RESUMEN
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. Genome-wide genotyping identified a CMT2 locus in 16q21-q23 between D16S3050 and D16S3106. The maximum two-point LOD score was 4.77 at theta = 0 for marker D16S3050. Sequencing of candidate genes identified a unique mutation, c.986G>A (p.Arg329His), affecting a totally conserved amino acid in the helical domain of cytoplasmic alanyl-tRNA synthetase (AlaRS). A second family with the same mutation and a different founder was then identified in a cohort of 91 CMT2 families. Although mislocation of mutant Arg329His-AlaRS in axons remains to be evaluated, experimental data point mostly to a quantitative reduction in tRNA(Ala) aminoacylation. Aminoacylation and editing functions closely cooperate in AlaRS, and Arg329His mutation could also lead to qualitative errors participating in neurodegeneration. Our report documents in 18 patients the deleterious impact of a mutation in human cytoplasmic AlaRS and broadens the spectrum of defects found in tRNA synthetases. Patients present with sensory-motor distal degeneration secondary to predominant axonal neuropathy, slight demyelination, and no atypical or additional CNS features.
Asunto(s)
Alanina-ARNt Ligasa/genética , Axones/metabolismo , Enfermedad de Charcot-Marie-Tooth/genética , Citoplasma/metabolismo , Mutación , Adolescente , Adulto , Secuencia de Aminoácidos , Aminoacilación , Niño , Estudios de Cohortes , Genes Dominantes , Humanos , Escala de Lod , Persona de Mediana Edad , Datos de Secuencia Molecular , Linaje , Homología de Secuencia de AminoácidoRESUMEN
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 kinetics of ceramide production was quantified by fluorescent microscopy or High-Performance-Liquid-Chromatogaphy and the sequence of events leading to apoptosis by flow cytometry. RESULTS: Regardless of the p53-status, both low and high-LET irradiation induced an early ceramide production in radiosensitive cells and late in the radioresistant. This production strongly correlated with the level of early apoptosis in radiosensitive cells and delayed apoptosis in the radioresistant ones, regardless of radiation quality, tumor type, radiosensitivity, or p53-status. Inhibition of caspase activity or ceramide production showed that, for both types of radiation, ceramide is essential for the initiation of early apoptosis in radiosensitive cells and late apoptosis following mitotic catastrophe in radioresistant cells. CONCLUSIONS: Ceramide is a determining factor in the onset of early and late apoptosis after low and high-LET irradiation and is the mediator of the p53-independent-apoptotic pathway. We propose that ceramide is the molecular bridge between mitotic catastrophe and the commitment phase of delayed apoptosis in response to irradiation.
Asunto(s)
Apoptosis/genética , Apoptosis/efectos de la radiación , Ceramidas/metabolismo , Radiación Ionizante , Proteína p53 Supresora de Tumor/genética , Carbono , Caspasas/metabolismo , Línea Celular Tumoral , Ceramidas/biosíntesis , Relación Dosis-Respuesta en la Radiación , Humanos , Cinética , Mitocondrias/metabolismo , Mitocondrias/efectos de la radiación , FotonesRESUMEN
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-BßCys65 and AαCys45-γCys23 disulfide bonds in reaching a normal fibrinogen plasma level. Decreased coagulant/antigen ratios indicate abnormal species secretion in heterozygous subjects which varies between individuals. However, in contrast to overexpression in experimental models, disruption of the AαCys36-BßCys65 disulfide bond did not result in the appearance of Aα-Bß-γ moieties in vivo. A 188 kDa molecule reacting only with anti Aα and anti Bß chains was found in the plasma of the AαCys45Tyr variant. Heterozygous carriers of Aα chain mutations usually have normal fibrinogen levels, in contrast to the AαCys36Gly, AαCys36Arg and AαCys45Tyr variants that are shown here to cause hypofibrinogenemia.
Asunto(s)
Disulfuros/química , Fibrinógeno/química , Adulto , Sustitución de Aminoácidos , Disulfuros/metabolismo , Femenino , Fibrinógeno/genética , Fibrinógeno/metabolismo , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Modelos Moleculares , Mutación/genética , Polimorfismo Genético , Conformación ProteicaRESUMEN
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.Val102Val but is co-transmitted with the disease in the first family. The second substitution, c.675+3dup, is an insertion of a T at position +3 of intron 5. To identify the functional impact of these nucleotide changes on splicing and because no RNA sample was available, we used in silico prediction and in vitro splicing assay. Mutation c.306G>A increases the strength of a preexisting cryptic donor site at position c.304 which becomes stronger than the normal donor site of intron 3. This variation creates a sequence that better matches the U1 small nuclear RNA (snRNA) binding consensus, and HeLa cells, transfected with the mutant minigene, produce a truncated exon 3 messenger RNA (mRNA). Mutation c.675+3dup was predicted to abolish the donor site of intron 5, and, indeed, HeLa cells transfected with the mutant minigene completely skip exon 5 from the transcript. The mutated sequence abolishes U1 snRNA binding and co-transfection of a mutated complementary U1 snRNA restored exon 5 inclusion in the mRNA. This work provides valuable information regarding the molecular basis of two forms of late onset of CMT1B, U1 snRNA mis-binding, and provides more evidence that a "silent" polymorphism may be a disease causing mutation.
Asunto(s)
Enfermedad de Charcot-Marie-Tooth/diagnóstico , Enfermedad de Charcot-Marie-Tooth/genética , Péptidos y Proteínas de Señalización Intracelular/genética , Fosfoproteínas/genética , ARN Nuclear Pequeño/metabolismo , Adulto , Exones , Femenino , Células HeLa , Humanos , Intrones , Masculino , Persona de Mediana Edad , Mutación , Linaje , Polimorfismo Genético , Empalme del ARNRESUMEN
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. In vivo, combining OGX-427 with local tumor irradiation (5 x 2 Gy) led to a significant regression of SQ20B tumors related to a high rate of apoptosis and decreased levels of glutathione antioxidant defenses. Increasing the total radiation dose (15 x 2 Gy) significantly amplified the radiosensitizing effect of OGX-427. Treatment of tumors with OGX-427 plus radiation resulted in a decrease in angiogenesis associated with a reduced activation of the Akt pathway. Furthermore, the combined treatment enhanced the survival of SQ20B-bearing mice and showed no signs of acute and delayed toxicity. Our findings demonstrate for the first time that Hsp27 knockdown enhances the cytotoxic effects of radiotherapy in vivo and provide preclinical proof of principle for clinical trials using Hsp27 antisense technology in the treatment of patients with HNSCC radioresistant cancers.
Asunto(s)
Proteínas de Choque Térmico HSP27/genética , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Oligonucleótidos Antisentido/uso terapéutico , Fármacos Sensibilizantes a Radiaciones/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Carcinoma de Células Escamosas/tratamiento farmacológico , Carcinoma de Células Escamosas/radioterapia , Línea Celular Tumoral , Femenino , Proteínas de Choque Térmico HSP27/farmacología , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Inmunohistoquímica , Ratones , Ratones Desnudos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/farmacología , Fármacos Sensibilizantes a Radiaciones/farmacología , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
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. Among the 18 non-SIDS cases, no LQTS mutation was identified. In contrast, our results led to a possible explanation for the death of at least three infants in the SIDS cohort. Half of the LQTS gene variants identified were located on the SCN5A gene. This study confirms that LQTS mutations may represent one of the leading genetic causes of SIDS. If autopsy fails to provide an explanation for an unexplained infant death, medicolegal investigation should be extended with a molecular screening of major LQTS genes. Identification of more LQTS mutations in SIDS cases could provide new insights into the pathophysiology of SIDS and, consequently, reduce the number of unexplained sudden infant deaths.
Asunto(s)
Síndrome de QT Prolongado/genética , Polimorfismo Genético , Muerte Súbita del Lactante/genética , Femenino , Humanos , Recién Nacido , Síndrome de QT Prolongado/complicaciones , Masculino , Muerte Súbita del Lactante/etiologíaRESUMEN
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. The radiosensitive Jurkat cell line, which does not express Hsp27 constitutively or in response to gamma-rays, was stably transfected with Hsp27 complementary DNA. Attenuation of Hsp27 expression was accomplished by antisense or RNAi (interfering RNA) strategies in SQ20B head-and-neck squamous carcinoma, PC3 prostate cancer, and U87 glioblastoma radioresistant cells. RESULTS: We measured concentration-dependent protection against the cytotoxic effects of radiation in Jurkat-Hsp27 cells, which led to a 50% decrease in apoptotic cells at 48 hours in the highest expressing cells. Underlying mechanisms leading to radiation resistance involved a significant increase in glutathione levels associated with detoxification of reactive oxygen species, a delay in mitochondrial collapse, and caspase activation. Conversely, attenuation of Hsp27 in SQ20B cells, characterized by their resistance to apoptosis, sensitizes cells to irradiation. This was emphasized by increased apoptosis, decreased glutathione basal level, and clonogenic cell death. Sensitization to irradiation was confirmed in PC3 and U87 radioresistant cells. CONCLUSION: Hsp27 gene therapy offers a potential adjuvant to radiation-based therapy of resistant tumors.
Asunto(s)
Apoptosis/efectos de la radiación , Silenciador del Gen/fisiología , Proteínas de Choque Térmico/fisiología , Proteínas de Neoplasias/fisiología , Tolerancia a Radiación/fisiología , Apoptosis/fisiología , Caspasas/metabolismo , Línea Celular Tumoral/efectos de la radiación , Regulación hacia Abajo , Activación Enzimática/efectos de la radiación , Rayos gamma , Glioblastoma/metabolismo , Glioblastoma/radioterapia , Glutatión/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Humanos , Células Jurkat/efectos de la radiación , Masculino , Mitocondrias/fisiología , Mitocondrias/efectos de la radiación , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Estrés Oxidativo/fisiología , Estrés Oxidativo/efectos de la radiación , Fotones , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/radioterapia , ARN sin Sentido/uso terapéutico , ARN Interferente Pequeño/uso terapéutico , Tolerancia a Radiación/genética , Especies Reactivas de Oxígeno/metabolismo , Factores de Tiempo , Transfección/métodos , Ensayo de Tumor de Célula MadreRESUMEN
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. For the first time, we show that this structural rearrangement is defective in the radioresistant SQ20B cells and associated with the lack of A-SMase activation and translocation, a result which could explain in part their resistance to apoptosis following ionizing radiation. Moreover, we show that SQ20B are protected against radiation injury through a fivefold upper level of endogenous glutathione compared to SCC61. Overcoming the endogenous antioxidant defenses of SQ20B through either H(2)O(2) treatment or GSH depletion triggers A-SMase activation and translocation, raft coalescence, and apoptosis. On the contrary, ROS scavengers abolished these events in radiosensitive SCC61 cells. Translation of this concept to tumor biology suggests that manipulation of rafts through redox equilibrium may provide opportunities for radiosensitization of tumor cells.
Asunto(s)
Carcinoma/patología , Microdominios de Membrana/metabolismo , Microdominios de Membrana/efectos de la radiación , Carcinoma/metabolismo , Caveolas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de la radiación , Ceramidas/biosíntesis , Colesterol/metabolismo , Regulación hacia Abajo/efectos de la radiación , Activación Enzimática/efectos de los fármacos , Activación Enzimática/efectos de la radiación , Rayos gamma , Glutatión/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Oxidación-Reducción , Transporte de Proteínas , Tolerancia a Radiación , Esfingomielina Fosfodiesterasa/clasificación , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/metabolismoRESUMEN
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. A second study group included 22 randomly selected patients who had AV block and a QT interval <600 ms. Normal controls were 100 consecutive individuals without medical history. Genetic studies screening for HERG, KCNQ1 KCNE1, KCNE2, and SCN5A mutations were performed. RESULTS: We identified four mutations on genes encoding potassium channels in five patients with AV block and acquired LQTS. These mutations were not found among patients with AV block and a QT interval <600 ms in duration or in healthy volunteers. Functional expression of three HERG mutations (R328C, R696C, and R1047L) had a dominant negative effect on wild-type I(Kr). One KCNE2 mutation (R77W) identified in a patient treated with flecainide did not alter I(Kr). CONCLUSIONS: This study showed that complete AV block complicated by LQTS was associated with HERG mutations in 17% of cases. Further studies are needed to identify factors, genetic or environmental, which may be implicated in bradycardia-related abnormalities of ventricular repolarization.
Asunto(s)
Predisposición Genética a la Enfermedad , Bloqueo Cardíaco/genética , Mutación/genética , Canales de Potasio/genética , Torsades de Pointes/genética , Anciano , Femenino , Genotipo , Bloqueo Cardíaco/complicaciones , Humanos , Masculino , Estudios Retrospectivos , Torsades de Pointes/etiologíaRESUMEN
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. Complicated postoperative evolution was defined as patients requiring mechanical ventilation or presenting low cardiac output syndrome for more than 72 h. We studied the ability of postoperative BNP concentrations to predict complicated evolution. RESULTS: Intubation duration, inotropic support duration, and intensive care unit stay were 68 (48-121) h, 78 (69-141) h, and 96 (76-149) h respectively. Patients with complicated evolution had higher 6 and 12-h BNP concentrations than patients with simple evolution (459 (210-897) vs 137 (67-248) ng/L and 547 (193-868) vs 185 (79-354) ng/L respectively; P < 0.05) and had longer intubation, inotropic support, and intensive care unit stay (96 (70-190) vs 50 (48-66) h, 100 (83-190) vs 70 (59-72) h, and 120 (90-240) vs 84 (72-96) h, P < 0.05). A 6-h BNP concentration >160 ng/L was able to predict complicated evolution with a sensitivity of 93% and a specificity of 67%. CONCLUSION: In neonates, BNP concentrations can predict adverse outcome in the postoperative period after ASO for TGA. This marker has potential clinical applications.
Asunto(s)
Procedimientos Quirúrgicos Cardiovasculares , Péptido Natriurético Encefálico/sangre , Complicaciones Posoperatorias/sangre , Complicaciones Posoperatorias/diagnóstico , Biomarcadores/sangre , Femenino , Humanos , Recién Nacido , Masculino , Pronóstico , Estudios Prospectivos , Factores de TiempoRESUMEN
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. Assays for BNP were performed with a Triage reagent pack (Biosite, Inc) on an Access 2 immunoanalyzer (Beckman-Coulter); NTproBNP assays were performed with a Roche reagent pack on an Elecsys 20.10 immunoanalyzer (Roche Diagnostics). Plasma samples were collected from consecutive patients hospitalized for cardiac disorders at our institution. Nonparametric tests were used for statistical analyses. The results show that alterations of renal function had less impact on BNP (p = 0.9) than on NTproBNP concentrations (p <0.0001). BNP and NTproBNP levels were lower in obese patients with CHF (515 +/- 61 ng/L and 1652 +/- 124 ng/L, respectively) than in lean patients (900 +/- 85 ng/L and 6686 +/- 749 ng/L). Although NTproBNP levels averaged about 10 times higher than BNP levels, there was significant correlation between these 2 markers (Deming regression r2 = 0.40, IC: 0.95). In conclusion, plasma BNP and NTproBNP assays are both useful for the diagnosis of CHF and left ventricular dysfunction. However, renal function and obesity must be taken into account for clinical interpretation. These assays have good analytical performance and the choice between them depends on local preference.
Asunto(s)
Insuficiencia Cardíaca/diagnóstico , Inmunoensayo/métodos , Péptido Natriurético Encefálico/sangre , Fragmentos de Péptidos/sangre , Insuficiencia Renal/sangre , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Creatinina/sangre , Femenino , Insuficiencia Cardíaca/sangre , Humanos , Inmunoensayo/instrumentación , Masculino , Persona de Mediana Edad , Obesidad/fisiopatología , Estadísticas no ParamétricasRESUMEN
Recent strategies to sensitize radioresistant tumours are based on combining gamma-irradiation with inducers of apoptosis. We report that the combination of three inhibitors of sphingolipid metabolism, DL-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol.HCl(DL-PDMP)+imipramine +/- D-erythro-2-(N-myristoylamino)-1-phenyl-1-propanol (D-MAPP), with 10-Gy irradiation triggers both mitotic and apoptotic killing in radioresistant SQ20B squamous carcinoma cells. In these cells, apoptosis is defective due to a lack of ceramide generation upstream, which cannot be explained by sphingomyelinase (neutral and acidic) deficiency or rapid derivation to the sphingolipid pathway. We present evidence of a functional transduction death pathway when ceramide generation is restored, which involves the mitochondrial-mediated pathway coupled to alterations in redox status and to executive caspases activation. The poly-drug treatment restored apoptosis to levels similar to those observed in radiosensitive SCC61 squamous carcinoma cells. Simultaneous exposure to gamma-irradiation and poly-drug treatment acted synergistically in SQ20B cells to produce a marked increase in both mitochondrial dysfunction and caspase cleavage, which led to a 7.8-fold increase in apoptosis within 48 h, relative to irradiated cells. Moreover, the results suggest that the ceramide released by irradiation or poly-drug treatment converges upon common cellular targets. Modulation of endogenous ceramide levels by inhibitors of sphingolipid metabolism may represent a new cellular target for the sensitization of radioresistant tumours to gamma-ray therapy.
Asunto(s)
Carcinoma de Células Escamosas/radioterapia , Ceramidas/farmacología , Apoptosis , Western Blotting , Carcinoma de Células Escamosas/metabolismo , Caspasa 3 , Caspasa 8 , Inhibidores de Caspasas , Caspasas/biosíntesis , Caspasas/metabolismo , Línea Celular Tumoral , Ceramidas/metabolismo , Cromatografía Líquida de Alta Presión , Relación Dosis-Respuesta en la Radiación , Activación Enzimática , Citometría de Flujo , Rayos gamma , Glucosiltransferasas/metabolismo , Glutatión/metabolismo , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/radioterapia , Humanos , Cinética , Mitocondrias/metabolismo , Mitosis , Miristatos/farmacología , Ácido N-Acetilneuramínico/metabolismo , Oxidación-Reducción , Propanolaminas/farmacología , Especies Reactivas de Oxígeno , Esfingolípidos/antagonistas & inhibidores , Factores de TiempoRESUMEN
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. A cohort of 75 previously studied patients was screened to evaluate this strategy in terms of sensibility, specificity, practicability and cost. In silico analysis was performed using the NextGENe® software. RESULTS: Our AmpliSeq custom panel allowed us to efficiently explore 96% of targeted sequences. Using adjusted alignment settings, all genetic variants (57 substitutions, 34 indels) present in covered regions and previously detected by HRM/sequencing were readily identified except a 73-bp MYBPC3 deletion (analytical sensitivity: 98.9%). Uncovered targeted regions were further analysed by a HRM/sequencing strategy. Complete molecular investigation was performed faster and cheaper than with previously used mutation detection methods. CONCLUSION: Finally, these results suggested that our new NGS approach based on Ampliseq libraries and Ion PGM sequencing is a highly efficient, fast and cheap high-throughput mutation detection method that is ready to be deployed in clinical laboratories.
Asunto(s)
Cardiomiopatías/genética , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Análisis de Secuencia de ADN/métodos , Estudios de Cohortes , Biología Computacional , Humanos , Programas Informáticos , Factores de TiempoRESUMEN
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. Mutational screening is crucial as this may have implications for therapy and management of LQTS patients. METHODS: Next-generation sequencing (NGS) workflow based on a custom AmpliSeq™ panel was designed for sequencing the five most prevalent cardiomyopathy-causing genes (KCNQ1, KCNH2, SCN5A, KCNE1, KCNE2) on Ion PGM™ Sequencer. A cohort of 30 previously studied patients was screened to evaluate this strategy in terms of sensitivity, specificity, practicability, and cost. In silico analysis was performed using NextGENe(®) software. RESULTS: Our AmpliSeq™ custom panel allowed us to explore 86 % of targeted sequences efficiently. Using adjusted alignment settings, all genetic variants (40 substitutions, 17 indels) present in covered regions and previously detected by high-resolution melt (HRM)/sequencing were readily identified. Uncovered targeted regions, which were mainly located in KCNH2, were further analyzed by HRM/sequencing strategy. Complete molecular investigation was performed faster and cheaper than with previously used mutation detection methods. CONCLUSION: Finally, these results suggested that our new NGS approach based on AmpliSeq™ libraries and Ion PGM™ sequencing is a highly efficient, fast, and cheap high-throughput mutation detection method that is ready to be deployed in clinical laboratories. This method will allow fast identification of LQTS mutations that will have further implications for therapeutics.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Síndrome de QT Prolongado/diagnóstico , Síndrome de QT Prolongado/genética , Análisis Mutacional de ADN/economía , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/economía , Humanos , Técnicas de Diagnóstico Molecular/economía , Técnicas de Diagnóstico Molecular/métodos , MutaciónRESUMEN
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. Several genetic causes of LVNC have been reported, with variable modes of inheritance, including autosomal dominant and X-linked inheritance, but relatively few responsible genes have been identified. In this report, we describe a case of a severe form of LVNC leading to death at 6 months of life. NGS sequencing using a custom design for hypertrophic cardiomyopathy panel allowed us to identify compound heterozygosity in the MYBPC3 gene (p.Lys505del, p.Pro955fs) in 3 days, confirming NGS sequencing as a fast molecular diagnosis tool. Other studies have reported neonatal presentation of cardiomyopathies associated with compound heterozygous or homozygous MYBPC3 mutations. In this family and in families in which parental truncating MYBPC3 mutations are identified, preimplantation or prenatal genetic screening should be considered as these genotypes leads to neonatal mortality and morbidity.
Asunto(s)
Proteínas Portadoras/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , No Compactación Aislada del Miocardio Ventricular/diagnóstico , No Compactación Aislada del Miocardio Ventricular/genética , Técnicas de Diagnóstico Molecular/métodos , Mutación , Secuencia de Bases , Análisis Mutacional de ADN , Salud de la Familia , Resultado Fatal , Femenino , Humanos , Lactante , Masculino , LinajeRESUMEN
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. METHODS: Efficiency of high resolution melting (HRM) analysis was evaluated for the most prevalent LQTS-causing genes (KCNQ1, KCNH2) using control DNAs and DNAs carrying previously identified gene variants. A cohort of 34 patients with a suspicion of LQTS was further blindly screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC strategy. RESULTS: HRM analysis was successfully optimized for KCNQ1 but optimisation of KCNH2 was more laborious as only 3 KCNH2 exons could be finally optimized. Remaining KCNH2 exons were analysed by direct sequencing. This molecular approach, which combined HRM and direct sequencing, was applied on the cohort of 34 cases and 9 putative mutations were identified. Using this approach, molecular investigation was completed faster and cheaper than using DHPLC strategy. CONCLUSIONS: This HRM/sequencing procedure represents an inexpensive, highly sensitive and high-throughput method to allow identification of mutations in the coding sequences of prevalent LQTS genes.
Asunto(s)
Análisis Mutacional de ADN/métodos , Síndrome de QT Prolongado/genética , Mutación , Temperatura de Transición , Cromatografía Líquida de Alta Presión , Estudios de Cohortes , Análisis Mutacional de ADN/economía , Canal de Potasio ERG1 , Canales de Potasio Éter-A-Go-Go/genética , Humanos , Canal de Potasio KCNQ1/genética , Desnaturalización de Ácido Nucleico , Reacción en Cadena de la PolimerasaRESUMEN
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. The sequence of biochemical events after GSH depletion and irradiation included ASK-1 followed by JNK activation which resulted in the triggering of the intrinsic apoptotic pathway through Bax translocation to mitochondria. CONCLUSIONS: This transient GSH depletion also triggered radiation-induced cell death in SQ20B stem cells, a key event to overcome locoregional recurrence of HNSCC. Finally, our in vivo data highlight the relevance for further clinical trials of endogenous redox modulation to enhance the cytotoxic effects of radiotherapy.
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Apoptosis , Carcinoma de Células Escamosas , Neoplasias de Cabeza y Cuello , Células Madre Neoplásicas , Adaptación Fisiológica , Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Tampones (Química) , Butionina Sulfoximina/farmacología , Carcinoma/patología , Carcinoma/terapia , Línea Celular Tumoral , Dimetilfumarato , Fumaratos/farmacología , Glutatión/antagonistas & inhibidores , Neoplasias de Cabeza y Cuello/patología , Neoplasias de Cabeza y Cuello/terapia , Humanos , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Neoplasias de Células Escamosas/patología , Neoplasias de Células Escamosas/terapia , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/patología , Células Madre Neoplásicas/efectos de la radiación , Oxidación-Reducción , Carcinoma de Células Escamosas de Cabeza y Cuello , Proteína X Asociada a bcl-2/metabolismoRESUMEN
Dilated Cardiomyopathy (DCM) is one of the leading causes of heart failure with high morbidity and mortality. More than 30 genes have been reported to cause DCM. To provide new insights into the pathophysiology of dilated cardiomyopathy, a mutational screening on 4 DCM-causing genes (MYH7, TNNT2, TNNI3 and LMNA) was performed in a cohort of 105 unrelated DCM (64 familial cases and 41 sporadic cases) using a High Resolution Melting (HRM)/sequencing strategy. Screening of a highly conserved arginine/serine (RS)-rich region in exon 9 of RBM20 was also performed. Nineteen different mutations were identified in 20 index patients (19%), including 10 novels. These included 8 LMNA variants in 9 (8.6%) probands, 5 TNNT2 variants in 5 probands (4.8%), 4 MYH7 variants in 3 probands (3.8%), 1 TNNI3 variant in 1 proband (0.9%), and 1 RBM20 variant in 1 proband (0.9%). One proband was double-heterozygous. LMNA mutations represent the most prevalent genetic DCM cause. Most patients carrying LMNA mutations exhibit conduction system defects and/or cardiac arrhythmias. Our study also showed than prevalence of mutations affecting TNNI3 or the (RS)-rich region of RBM20 is lower than 1%. The discovery of novel DCM mutations is crucial for clinical management of patients and their families because pre-symptomatic diagnosis is possible and precocious intervention could prevent or ameliorate the prognosis.
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Cardiomiopatía Dilatada/diagnóstico , Cardiomiopatía Dilatada/genética , Lamina Tipo A/genética , Mutación , Análisis de Secuencia de ADN/métodos , Adulto , Arritmias Cardíacas/etiología , Cardiomiopatía Dilatada/complicaciones , Cardiomiopatía Dilatada/fisiopatología , Estudios de Cohortes , Análisis Mutacional de ADN , Diagnóstico Precoz , Exones , Femenino , Francia , Pruebas Genéticas , Genotipo , Sistema de Conducción Cardíaco/patología , Heterocigoto , Humanos , Masculino , Persona de Mediana Edad , Linaje , FenotipoRESUMEN
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. A cohort of 34 HCM patients was further blindly screened. To evaluate HRM sensitivity, this cohort was also screened using an optimized DHPLC methodology. RESULTS: All gene variants detected by DHPLC were also readily identified as abnormal by HRM analysis. Mutational screening of a cohort of 34 HCM cases led to identification of 19 mutated alleles. Complete molecular investigation was completed two times faster and cheaper than using DHPLC strategy. CONCLUSIONS: HRM analysis represents an inexpensive, highly sensitive and high-throughput method to allow identification of mutations in the coding sequences of prevalent HCM genes. Identification of more HCM mutations will provide new insights into genotype/phenotype relationships and will allow a better knowledge of the HCM physiopathology.
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Cardiomiopatía Hipertrófica/genética , Congelación , Variación Genética , Cromatografía Líquida de Alta Presión , Humanos , Desnaturalización de Ácido Nucleico , Análisis de Secuencia de ADNRESUMEN
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. Genotyping of 6 gene polymorphisms previously reported as putative HCM modifiers (5 RAAS polymorphisms and TNF-α -308 G/A) was also performed. Seventy-five mutations were identified in 92 index patients (48%); 32 were novel. MYBPC3 mutations (25%) represent the most prevalent cause of inherited HCM whereas MYH7 mutations (12%) rank second in the pathogenesis. The onset age was older in patients carrying MYBPC3 mutations than in those with MYH7 mutations. The MYBPC3 IVS20-2A>G splice mutation was identified in 7% of our HCM population. Multiple gene mutations were identified in 9 probands (5%), highlighting the importance of screening other HCM-causing genes even after a first mutation has been identified, particularly in young patients with a severe phenotype. No single or cumulative genetic modifier effect could be evidenced in this HCM cohort.