Inactivation Of Mutant Research Articles

Functional analysis of novel SCN5A mutations related to Brugada syndrome

Abstract Brugada syndrome (BrS) is an arrhythmogenic disorder that has been linked to mutations in SCN5A, the gene encoding for the pore-forming α-subunit of the cardiac Na+ channel. Recently, novel SCN5A missense mutations (A385T and R504T) were identified in a BrS patient. Since the mutations are in the loop connecting transmembrane segments 5 and 6 in domain 1 (S5-S6 in DI) and segments 6 and 1 between domain 1 and 2 (DI-DII linker), it can lead to dysfunctional property of the Na+ channel. Here we aimed to characterize the electrophysiological properties of A385T and R504T. The wild-type (WT) and mutant SCN5A were transiently transfected in HEK293 cells, and the Na+ channel was analyzed using the whole-cell patch-clamp technique. WT, A385T, R504T, and double mutant (A385T/R504T) showed no significant differences in the current density and the voltage-dependent activation. Unexpectedly, a rightward shift of the voltage-dependent inactivation was identified in the three groups of mutation. Besides, the recovery from inactivation of double mutant was faster than that of WT. These results suggest that, contrary to the expected mechanism of BrS, the mutations cause a gain-of-function of NaV1.5. However, the current densities of R504T and double mutant transfected with β-subunit SCN1B were significantly suppressed but A385T was not different from WT. The voltage dependent activation and inactivation of all mutants were not significantly different from WT. The recovery from inactivation of all mutants were slower than that of WT. These results suggest that R504T mutation of α-subunit SCN5A interacting with β-subunit SCN1B is responsible to pathophysiological function of novel BrS. Funding Acknowledgement Type of funding sources: None.

Abstract 1752: BRCA-mediated tumorigenesis is origin and cell-type dependent

Abstract BRCA1 and BRCA2 mutations predispose to select cancers, yet the interplay between germline and somatic BRCA alterations in driving tumorigenesis and conferring drug sensitivity remain poorly understood. To determine which tumors are dependent on mutant BRCA, we integrated the prospective clinical sequencing of germline blood and matched tumor specimens from 17,152 advanced cancer patients with zygosity analysis, broader somatic molecular features, and treatment outcomes. Tumor lineage dictated BRCA dependence in cancers of both the 2.7% of carriers with germline pathogenic variants and the 1.8% of patients with somatic loss-of-function mutations in BRCA1 and BRCA2 across 38 affected cancer types. The rate of biallelic inactivation of mutant BRCA1/2 varied by mutational origin and tumor lineage. Consequently, BRCA-mediated phenotypes such as homologous recombination deficiency (HRD) were associated with BRCA1/2 mutations in a cell type- and zygosity-dependent manner. Phenotypic penetrance was greatest in tumors of high-risk cancer types and in tumors with biallelic inactivation of mutant BRCA, independent of its germline or somatic origin. Conversely, heterozygous BRCA mutations in other cancer types conferred no HRD phenotype. These lineage-specific differences among hallmarks of BRCA dependence also predicted differential response to PARP-inhibitor therapy. Collectively, only BRCA mutations in tumors of high BRCA penetrance had a strong selective pressure for somatic biallelic inactivation, conferred dose-dependent somatic phenotypic consequences, and PARP inhibitor sensitivity. In contrast, BRCA1/2-mutant patients with cancers not traditionally associated with BRCA susceptibility generally had tumorigenesis independent of mutant BRCA. Overall, mutant BRCA was a founding pathogenic event on which some tumors depended while in others it was likely a dispensable and biologically neutral passenger mutation unrelated to tumorigenesis. This difference was conditioned by lineage, mutational origin, and zygosity, an understanding of which requires integrated germline and somatic molecular characterization in cancer patients with implications for screening, disease pathogenesis, clinical trial design, and therapy. Citation Format: Philip Jonsson, Michael L. Cheng, Chaitanya Bandlamudi, Preethi Srinivasan, Shweta S. Chavan, Noah D. Friedman, Ezra Y. Rosen, Allison L. Richards, Nancy Bouvier, S. Duygu Selcuklu, Craig Bielski, Wassim Abida, Ahmet Zehir, Nikolaus Schultz, Mark T. Donoghue, Jose Baselga, Kenneth Offit, Marc Ladanyi, Eileen M. O’Reilly, Howard I. Scher, Zsofia K. Stadler, Mark E. Robson, David M. Hyman, Michael F. Berger, David B. Solit, Barry S. Taylor. BRCA-mediated tumorigenesis is origin and cell-type dependent [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 1752.

Meropenem-Vaborbactam Resistance Selection, Resistance Prevention, and Molecular Mechanisms in Mutants of KPC-Producing Klebsiella pneumoniae.

ABSTRACTVaborbactam (formerly RPX7009) is a new β-lactamase inhibitor based on a cyclic boronic acid pharmacophore with potent inhibitory activity against Klebsiella pneumoniae carbapenemases (KPC). It has been developed in combination with meropenem. The objective of these studies was to identify the concentrations of both agents associated with the selection or prevention of single-step mutations leading to reduced sensitivity to the combination and to characterize the selected mutations. Eighteen strains of KPC-producing Klebsiella pneumoniae with various degrees of sensitivity to meropenem (MICs, 8 to 512 μg/ml) and meropenem-vaborbactam (MICs, ≤0.06 to 32 μg/ml) and preexisting resistance mechanisms were selected from a worldwide collection of isolates recovered from surveillance studies, emphasizing strains for which MICs were in the upper range of the meropenem-vaborbactam MIC distribution. Meropenem and vaborbactam at 8 μg/ml each suppressed the drug resistance mutation frequency to <1 × 10−8 in 77.8% (14/18) of strains, and all strains were inhibited when the meropenem concentration was increased to 16 μg/ml. Mutants selected at lower drug concentrations showed phenotypes associated with previously described carbapenem resistance mechanisms, including ompK36 inactivation in mutants selected from OmpK36-proficient strains and an increased blaKPC gene copy number in strains with partially functional ompK36. No mutations in the coding region of blaKPC were identified. These data indicate that the selection of mutants with reduced sensitivity to meropenem-vaborbactam from KPC-producing Klebsiella pneumoniae strains is associated with previously described mechanisms involving porin mutations and the increase in the blaKPC gene copy number and not changes in the KPC enzyme and can be prevented by the drug concentrations achieved with optimal dosing of the combination.

Analyses of a novel SCN5A mutation (C1850S): conduction vs. repolarization disorder hypotheses in the Brugada syndrome

Brugada syndrome (BrS) is characterized by arrhythmias leading to sudden cardiac death. BrS is caused, in part, by mutations in the SCN5A gene, which encodes the sodium channel alpha-subunit Na(v)1.5. Here, we aimed to characterize the biophysical properties and consequences of a novel BrS SCN5A mutation. SCN5A was screened for mutations in a male patient with type-1 BrS pattern ECG. Wild-type (WT) and mutant Na(v)1.5 channels were expressed in HEK293 cells. Sodium currents (I(Na)) were analysed using the whole-cell patch-clamp technique at 37 degrees C. The electrophysiological effects of the mutation were simulated using the Luo-Rudy model, into which the transient outward current (I(to)) was incorporated. A new mutation (C1850S) was identified in the Na(v)1.5 C-terminal domain. In HEK293 cells, mutant I(Na) density was decreased by 62% at -20 mV. Inactivation of mutant I(Na) was accelerated in a voltage-dependent manner and the steady-state inactivation curve was shifted by 11.6 mV towards negative potentials. No change was observed regarding activation characteristics. Altogether, these biophysical alterations decreased the availability of I(Na). In the simulations, the I(to) density necessary to precipitate repolarization differed minimally between the two genotypes. In contrast, the mutation greatly affected conduction across a structural heterogeneity and precipitated conduction block. Our data confirm that mutations of the C-terminal domain of Na(v)1.5 alter the inactivation of the channel and support the notion that conduction alterations may play a significant role in the pathogenesis of BrS.

Erratum: Restoration of Inactivation in Mutants of Shaker Potassium Channels by a Peptide Derived from ShB

In the report "Restoration of inactivation in mutants of Shaker potassium channels by a peptide derived from ShB" by W. N. Zagotta et al. (26 Oct., p. 568), in figure 3A the records labeled ""after wash"" were mistakenly duplicated in the control records. The correct control records are shown here. [See figure in the PDF file]

Erratum: Restoration of Inactivation in Mutants of Shaker Potassium Channels by a Peptide Derived from ShB

Erratum: Restoration of Inactivation in Mutants of <i>Shaker</i> Potassium Channels by a Peptide Derived from ShB

Response : Optical Crystallization Work

<i>Response</i> : Optical Crystallization Work

Restoration of Inactivation in Mutants of Shaker Potassium Channels by a Peptide Derived from ShB

Site-directed mutagenesis experiments have suggested a model for the inactivation mechanism of Shaker potassium channels from Drosophila melanogaster. In this model, the first 20 amino acids form a cytoplasmic domain that interacts with the open channel to cause inactivation. The model was tested by the internal application of a synthetic peptide, with the sequence of the first 20 residues of the ShB alternatively spliced variant, to noninactivating mutant channels expressed in Xenopus oocytes. The peptide restored inactivation in a concentration-dependent manner. Like normal inactivation, peptide-induced inactivation was not noticeably voltage-dependent. Trypsin-treated peptide and peptides with sequences derived from the first 20 residues of noninactivating mutants did not restore inactivation. These results support the proposal that inactivation occurs by a cytoplasmic domain that occludes the ion-conducting pore of the channel.

Antibiotic resistant group A streptococci. 3. Penicillin resistance and mitomycin C inactivation of mutants which lose soluble hemolysin production.

ABSTRACTGroup A streptococcus mutants, which lost the ability to produce soluble hemolysins, either β or α hemolytic on the horse blood agar, showed penicillin‐mitomycin C double resistance. Penicillin resistance was high and stable, but mitomycin C resistance was not remarkably high. Mitomycin C resistance was always accompanied with inactivation of the antibacterial activity of the drug, while mitomycin C and penicillin sensitive group A streptococci never showed mitomycin inactivation. The component affecting the mitomycin C was not found in the sterile filtrate of mutant cell cultures, nor could it be extracted by sonic oscillation. The activity of the factor diminished, when living mutant cells were heated at 60 C for 30 min, but activity was not diminished at 56 C. The intensity of activity was a function of the bacterial quantities used. Mutant cells suspended in heart infusion broth, proteose peptone, casamino acid and horse serum were able to render the factor which inactivated mitomycin C, while mutant cells suspended in bovine serum albumin, RNA, or saline did not show the phenomenon.