Síndrome de Brugada: a Propósito de Cinco Casos

El síndrome de Brugada es causa frecuente de muerte súbita en pacientes sin cardiopatía estructural demostrable. Está caracterizado por aparente bloqueo de rama derecha, elevación del segmento ST en V1-V3 y probabilidad de presentar síncope o paro cardiaco por arritmias ventriculares malignas. Se diagnosticaron cinco pacientes en los últimos 5 años con exclusión de patología estructural cardiaca; cuatro debutaron con una parada cardiaca en fibrilación ventricular y uno con síncope. En un paciente se detectó alternancia de la morfología del ST, complejo a complejo en V1-V2, en la prueba de ajmalina, rechazó implantarse un desfibrilador automático, se indicó amiodarona y propranolol, evolucionó sin arritmias durante 4 años y 9 meses. A tres se les implantó el desfibrilador; uno recibió 12 choques adecuados, 9 de ellos en 72 horas en el mes 18 del implante, debido a un

Síndrome de Brugada: a Propósito de Cinco Casos

El síndrome de Brugada es causa frecuente de muerte súbita en pacientes sin cardiopatía estructural demostrable. Está caracterizado por aparente bloqueo de rama derecha, elevación del segmento ST en V1-V3 y probabilidad de presentar síncope o paro cardiaco por arritmias ventriculares malignas. Se diagnosticaron cinco pacientes en los últimos 5 años con exclusión de patología estructural cardiaca; cuatro debutaron con una parada cardiaca en fibrilación ventricular y uno con síncope. En un paciente se detectó alternancia de la morfología del ST, complejo a complejo en V1-V2, en la prueba de ajmalina, rechazó implantarse un desfibrilador automático, se indicó amiodarona y propranolol, evolucionó sin arritmias durante 4 años y 9 meses. A tres se les implantó el desfibrilador; uno recibió 12 choques adecuados, 9 de ellos en 72 horas en el mes 18 del implante, debido a un

Identification of hereditary breast and ovarian cancer germline variants in Granada (Spain): NGS perspective

Funding for open access charge: Universidad de Granada/CBUA. Maria Molina-Zayas has been a recipient of the 2018 AEFA Post-residency Grant (Spanish Association of Clinical Laboratory) and Dr. Carmen Garrido-Navas holds a postdoctoral fellowship from the Ministry of Economy, Competitiveness, Enterprises and Universities (DOC_01682).The aim of this study was to assess the prevalence of germline variants in cancer-predisposing genes by either targeted (BRCA1/2) or multigene NGS panel in a high-risk Hereditary Breast and Ovarian Cancer (HBOC) cohort. Samples from 824 Caucasian probands were retrospectively collected and the impact of genetic diagnosis and genetic variants epidemiology in this cohort was evaluated. Performance of risk-reducing prophylactic measures, such as prophylactic mastectomy and/or prophylactic oophorectomy, was assessed through clinical follow-up of patients with a positive genetic result. Pathogenic variants predisposing to HBOC were identified in 11.9% (98/824) individuals at BRCA2 (47/98), BRCA1 (24/98), PALB2 (8/51), ATM (7/51), CHEK2 (6/51) MSH6, (2/51), RAD51C (2/51) and TP53 (2/386). Of them, 11 novel pathogenic variants and 12 VUS were identified, characterized, and submitted to ClinVar. Regarding clinical impact, the risk of developing basal or Her2 breast cancer was increased 15.7 times or 37.5 times for BRCA1 and MSH6 pathogenic variants respectively. On the contrary, the risk of developing basal or luminal A breast cancer was reduced to 81% or 77% for BRCA2 and BRCA1 pathogenic variants, respectively. Finally, 53.2% of individuals testing positive for class IV/V variants underwent prophylactic surgery (mastectomy, oophorectomy or both) being significantly younger at the cancer diagnosis than those undertaking prophylactic measures (p = 0.008). Of them, 8 carried a pathogenic/likely pathogenic variant in other genes different from BRCA1 and BRCA2, and the remaining (46.7%) decided to continue with clinical follow-up. No differences in pathogenicity or risk of developing cancer were found for BRCA1/2 between targeted and multigene sequencing strategies; however, NGS was able to resolve a greater proportion of high-risk patients.Universidad de Granada/CBUA2018 AEFA Post-residency Grant (Spanish Association of Clinical Laboratory)Ministry of Economy, Competitiveness, Enterprises and Universities DOC_0168

Arritmias Supraventriculares y Síndrome de Brugada

El síndrome de Brugada está constituido por un patrón electrocardiográfico característico y tendencia a presentar síncopes o paro cardiaco por arritmias ventriculares malignas. La asociación de fibrilación auricular es de un 10%. En los últimos 6 años atendimos 15 pacientes (13 masculinos), portadores de esa entidad; en dos se constataron episodios de fibrilación auricular y otro tenía documentado una taquicardia intranodal. El primero había presentado un síncope, la estimulación eléctrica programada desde ventrículo derecho desencadenó fibrilación ventricular autolimitada, se le implantó un desfibrilador automático; el segundo aquejaba palpitaciones irregulares, de corta duración, se le indujo fibrilación auricular por manipulación de catéteres, la estimulación eléctrica programada no provocó arritmias. Al paciente con taquicardia intranodal se le realizó ablación exitosa de la vía lenta, previa estimulación eléctrica programada desde ventrículo derecho negativa. Ninguno de ellos tenía antecedentes familiares de muerte súbita. En el seguimiento entre 3 y 15 meses, el primer paciente al que se implantó el desfibrilador automático presentó dos descargas eléctricas del mismo por episodios nocturnos de fibrilación ventricular, el segundo paciente presentó otro episodio similar de palpitaciones autolimitadas y el tercero se mantiene asintomático, sin fármacos. La incidencia de arritmias supraventriculares en esta entidad es elevada. Las manifestaciones clínicas, la documentación de la taquicardia y la estimulación eléctrica programada nos permitirán diagnosticarlas y tratarlas adecuadamente

Arritmias Supraventriculares y Síndrome de Brugada

El síndrome de Brugada está constituido por un patrón electrocardiográfico característico y tendencia a presentar síncopes o paro cardiaco por arritmias ventriculares malignas. La asociación de fibrilación auricular es de un 10%. En los últimos 6 años atendimos 15 pacientes (13 masculinos), portadores de esa entidad; en dos se constataron episodios de fibrilación auricular y otro tenía documentado una taquicardia intranodal. El primero había presentado un síncope, la estimulación eléctrica programada desde ventrículo derecho desencadenó fibrilación ventricular autolimitada, se le implantó un desfibrilador automático; el segundo aquejaba palpitaciones irregulares, de corta duración, se le indujo fibrilación auricular por manipulación de catéteres, la estimulación eléctrica programada no provocó arritmias. Al paciente con taquicardia intranodal se le realizó ablación exitosa de la vía lenta, previa estimulación eléctrica programada desde ventrículo derecho negativa. Ninguno de ellos tenía antecedentes familiares de muerte súbita. En el seguimiento entre 3 y 15 meses, el primer paciente al que se implantó el desfibrilador automático presentó dos descargas eléctricas del mismo por episodios nocturnos de fibrilación ventricular, el segundo paciente presentó otro episodio similar de palpitaciones autolimitadas y el tercero se mantiene asintomático, sin fármacos. La incidencia de arritmias supraventriculares en esta entidad es elevada. Las manifestaciones clínicas, la documentación de la taquicardia y la estimulación eléctrica programada nos permitirán diagnosticarlas y tratarlas adecuadamente

Essential Role of Cyclophilin A for Hepatitis C Virus Replication and Virus Production and Possible Link to Polyprotein Cleavage Kinetics

Viruses are obligate intracellular parasites and therefore their replication completely depends on host cell factors. In case of the hepatitis C virus (HCV), a positive-strand RNA virus that in the majority of infections establishes persistence, cyclophilins are considered to play an important role in RNA replication. Subsequent to the observation that cyclosporines, known to sequester cyclophilins by direct binding, profoundly block HCV replication in cultured human hepatoma cells, conflicting results were obtained as to the particular cyclophilin (Cyp) required for viral RNA replication and the underlying possible mode of action. By using a set of cell lines with stable knock-down of CypA or CypB, we demonstrate in the present work that replication of subgenomic HCV replicons of different genotypes is reduced by CypA depletion up to 1,000-fold whereas knock-down of CypB had no effect. Inhibition of replication was rescued by over-expression of wild type CypA, but not by a mutant lacking isomerase activity. Replication of JFH1-derived full length genomes was even more sensitive to CypA depletion as compared to subgenomic replicons and virus production was completely blocked. These results argue that CypA may target an additional viral factor outside of the minimal replicase contributing to RNA amplification and assembly, presumably nonstructural protein 2. By selecting for resistance against the cyclosporine analogue DEBIO-025 that targets CypA in a dose-dependent manner, we identified two mutations (V2440A and V2440L) close to the cleavage site between nonstructural protein 5A and the RNA-dependent RNA polymerase in nonstructural protein 5B that slow down cleavage kinetics at this site and reduce CypA dependence of viral replication. Further amino acid substitutions at the same cleavage site accelerating processing increase CypA dependence. Our results thus identify an unexpected correlation between HCV polyprotein processing and CypA dependence of HCV replication

A Major Determinant of Cyclophilin Dependence and Cyclosporine Susceptibility of Hepatitis C Virus Identified by a Genetic Approach

Since the advent of genome-wide small interfering RNA screening, large numbers of cellular cofactors important for viral infection have been discovered at a rapid pace, but the viral targets and the mechanism of action for many of these cofactors remain undefined. One such cofactor is cyclophilin A (CyPA), upon which hepatitis C virus (HCV) replication critically depends. Here we report a new genetic selection scheme that identified a major viral determinant of HCV's dependence on CyPA and susceptibility to cyclosporine A. We selected mutant viruses that were able to infect CyPA-knockdown cells which were refractory to infection by wild-type HCV produced in cell culture. Five independent selections revealed related mutations in a single dipeptide motif (D316 and Y317) located in a proline-rich region of NS5A domain II, which has been implicated in CyPA binding. Engineering the mutations into wild-type HCV fully recapitulated the CyPA-independent and CsA-resistant phenotype and four putative proline substrates of CyPA were mapped to the vicinity of the DY motif. Circular dichroism analysis of wild-type and mutant NS5A peptides indicated that the D316E/Y317N mutations (DEYN) induced a conformational change at a major CyPA-binding site. Furthermore, nuclear magnetic resonance experiments suggested that NS5A with DEYN mutations adopts a more extended, functional conformation in the putative CyPA substrate site in domain II. Finally, the importance of this major CsA-sensitivity determinant was confirmed in additional genotypes (GT) other than GT 2a. This study describes a new genetic approach to identifying viral targets of cellular cofactors and identifies a major regulator of HCV's susceptibility to CsA and its derivatives that are currently in clinical trials

Production of Infectious Genotype 1b Virus Particles in Cell Culture and Impairment by Replication Enhancing Mutations

With the advent of subgenomic hepatitis C virus (HCV) replicons, studies of the intracellular steps of the viral replication cycle became possible. These RNAs are capable of self-amplification in cultured human hepatoma cells, but save for the genotype 2a isolate JFH-1, efficient replication of these HCV RNAs requires replication enhancing mutations (REMs), previously also called cell culture adaptive mutations. These mutations cluster primarily in the central region of non-structural protein 5A (NS5A), but may also reside in the NS3 helicase domain or at a distinct position in NS4B. Most efficient replication has been achieved by combining REMs residing in NS3 with distinct REMs located in NS4B or NS5A. However, in spite of efficient replication of HCV genomes containing such mutations, they do not support production of infectious virus particles. By using the genotype 1b isolate Con1, in this study we show that REMs interfere with HCV assembly. Strongest impairment of virus formation was found with REMs located in the NS3 helicase (E1202G and T1280I) as well as NS5A (S2204R), whereas a highly adaptive REM in NS4B still allowed virus production although relative levels of core release were also reduced. We also show that cells transfected with the Con1 wild type genome or the genome containing the REM in NS4B release HCV particles that are infectious both in cell culture and in vivo. Our data provide an explanation for the in vitro and in vivo attenuation of cell culture adapted HCV genomes and may open new avenues for the development of fully competent culture systems covering the therapeutically most relevant HCV genotypes