Evaluation of a new NGS method based on a custom AmpliSeq library and Ion Torrent PGM sequencing for the fast detection of genetic variations in cardiomyopathies.

Background: Hypertrophic and dilated cardiomyopathies are common genetic cardiac diseases. Due to large cohorts to investigate, large number of causative genes and high rate of private mutations, mutational screening must be performed using an extremely sensitive and specific detection method.

Methods: NGS workflow based on a custom AmpliSeq panel was designed for sequencing most prevalent cardiomyopathy-causing genes on the Ion PGM™ Sequencer.

Evaluation of a new high-throughput next-generation sequencing method based on a custom AmpliSeq™ library and ion torrent PGM™ sequencing for the rapid detection of genetic variations in long QT syndrome.

Background And Objective: Inherited long QT syndrome (LQTS) is a cardiac channelopathy associated with a high risk of sudden death. The prevalence has been estimated at close to 1:2,000. Due to large cohorts to investigate and high rate of private mutations, mutational screening must be performed using an extremely sensitive and specific detection method.

Next-generation sequencing (NGS) as a fast molecular diagnosis tool for left ventricular noncompaction in an infant with compound mutations in the MYBPC3 gene.

Left ventricular noncompaction (LVNC) is a clinically heterogeneous disorder characterized by a trabecular meshwork and deep intertrabecular myocardial recesses that communicate with the left ventricular cavity. LVNC is classified as a rare genetic cardiomyopathy. Molecular diagnosis is a challenge for the medical community as the condition shares morphologic features of hypertrophic and dilated cardiomyopathies.

p53-independent early and late apoptosis is mediated by ceramide after exposure of tumor cells to photon or carbon ion irradiation.

Background: To determine whether ceramide is responsible for the induction of p53-independent early or late apoptosis in response to high- and low-Linear-Energy-Transfer (LET) irradiation.

Methods: Four cell lines displaying different radiosensitivities and p53-protein status were irradiated with photons or 33.4 or 184 keV/μm carbon ions.

The natural occurrence of human fibrinogen variants disrupting inter-chain disulfide bonds (A{alpha}Cys36Gly, A{alpha}Cys36Arg and A{alpha}Cys45Tyr) confirms the role of N-terminal A{alpha} disulfide bonds in protein assembly and secretion.

Analyses of site-directed fibrinogen mutants expressed in several recombinant models have previously shown that both inter- and intra-chain disulfide bonds are critical for fibrinogen assembly and secretion. Four naturally occurring mutations on AαCys36 and AαCys45 residues are reported here to be associated with decreased fibrinogen levels. This confirms the main role of the AαCys36-BβCys65 and AαCys45-γCys23 disulfide bonds in reaching a normal fibrinogen plasma level.

Transient alteration of cellular redox buffering before irradiation triggers apoptosis in head and neck carcinoma stem and non-stem cells.

Background: Head and neck squamous cell carcinoma (HNSCC) is an aggressive and recurrent malignancy owing to intrinsic radioresistance and lack of induction of apoptosis. The major focus of this work was to design a transient glutathione depleting strategy during the course of irradiation of HNSCC in order to overcome their radioresistance associated with redox adaptation.

Methodology/principal Findings: Treatment of SQ20B cells with dimethylfumarate (DMF), a GSH-depleting agent, and L-Buthionine sulfoximine (BSO), an inhibitor of GSH biosynthesis 4 h before a 10 Gy irradiation led to the lowering of the endogenous GSH content to less than 10% of that in control cells and to the triggering of radiation-induced apoptotic cell death.

Development of a high resolution melting method for the detection of genetic variations in Long QT Syndrome.

Background: Inherited Long QT Syndrome (LQTS) is a cardiac channelopathy associated with a high risk of sudden death. The prevalence has been estimated at close to 1:2000. Due to large cohorts to investigate, the size of the 3 prevalent mutated genes, and the presence of a large spectrum of private mutations, mutational screening requires an extremely sensitive and specific scanning method.

Development of a high resolution melting method for the detection of genetic variations in hypertrophic cardiomyopathy.

Background: Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiac disease affecting 1 in 500 people. Due to large cohorts to investigate, the number of disease-causing genes, the size of the 2 prevalent mutated genes, and the presence of a large spectrum of private mutations, mutational screening must be performed using an extremely sensitive and specific scanning method.

Methods: High Resolution Melting (HRM) analysis was developed for prevalent HCM-causing genes (MYBPC3, MYH7, TNNT2, and TNNI3) using control DNAs and DNAs carrying previously identified gene variants.

Prevalence and spectrum of mutations in a cohort of 192 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 sarcomeric genes but modifiers genes may also modulate the phenotypic expression of HCM mutations. The aim of the current study was to report the frequency of single and multiple gene mutations in a large French cohort of HCM patients and to evaluate the influence of polymorphisms previously suggested to be potential disease modifiers in this myocardial pathology. We report the molecular screening of 192 unrelated HCM patients using denaturing high-performance liquid chromatography/sequencing analysis of the MYBPC3, MYH7, TNNT2 and TNNI3 genes.

A major determinant for binding and aminoacylation of tRNA(Ala) in cytoplasmic Alanyl-tRNA synthetase is mutated in dominant axonal Charcot-Marie-Tooth disease.

Charcot-Marie-Tooth disease (CMT) is the most common cause of inherited peripheral neuropathy, with an estimated frequency of 1/2500. We studied a large family with 17 patients affected by the axonal form of CMT (CMT2). Analysis of the 15 genes or loci known to date was negative.

U1 snRNA mis-binding: a new cause of CMT1B.

We report the molecular characterization of two splice mutations in two different French families affected with a late onset form of Charcot-Marie-Tooth disease type 1B (CMT1B), an autosomal dominant inherited disorder caused by mutations in the myelin protein zero gene. The first substitution, c.306G>A, located in exon 3, does not change the codon p.

Heat shock protein 27 as a new therapeutic target for radiation sensitization of head and neck squamous cell carcinoma.

In a wide range of human cancers, increased levels of heat shock protein 27 (Hsp27) are closely associated with tumorigenesis, metastasis, resistance to anticancer therapeutics, and thus poor prognosis. In this study, we evaluate the radiosensitizing effects of Hsp27 gene silencing using OGX-427, a second-generation antisense oligonucleotide (ASO), on the radioresistant head and neck squamous cell carcinoma (HNSCC) SQ20B cells. In vitro, the downregulation of Hsp27 significantly enhanced radiation-induced apoptotic and clonogenic death, and promoted Akt inactivation.

Contribution of long-QT syndrome genetic variants in sudden infant death syndrome.

A cohort of 52 French unrelated infant cases who died unexpectedly before they reached 12 months of age was blindly investigated to better quantify the contribution of long-QT syndrome (LQTS) genetic variants in French cases of sudden infant death syndrome (SIDS). After a standardized autopsy protocol, a blinded molecular screening of the KCNQ1, KCNH2, SCN5A, KCNE1, and KCNE2 genes was performed on each case. These postmortem investigations enabled us to reclassify 18 as non-SIDS cases, 32 as SIDS cases, and 2 as suspected SIDS cases.

Validation of high-resolution DNA melting analysis for mutation scanning of the LMNA gene.

Objectives: LMNA mutations lead to a wide spectrum of disorders now called laminopathies. Due to large cohorts to investigate, mutational screening must be performed using an extremely sensitive and specific scanning method.

Design And Methods: High Resolution Melting (HRM) analysis was developed for LMNA mutation detection.

Biophysical characterization of a new SCN5A mutation S1333Y in a SIDS infant linked to long QT syndrome.

Various entities and genetic etiologies, including inherited long QT syndrome type 3 (LQT3), contribute to sudden infant death syndrome (SIDS). The goal of our research was to biophysically characterize a new SCN5A mutation (S1333Y) in a SIDS infant. S1333Y channels showed the gain of Na(+) channel function characteristic of LQT3, including a persistent inward Na(+) current and an enhanced window current that was generated by a -8 mV shift in activation and a +7 mV shift in inactivation.

Diversity and complexity of ceramide generation after exposure of jurkat leukemia cells to irradiation.

Purpose: To define which intracellular pools of sphingomyelin and ceramide are involved in the triggering of apoptosis of Jurkat leukemia cells in response to gamma-ray exposure.

Methods And Materials: We examined the kinetics of ceramide generation at the whole-cell level and in different subcellular compartments (plasma membrane rafts, mitochondria, and endoplasmic reticulum) after irradiation with photons. Ceramide was measured by high-performance liquid chromatography or after pulse labeling experiments, and the presence of sphingomyelinase within mitochondria was assessed by electron microscopy.

Rapid, sensitive and inexpensive detection of SCN5A genetic variations by high resolution melting analysis.

Objectives: SCN5A mutations lead to a wide spectrum of cardiovascular disorders. Due to large cohorts to investigate and the large gene size, mutational screening must be performed using an extremely sensitive and specific scanning method.

Design And Methods: High Resolution Melting (HRM) analysis was developed for SCN5A mutation detection using control DNAs and DNAs carrying previously identified gene variants.

A new C-terminal hERG mutation A915fs+47X associated with symptomatic LQT2 and auditory-trigger syncope.

Background: A novel mutation of hERG (A915fs+47X) was discovered in a 32-year-old woman with torsades de pointes, long QTc interval (515 ms), and syncope upon auditory trigger.

Objective: We explored whether the properties of this mutation could explain the pathology.

Methods: Whole-cell A915fs+47X (del) and wild-type (WT) currents were recorded in transiently transfected COS7 cells or Xenopus oocytes.

Protective role of Hsp27 protein against gamma radiation-induced apoptosis and radiosensitization effects of Hsp27 gene silencing in different human tumor cells.

Purpose: The ability of heat shock protein 27 (Hsp27) to protect cells from stressful stimuli and its increased levels in tumors resistant to anticancer therapeutics suggest that it may represent a target for sensitization to radiotherapy. In this study, we investigate the protective role of Hsp27 against radiation-induced apoptosis and the effect of its attenuation in highly expressing radioresistant cancer cell lines.

Methods And Materials: We examined clonogenic death and the kinetics of apoptotic events in different tumor cell lines overexpressing or underexpressing Hsp27 protein irradiated with photons.

Radioresistance of human carcinoma cells is correlated to a defect in raft membrane clustering.

In addition to DNA damage, exposure to irradiation involves the plasma membrane in the early phases of gamma-ray-induced cell death. The involvement of raft microdomains following gamma-radiation derives essentially from the role of ceramide as a critical component leading to apoptosis. It is demonstrated here that gamma-irradiation of a radiosensitive human head and neck squamous carcinoma cell line (SCC61) results in the triggering of raft coalescence to larger membrane platforms associated with the externalization of an acid sphingomyelinase (A-SMase), leading to ceramide release in raft, 30 min postirradiation.

Time course and prognostic value of plasma B-type natriuretic peptide concentration in neonates undergoing the arterial switch operation.

Background: Plasma B-type natriuretic peptide (BNP) can predict postoperative complications after cardiac surgery in adults. Our aim was to investigate BNP kinetics and prognostic value in neonates undergoing the arterial switch operation (ASO) for transposition of the great arteries (TGA).

Methods: We measured BNP concentrations in 30 neonates before, immediately after, and 6, 12, 24, and 48 h after ASO for TGA.

Torsades de pointes complicating atrioventricular block: evidence for a genetic predisposition.

Background: The prevalence of genetic risk factors has not been systematically evaluated in the setting of complete atriventricular (AV) block complicated by long QT syndrome (LQTS).

Objective: This study was performed to determine to what extent acquired LQTS in the context of AV block has a genetic substrate.

Methods: Among 420 recipients of pacemakers implanted over a 3-year period, we identified retrospectively 29 patients with complete AV block and a QT interval >600 ms in duration.

Plasma BNP and NT-proBNP assays by automated immunoanalyzers: analytical and clinical study.

The plasma concentrations of natriuretic peptides, BNP and NTproBNP, have been shown to be markers for the diagnosis of congestive heart failure (CHF). In this study, plasma BNP and NTproBNP concentrations were evaluated and stratified according to renal function, body mass index (BMI), and New York Heart Association (NYHA) classification. Comparison studies between the 2 natriuretic peptide markers were performed.