardiomyopathy and arrhythmogenic cardiomyopathy of the right ventricle. Hypertrophic cardiomyopathy was the first identified at the molecular level and then the first to benefit of molecular testing. The molecular analyses were then extended the following years to the dilated cardiomyopathy and restrictive cardiomyopathy. The arrhythmogenic right ventricular cardiomyopathy was the latest to be analyzed at the molecular level because the identification of genes involved in that phenotype was published only in 2002 to 2006. The genetics analysis of these diseases has developed over the past decade and, although still complex, is now available in current hospital practice. The objectives of these tests are to confirm a diagnosis difficult to achieve by classic clinical approach and to perform predictive and presymptomatic diagnosis in families when the mutation was identified. This allows for appropriate care of patients at risk, and may respond to a request for prenatal diagnosis in particularly serious forms. These tests are framed in the context of genetic counselling consultation and patients are the subjects of a multidisciplinary care in reference centres.

[Biochemical exploration of energetic metabolism and oxidative stress in low grade gliomas: central and peripheral tumor tissue analysis]. / Exploration biochimique du métabolisme énergétique et du stress oxydant des gliomes de bas grade: analyse du tissu tumoral central et périphérique.

Gliomas represent 50% of primary brain tumors, and their prognosis remains poor despite the advances in diagnosis and therapeutic strategies. Low grade gliomas (LGG) are infiltrative tumors and they constantly undergo malignant transformation. Metabolic exploration of human gliomas in vivo, in animals and by using cell culture models showed important differences between tumor tissues and normal brain tissues, which can provide new markers for diagnosis, prognosis and therapeutic targets. In this study, energetic and oxidant metabolisms were explored in biopsy extracts of LGG obtained from the centre and the periphery of tumors. Metabolic pattern of these tumors was explored and the differences between the centre and the periphery pointed. Our study showed a metabolic heterogeneity between tumors, with hypermetabolic and hypometabolic profiles. Lactate to pyruvate ratio was>1, suggesting that the energy metabolism in LGG is glycolytic in nature, particularly in the centre of the tumors. Peripheral samples of tumors showed increased glucose consumption and cytochrome c oxidase activity. Lipid peroxidation and catalase activity were also increased in the periphery compared to the centre of tumors. A relationship between the main antioxidant and energy metabolism enzymes activities was observed, suggesting that periphery of tumors is more active metabolically and more resistant to free radical injury.

[Cellular energetic metabolism of cerebral tissue: metabolic characteristics of glial tumours]. / Métabolisme énergétique cellulaire du tissu cérébral: spécificités métaboliques des tumeurs gliales.

This review reports recent observations concerning specificities of the cellular energy metabolism in cerebral tissues that highlight on characteristics of that of glial tumours, such as the association of metabolic alterations aggressiveness of these tumours. Compared to normal cerebral tissue, glial tissue exhibits both a relative independence towards oxygen and substrate furnitures and thus vascularization, as well as the metabolic co-operation of neurons and glial cells within the tumour. Occurrence of a Warburg effect could explain such metabolic autonomy that might be associated to genetic changes observed in gliomas. Characteristics of the glycolytic metabolism within glioma tissue therefore may be novel land therapeutic approaches for the treatment of these tumours.

Increased adiponectin receptor-1 expression in adipose tissue of impaired glucose-tolerant obese subjects during weight loss.

OBJECTIVE: To investigate the mRNA expression of adiponectin, AdipoR1 and AdipoR2, the two recently cloned adiponectin receptors and peroxisome proliferator activated receptor (PPAR)gamma2 in adipose tissue of obese individuals before and during a very low calorie diet (VLCD) inducing weight loss. METHODS: Twenty-three non-diabetic obese subjects with normal (NGT, n = 11) or impaired glucose tolerance (IGT, n = 12) (age, 47 +/- 3 years; body mass index, 39.3 +/- 1.3 kg/m2) were studied before and after a 3-week 3.9 MJ diet daily without exercise. mRNA levels of nine IGT and six NGT subjects were measured by real-time PCR in s.c. abdominal adipose tissue. RESULTS: Metabolic parameters and insulin sensitivity were improved by VLCD in the IGT group, but minimally affected in the NGT group. VLCD increased expression of AdipoR1 in the IGT (P = 0.02), but not in the NGT group. Adiponectin, AdipoR2 and PPARgamma2 mRNA levels did not change during VLCD in any group. In the IGT, but not in the NGT group, AdipoR1 and AdipoR2 expressions were positively related to that of PPARgamma2 and, after VLCD, AdipoR1 and AdipoR2 expressions were positively related to each other and to that of adiponectin. CONCLUSION: In the NGT group, the 3-week VLCD inducing weight loss did not modify metabolic parameters, insulin sensitivity and the expression of the adiponectin system in adipose tissue. By contrast, in the IGT group, AdipoR1 expression increased and we found a coordinate regulation of the expression of adiponectin and its receptors. These modifications could participate, through adiponectin action on adipocytes, to the improved metabolic parameters observed in IGT subjects.

[Alpha 2 macroglobulin immunoturbidimetric assays (DakoCytomation reagents) on Roche Diagnostic analysers (Modular P, Cobas Integra). Application to FibroTest-Actic-Test]. / Dosage immunoturbidimétrique de l'alpha 2 macroglobuline (réactifs DakoCytomation) sur les automates Roche Diagnostics (Modular P, Cobas Intégra). Application au FibroTest-ActiTest).

BACKGROUND: Alpha2Macroglobulin (A2M) measure showed a revival since it was introduced into FibroTest-ActiTest-Fibro (FT-AT-Fibro) algorithm. More often than not, this assay is performed in immunonephelemetry. Progresses in the comprehension of fibrosis dynamics and better treatment efficacy follow-up will increase FT-AT-Fibro prescriptions. Despite efforts to standardize methods of enzymatic activity measure and proteins measure, we still observe important interlaboratory and intersystem variability. AIM: The primary aim of the study is to validate immunoturbidimetric measure of A2M on Modular P and Cobas Integra analysers (Roche Diagnostics) in utility channel using DakoCytomation reagents in order to extend the analytical system range allowed to measure A2M. The secondary aim of the study is to verify transferability of the six FT-AT composants (A2M, haptoglobin, apolipoprotein A1, total bilirubin, GGT and ALT) to Roche Diagnostics equipment by comparing with results measured on the reference system. RESULTS: A2M measures (n = 146) showed linearity, repetitiveness and were reproducible. Readjustments to adapt A2M measures were required. A corrector factor of 0.84 for Modular P and of 0.87 for Cobas Integra was introduced in order to readjust the immunoturbidimetric method to the immunonephelemetric method. The rationale of proposed corrector factors is based on the use of Dade Behring and DakoCytomation reagents (antisera and calibrant). Biologist vigilance is required to point out modifications or variations in reagents that could be done by the company. The six parameters results transferability from the reference system to Roche Diagnostics was demonstrated by statistic analysis. FT-AT showed excellent correlations to the reference system for Modular P and Cobas Integra analysers. In this study no difference more than 0.11 was recorded and only few subjects had differences between 0.05 and 0.10. Therefore this very low inter-analysers variability has no significant clinical impact. CONCLUSION: This study showed that the analytical system made of Modular P, Cobas Integra, Roche Diagnostics and DakoCytomation reagents can be used for FibroTest-ActiTest-Fibro parameters assessment. Their statistical and clinical variability were acceptable compared to the reference system.

[Results transferability on RXL, ARX, X-Pand, BN2 (Dade Behring) and modular DP (Roche Diagnostics) analysers: application to component assays of fibrotest and Actitest]. / Transférabilité des résultats entre les automates RXL, ARX, X-Pand, BN2 (Dade Behring) et le Modular DP (Roche Diagnostics): application aux paramètres des Fibrotest et Actitest.

The follow up of patients with chronic liver diseases and the data from multicentric clinical studies are affected by the variability of assay results for the same parameter between the different laboratories. Today, the main objective in clinical chemistry throughout the world is to harmonise the assay results between the laboratories after the confirmation of their traceability, in relation to defined reference systems. In this context, the purpose of our study was to verify the homogeneity of haptoglobin, apolipoprotein A1, total bilirubin, GGT activity, ALAT activity results, which are combined in Fibrotest and Actitest, between Dimension Analysers RXL, ARX and X-PAND (Dade Behring Society). Moreover, we verified the transferability of Fibrotest and Actitest results between the RXL, and either the BN2 (haptoglobin and apolipoprotein A1) or the Modular DP (total bilirubin, GGT and ALAT activity concentrations). The serum samples from 150 hospitalised patients were analysed on the different analysers. Specific protein assays were calibrated using solutions standardised against reference material on Dimension and BN2 analysers. Total bilirubin assays were performed by a diazoreaction on Dimension and Modular DP analysers. The GGT and ALAT activity measurements on the Dimension analysers were performed in accordance with the reference methods defined by the International Federation of Clinical Chemisty and Laboratory Medicine (IFCC). On the Modular, enzyme activity measurements were performed according to the Szasz method (L-gamma- glutamyl-4-nitroanilide as substrate) modified by Persijn and van der Slik (L-gamma- glutamyl-3-carboxy- 4-nitroanilide as substrat) for GGT and according to the IFCC specifications for ALAT. The methods of enzymatic activity measurement were calibrated on the Modular only. Liver fibrosis and necroinflammatory activity indices were determined using calculation algorithms, after having adjusted each component's result of Fibrotest and Actitest for gender and age. Our study has shown, for each parameter, harmonious results between the Dimension analysers and between RXL and BN2- Modular DP. Disregarding alpha-2 macroglobulin which cannot be assayed on RXL, the results of Fibrotest and Actitest were similar as performed on BN2- Modular DP and RXL.

[X-linked adrenoleukodystrophy in a female proband: clinical presentation, biological diagnosis and family consequences]. / Adrénoleucodystrophie liée à l'X chez une proposante symptomatique: présentation clinique, diagnostic biologique et conséquences familiales.

INTRODUCTION: X-linked adrenoleukodystrophy (ALD) is the most frequent type of leukodystrophy (1/17 000 males). The phenotypic range in male patients varies from the severe cerebral presentations in children to the milder myeloneuropathy and to isolate adrenal insufficiency. More than a half of the carrier females display clinical symptoms over the age of 40 years. OBSERVATION: Diagnosis of ALD was raised in a 40 year-old female who presented with spastic paraparesis and sphincterian dysfunction, occurring after the delivery of her first child. There was no family history of ALD. Very long-chain fatty acids (VLFCA) were assayed in her one-year-old son in order to propose appropriate hormonal and neurological survey. His dosage was abnormal and an adrenal insufficiency was subsequently found. A brain MRI will be proposed biannually when he reaches to age of for years. The proband's mother had an increased level of VLCFA, showing that she was a carrier. Family screening was extended to the proband's sisters and maternal aunt who already had children, but also to her brother, who may express a mild form of the disease later on, and to her maternal uncles who might be asymptomatic carriers. A frameshift mutation was found in the ABCD1 gene and will allow accurate carrier identification and prenatal diagnosis in the family. CONCLUSION: ALD diagnosis should be evoked in a woman affected by myelopathy despite the lack of family history. Such a diagnosis has severe consequences since some of the related males may carry the mutation although they do not display any symptom at time of diagnosis, and because carrier females have a risk to both have a clinical expression of the disease and give birth to an affected boy.

New mutations of SCN4A cause a potassium-sensitive normokalemic periodic paralysis.

BACKGROUND: Periodic paralysis is classified into hypokalemic (hypoPP) and hyperkalemic (hyperPP) periodic paralysis according to variations of blood potassium levels during attacks. OBJECTIVE: To describe new mutations in the muscle sodium channel gene SCN4A that cause periodic paralysis. METHODS: A thorough clinical, electrophysiologic, and molecular study was performed of four unrelated families who presented with periodic paralysis. RESULTS: The nine affected members had episodes of muscle weakness reminiscent of both hyperPP and hypoPP. A provocative test with potassium chloride was positive in two patients. However, repeated and carefully performed tests of blood potassium levels during attacks resulted in normal potassium levels. Remarkably, two patients experienced hypokalemic episodes of paralysis related to peculiar provocative factors (corticosteroids and thyrotoxicosis). Similarly to hyperPP, electromyography in nine patients revealed increased compound muscle action potentials after short exercise and a delayed decline during rest after long exercise as well as myotonic discharges in one patient. With use of molecular genetic analysis of the gene SCN4A, three new mutations were found affecting codon 675. They resulted in an amino acid substitution of a highly conserved arginine (R) to either a glycine (G), a glutamine (Q), or a tryptophan (W). Interestingly, hypoPP is caused by both mutations affecting nearby codons as well as the change of an arginine into another amino acid. CONCLUSION: A potassium-sensitive and normokalemic type of periodic paralysis caused by new SCN4A mutations at codon 675 is reported.

[Influence of pyridoxal phosphate in measuring aminotransferases activities in patients with viral hepatitis]. / Effet du phosphate de pyridoxal dans la mesure des activités aminotransférases chez les patients avec hépatite virale.

Effect of a pyridoxal phosphate (PP) supplementation of reagents used for ALT and AST measurement was studied in serum of 20 patients suffering from viral hepatitis. Measurements of enzyme activities were carried out at 37 degrees C, using an automate (AU 600, Olympus). Significant differences (p < 0.0001) were observed both for ALT and AST, meanwhile they were more marked for ALT than for AST. This difference was associated with a strong interindividual variability regarding PP activation effect on ALT. In conclusion, aminotransferase measurements should be carried out with a reagent supplemented with PP, when the enzyme activity is used to evaluate a cytolysis. The same recommendation applies when ALT results are integrated into various combinations developed for the evaluation of liver status.

Low incidence of cardiac events with beta-blocking therapy in children with long QT syndrome.

AIMS: To evaluate the effect of beta-blockers in children with long QT syndrome (LQTS) we reviewed the outcome of 122 patients (pts). METHODS: LQTS was diagnosed in 24 neonates and in 98 pts aged 0.5-15 years. Diagnosis was made because of syncope in 51 pts, bradycardia in 10 neonates and family history in 61 pts. The longest QTc ranged from 400 to 700 ms. Thirteen pts had 2:1 atrioventricular block and/or ventricular arrhythmias. Screening for mutations was performed in 118 pts. All children were treated with beta-blockers, annually checked by exercise testing and/or 24 h ECG monitoring. RESULTS: Four pts died. Survivors were followed-up for 1-18 years (7.5 +/- 5.3 years). Five neonates and 3 older pts received a prophylactic pacemaker (1 death) so that only 111/122 pts survived and were followed-up with beta-blockers alone. None of them died and five experienced a non-fatal cardiac event. There was no cardiac event among pts who were diagnosed because of familial history and among symptomatic KCNQ1 pts who were effectively treated with beta-blockers. CONCLUSION: The outcome of children with LQTS under effective beta-blockers is favourable. Persisting arrhythmia or symptoms despite beta-blockers should aim at identifying other genotypes than KCNQ1.

Systemic low-grade inflammation is related to both circulating and adipose tissue TNFalpha, leptin and IL-6 levels in obese women.

OBJECTIVE: We assessed the relationships between four circulating acute phase proteins and the circulating and adipose tissue levels of three adipocytokines. SUBJECTS: In all, 15 nondiabetic obese women with a body mass index (BMI) above 32 kg/m(2) were investigated. METHOD: Circulating concentrations of C-reactive protein (CRP), alpha 1 acid glycoprotein (AAG), fibrinogen, alpha 1 antitrypsin and both circulating and adipose tissue levels of interleukin (IL)-6, tumor necrosis factor (TNF)alpha and leptin were measured by either nephelometry or enzyme-linked immunosorbent assay. RESULTS: We found a strong positive correlation between both circulating and adipose tissue levels of IL-6, TNFalpha and leptin and serum CRP levels. All these adipose tissue adipocytokines were also positively correlated with serum AAG levels. These correlations disappeared when adjusted for fat mass, suggesting that the relationship observed was dependent on fat amount. CONCLUSION: Our results indicate a strong relationship between adipocytokines and inflammatory markers, and suggest that cytokines secreted by adipose tissue in obese subjects could play a role in increased inflammatory proteins secretion by the liver.

[Neonatal forms of congenital long QT syndrome]. / Formes néonatales du syndrome de QT long congénital.

UNLABELLED: The neonatal congenital long QT syndrome (LQTS) is rare and of bad prognosis due to the presence of severe ventricular arrhythmia and conduction abnormalities. METHODS: we included 24 propositus newborns from our population with LQTS. Genetic study was possible in 19 cases. RESULTS: the diagnosis of LQTS was made according to a QT prolongation associated with a sinusal neonatal bradycardia (n=9) or a 2/1 AV block (n=15). The onset presentation consisted of syncope (n=2), torsades de pointes (n=7), cardiovascular collapse (n=5), cardiac arrest (n=1). The mean QTc was at 550+60 ms. During the neonatal period the treatment consisted of beta-blocking agents in all cases, associated with a definitive pacemaker implantation in 10 cases with 2/1 AV block. Three newborns with a 2/1 AV block died during the first month of life (one case due to a septecemia after implantation of a pacemaker, and two who were waiting for that implantation). All survivors remained asymptomatic during a follow-up period of 7 years. In all cases with a 2/1 AV block we identified mutations in HERG (n=8). Newborns with isolated sinusal bradycardia presented all a mutation in KCNQ1 (n=9). CONCLUSION: the LTQS with 2/1 AV block is preferably associated with mutation in HERO with a bad initial prognosis.

Lack of association of the potassium channel-associated peptide MiRP2-R83H variant with periodic paralysis.

A missense variant (R83H) of the gene (KCNE3) encoding a potassium channel-associated peptide, MinK-related peptide 2 (MiRP2), has been reported in periodic paralysis patients. In the current study, no difference in the frequency of the MiRP2-R83H variant between periodic paralysis patients and healthy individuals was found. Furthermore, there was no segregation of this gene variant with the disease. These observations weaken the proposal that MiRP2-R83H causes periodic paralysis.

Interpretation of circulating C-reactive protein levels in adults: body mass index and gender are a must.

OBJECTIVE: The recently demonstrated association between C-reactive protein (CRP) level and body mass index (BMI) raised the question of the link between CRP and the degree of obesity. In the present study, we measured CRP in a healthy population with a wide range of BMI in order to appreciate the influence of overweight in the interpretation of CRP results in clinical use. METHOD: Blood donors, aged from 19 to 65 years, were included in the study. According to BMI, subjects were classified into 3 groups: A (BMI<25 kg/m(2), n=611); B (25-30, n=147); C (> 30, n=34). RESULTS: CRP values were different among women and men. CRP progressively increased with BMI in women. These results clearly showed that average level of CRP was quite different according to BMI and gender of the subjects and generated different normal ranges of CRP expressed in mg/L (median, 75(th) percentile): Group A: women: 0.44, 0.93; men: 0.40, 0.79, Group B: women: 1.28, 1.84; men: 0.84, 2.17, Group C: women: 3.61, 7.21; men: 1.16, 3.08. CONCLUSION: Our results suggest that for an inflammatory disease diagnosis, a CRP concentration of 5 mg/L is normal for obese women but is five times the 75(th) percentile for normal people.

Genetic testing and genetic counselling in hypertrophic cardiomyopathy: the French experience.

AIMS: A major breakthrough in the molecular genetics of hypertrophic cardiomyopathy (HCM) has made genetic testing now available in clinical practice, raising new questions about its implications, potential benefits, and the organisation of the procedure. The aim of this work was (1) to discuss the different questions related to genetic testing in HCM, and propose guidelines for the different situations, (2) to report our preliminary experience with a specific procedure. METHODS AND RESULTS: The main questions asked by patients and relatives concern presymptomatic diagnosis and prenatal counselling/diagnosis, while clinicians sometimes discuss diagnostic and prognostic testing. To take into account the complex medical and psychological implications of this new approach, we developed a specific, multidisciplinary, and multiple step procedure, including a cardiologist, a geneticist, and a psychologist. Seventy subjects were examined, including (1) 29 adults for presymptomatic diagnosis (of whom 10 left the procedure after the first visit and 19 continued, among whom six had a mutation and two experienced negative psychological impact, observed during follow up), (2) nine couples of parents for presymptomatic diagnosis in their children (the procedure was stopped after the first visit in eight and continued in one), (3) 22 couples for prenatal counselling (no prenatal genetic testing was asked for after the first visit), and (4) 10 subjects for diagnostic testing. We decided to perform no prognostic testing. CONCLUSION: Our preliminary experience confirms the complexity of the situation and suggests the necessity for a specific procedure to ensure good practice in genetic testing of HCM.

[T wave abnormalities on Holter monitoring of congenital long QT syndrome: phenotypic marker of a mutation of LQT2 (HERG)]. / Anomalies de l'onde T au Holter dans le syndrome du QT long congénital: marqueur phénotypique d'une mutation dans LQT2 (HERG).

The two genes which code for the potassium channels, KCNQ1 and HERG, are responsible for the most common forms of the long QT syndrome, LQT1 and LQT2. Abnormalities of duration and morphology of the ventricular repolarisation are amongst the diagnostic criteria of this syndrome. The morphology of the T waves was studied by 24 hour Holter monitoring in 190 subjects with a long QT syndrome due to KCNQ1 (LQT1) [N = 133] or HERG (N = 57) and in 100 controls, and it was compared with the ECG T wave. The T wave was characterised according to 3 morphological features: grade 0 (G0) = normal, grade 1 (G&) = slight ST depression and grade 2 (G2) = presence of ST elevation of the descending phase of the T wave. The T wave morphology on Holter ECG was normal for most LQT1 and control subjects compared with LQT2 (92%, 96% and 19% respectively, p < 0.01). Grade 1 appearances were observed more often in LQT2 (18 vs 8% for LQT1 and 4% for controls, p < 0.01). Grade 2 appearances were only observed in the cases of LQT2 (63%). The predictive factors of G2 were young age and an anti-sense mutation of the transmembrane domaines of HERG. The authors conclude that Holter monitoring improves detection of T wave changes compared with the ECG. Grade 2 changes seem to be a phenotype marker for a HERG mutation, especially those situated in the transmembrane domaines.