SCN5A compound heterozygosity mutation in Brugada syndrome: Functional consequences and the implication for pharmacological treatment.

AIMS: SCN5A gene encodes the α-subunit of Nav1.5, mainly found in the human heart. SCN5A variants are the most common genetic alterations associated with Brugada syndrome (BrS). In rare cases, compound heterozygosity is observed; however, its functional consequences are poorly understood. We aimed to analyze the functional impact of de novo Nav1.5 mutations in compound heterozygosity in distinct alleles (G400R and T1461S positions) previously found in a patient with BrS. Moreover, we evaluated the potential benefits of quinidine to improve the phenotype of mutant Na+ channels in vitro. MATERIALS AND METHODS: The functional properties of human wild-type and Nav1.5 variants were evaluated using whole-cell patch-clamp and immunofluorescence techniques in transiently expressed human embryonic kidney (HEK293) cells. KEY FINDINGS: Both variants occur in the highly conservative positions of SCN5A. Although all variants were expressed in the cell membrane, a significant reduction in the Na+ current density (except for G400R alone, which was undetected) was observed along with abnormal biophysical properties, once the variants were expressed in homozygosis and heterozygosis. Interestingly, the incubation of transfected cells with quinidine partially rescued the biophysical properties of the mutant Na+ channel. SIGNIFICANCE: De novo compound heterozygosis mutations in SNC5A disrupt the Na+ macroscopic current. Quinidine could partially reverse the in vitro loss-of-function phenotype of Na+ current. Thus, our data provide, for the first time, a detailed biophysical characterization of dysfunctional Na+ channels linked to compound heterozygosity in BrS as well as the benefits of the pharmacological treatment using quinidine on the biophysical properties of Nav1.5.

In Silico Study of the Resistance to Organophosphorus Pesticides Associated with Point Mutations in Acetylcholinesterase of Lepidoptera: B. mandarina, B. mori, C. auricilius, C. suppressalis, C. pomonella, H. armígera, P. xylostella, S. frugiperda, and S. litura.

An in silico analysis of the interaction between the complex-ligands of nine acetylcholinesterase (AChE) structures of Lepidopteran organisms and 43 organophosphorus (OPs) pesticides with previous resistance reports was carried out. To predict the potential resistance by structural modifications in Lepidoptera insects, due to proposed point mutations in AChE, a broad analysis was performed using computational tools, such as homology modeling and molecular docking. Two relevant findings were revealed: (1) Docking results give a configuration of the most probable spatial orientation of two interacting molecules (AChE enzyme and OP pesticide) and (2) a predicted ΔGb. The mutations evaluated in the form 1 acetylcholinesterase (AChE-1) and form 2 acetylcholinesterase (AChE-2) structures of enzymes do not affect in any way (there is no regularity of change or significant deviations) the values of the binding energy (ΔGb) recorded in the AChE-OPs complexes. However, the mutations analyzed in AChE are associated with a structural modification that causes an inadequate interaction to complete the phosphorylation of the enzyme.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species.

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candida species known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei--A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. krusei demands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

Novel point mutations in the ERG11 gene in clinical isolates of azole resistant Candida species

The azoles are the class of medications most commonly used to fight infections caused by Candida sp. Typically, resistance can be attributed to mutations in ERG11 gene (CYP51) which encodes the cytochrome P450 14α-demethylase, the primary target for the activity of azoles. The objective of this study was to identify mutations in the coding region of theERG11 gene in clinical isolates of Candidaspecies known to be resistant to azoles. We identified three new synonymous mutations in the ERG11 gene in the isolates of Candida glabrata (C108G, C423T and A1581G) and two new nonsynonymous mutations in the isolates of Candida krusei - A497C (Y166S) and G1570A (G524R). The functional consequence of these nonsynonymous mutations was predicted using evolutionary conservation scores. The G524R mutation did not have effect on 14α-demethylase functionality, while the Y166S mutation was found to affect the enzyme. This observation suggests a possible link between the mutation and dose-dependent sensitivity to voriconazole in the clinical isolate of C. krusei. Although the presence of the Y166S in phenotype of reduced azole sensitivity observed in isolate C. kruseidemands investigation, it might contribute to the search of new therapeutic agents against resistant Candida isolates.

mtDNA mutations, hearing loss and aminoglycoside treatment in Mexicans.

UNLABELLED: Streptomycin and aminoglycoside derivatives are commonly used to treat tuberculosis and other stubborn infections; these drugs may alter auditory and/or vestibular function. Mutations in mitochondrial DNA have been associated with hypersensitivity to aminoglycosides; no studies have been conducted in Mexicans, which are very prone to such alterations because aminoglycosides have been prescribed carelessly for many years, irrespective of the ailment to be treated. AIM: We investigated "hot spot" mutations described previously as causing inner ear alterations. METHODS: Hot spot mutations at the 12S rRNA gene and the tRNA Serine (UCN) gene were screened by PCR-RFLP and sequencing in 65 subjects undergoing audiological and vestibular testing. STUDY DESIGN: Experimental. RESULTS: 32 individuals had healthy auditory and vestibular function, whereas 33 subjects had auditory affections. We found none of the previously reported mutations related to aminoglycoside hypersensitivity, or non-syndromic hearing loss. Two hearing-impaired patients that had been treated with streptomycin had the T1189C variant of the mitochondrial 12S rRNA region. CONCLUSION: Mutations related to hearing loss in other ethnic backgrounds were not found in Mexicans. However, the T1189C variant is possibly a putative mutation related to aminoglycoside hypersensitivity and was present in 2 patients.

mtDNA mutations, hearing loss and aminoglycoside treatment in Mexicans / Mutações em DNA mitocondrial, hipoacusia e tratamento de mexicanos com aminoglicosídeos

Streptomycin and aminoglycoside derivatives are commonly used to treat tuberculosis and other stubborn infections; these drugs may alter auditory and/or vestibular function. Mutations in mitochondrial DNA have been associated with hypersensitivity to aminoglycosides; no studies have been conducted in Mexicans, which are very prone to such alterations because aminoglycosides have been prescribed carelessly for many years, irrespective of the ailment to be treated. AIM: We investigated "hot spot" mutations described previously as causing inner ear alterations. METHODS: Hot spot mutations at the 12S rRNA gene and the tRNA Serine (UCN) gene were screened by PCR-RFLP and sequencing in 65 subjects undergoing audiological and vestibular testing. STUDY DESIGN: Experimental. RESULTS: 32 individuals had healthy auditory and vestibular function, whereas 33 subjects had auditory affections. We found none of the previously reported mutations related to aminoglycoside hypersensitivity, or non-syndromic hearing loss. Two hearing-impaired patients that had been treated with streptomycin had the T1189C variant of the mitochondrial 12S rRNA region. CONCLUSION: Mutations related to hearing loss in other ethnic backgrounds were not found in Mexicans. However, the T1189C variant is possibly a putative mutation related to aminoglycoside hypersensitivity and was present in 2 patients.

Derivados de aminoglicosídeos e estreptomicina são comumente utilizados para tratar tuberculose e outras infecções mais resistentes; esses medicamentos podem alterar a função vestibular e/ou auditiva. Mutações no DNA mitocondrial têm sido associadas à hipersensibilidade a aminoglicosídeos; não há estudos conduzidos com mexicanos, que são muito predispostos a tais alterações, uma vez que aminoglicosídeos têm sido exageradamente prescritos há anos, sem associações à doença sendo tratada. OBJETIVO: investigamos mutações "hot spot" previamente descritas como causas de alterações no ouvido interno. MÉTODOS: Mutações hot spot no gene 12S rRNA e gene SerinatRNA (UCN) foram triados pela PCR-RFLP e sequenciados em 65 indivíduos sujeitos a exames audiométricos e vestibulares. Desenho do estudo: Experimental. RESULTADOS: 32 indivíduos com funções auditiva e vestibular normais, e 33 indivíduos com doenças auditivas. Não encontramos nenhuma das mutações previamente relatadas como associadas à hipersensibilidade aos aminoglicosídeos, ou perda auditiva não-sindrômica. Dois pacientes com hipoacusia que haviam sido tratados com estreptomicina tinham a variante T1189C na região 12S rRNA. CONCLUSÃO: Mutações associadas à hipoacusia em outras etnias não foram encontradas em mexicanos. Entretanto, a variante T1189C é possivelmente uma mutação associada à hipersensibilidade a aminoglicosídeos, e esteve presente em dois pacientes.

Genetic toxicology of dental composite resin extracts in somatic cells in vivo.

The aim of this study was to assess the potential genetic toxicity associated to nine aqueous extracts from dental composite resins (Charisma, Fill Magic, Fill Magic Flow, Durafill, TPH Spectrum, Concept, Natural Look, Filtek Z250 and Filtek P60) and one random extract. Homologous mitotic recombination, point and chromosomal mutation effects were determined in somatic proliferative cells of Drosophila melanogaster exposed to aqueous extracts of the clinically used composites. Reproducible increases in clone mutant spot frequencies induced by diluted extract of Fill Magic Flow were observed. These increments were exclusively associated to the induction of homologous recombination - a genetic phenomenon involved in the loss of heterozygosis. The other eight composite resins and the random extract had no statistically significant effect on total spot frequencies - suggesting that they are non-genotoxic in the somatic mutation and recombination test assay, which agrees with the applications they have in dentistry. These findings - supported by numerous studies showing a positive correlation between carcinogenicity in man and genotoxicity in the Drosophila wing spot test - point to the potential risks some composite resins pose to the health of patients and dentistry personnel.

Induced DNA damage by dental resin monomers in somatic cells.

The present in vivo study investigated the genotoxicity of four dental resin monomers: triethyleneglycoldimethacrylate (TEGDMA), hydroxyethylmethacrylate (HEMA), urethanedimethacrylate (UDMA) and bisphenol A-glycidylmethacrylate (BisGMA). The Somatic Mutation and Recombination Test (SMART) in Drosophila melanogaster was applied to analyse their genotoxicity expressed as homologous mitotic recombination, point and chromosomal mutation. SMART detects the loss of heterozygosity of marker genes expressed phenotypically on the fly's wings. This fruit fly has an extensive genetic homology to mammalians, which makes it a suitable model organism for genotoxic investigations. The present findings provide evidence that the mechanistic basis underlying the genotoxicity of UDMA and TEGDMA is related to homologous recombination and gene/chromosomal mutation. A genotoxic pattern can correspondingly be discerned for both UDMA and TEGDMA: their genotoxicity is attributed respectively to 49% and 44% of mitotic recombination, as well as 51% and 56% of mutational events, including point and chromosomal alterations. The monomer UDMA is 1.6 times more active than TEGDMA to induce mutant clones per treatment unit. BisGMA and HEMA had no statistically significant effect on total spot frequencies - suggesting no genotoxic action in the SMART assay. The clinical significance of these observations has to be interpreted for data obtained in other bioassays.

The mutation spectra of chlorinated drinking water samples using the base-specific TA7000 strains of Salmonella in the microsuspension assay.

Mutation spectra analysis can provide important information about the types of genotoxic compounds that can be present in environmental samples. In this study, we used the TA7000 base-specific Salmonella typhimurium tester strains to characterize water samples from two drinking water treatment plants (DWTPs) in São Paulo, Brazil. Because of the small sample sizes of these environmental samples, the use of the microsuspension protocol was necessary. Acidic extracts of drinking water samples from the two DWTPs gave similar responses in the TA7000 strains and caused primarily CG to AT transversions. It is likely that halogenated disinfection by-products, generated during the chlorination of water, are causing the response seen with the TA7000 strains.

Genotoxic effects of benzo[a]pyrene and dibenzo[a,l]pyrene in a human lung cell line.

Several studies have shown that polycyclic aromatic hydrocarbons (PAHs) produce genotoxic effects in assays performed in vivo and in vitro. This study was undertaken to investigate the ability of benzo[a]pyrene (BP) and dibenzo[a,l]pyrene (DBP) to induce DNA damage in a human lung fibroblast cell line (MRC-5), using sister-chromatid exchanges test (SCEs), the comet assay, and evaluating point mutations in codon 12 of the K-ras protooncogene by polymerase chain reaction-single-strand conformation polymorphisms (PCR-SSCPs) and restriction fragment length polymorphisms (RFLP)-enriched PCR methods. Sister-chromatid exchanges frequencies were significantly increased in cells exposed to benzo[a]pyrene and dibenzo[a,l]pyrene in relation to controls (p < .001). Using the standard alkaline comet assay, significant differences between groups were found for the variable comet moment (CM) when cells were exposed to BP (p < .001) and DBP (p < .001). Nevertheless, PCR-SSCP and RFLP-enriched PCR methods did not show any association between treatments with BP and DBP and K-ras point mutations. The data presented in this study indicated that BP and DBP induced both DNA strand breaks and sister-chromatid exchanges but not significant point mutations at codon 12 of K-ras gene in the MRC-5 cell line.