Helena Caria

Genotoxicity of quercetin in the micronucleus assay in mouse bone marrow erythrocytes, human lymphocytes, V79 cell line and identification of kinetochore-containing (CREST staining) micronuclei in human lymphocytes

Quercetin, a mutagenic flavonoid widely distributed in edible plants, was studied for the induction of micronuclei (MN). We have carried out the MN assay in bone marrow polychromatic erythrocytes in mice, in cytokinesis-blocked human lymphocytes and in cytokinesis-blocked V79 cells. MN assay in vitro was performed in the presence and in the absence of S9. To further extend the study, an antikinetochore antibody (CREST staining) was used to distinguish MN containing whole chromosomes (kinetochore positive) from those containing acentric fragments (kinetochore negative). When tested in vivo quercetin failed to induce micronuclei, a result which is in agreement with other published reports. When tested in vitro in V79 cells quercetin clearly induces micronuclei in the absence of S9 and also in the presence of S9 for the highest dose used. When tested in vitro in human lymphocytes quercetin shows a significant induction of micronuclei in the absence and in the presence of S9. The presence of S9 compared to its absence is not significant for any of the systems used. Both in the presence and absence of S9, quercetin appears to behave as a clastogenic agent in human lymphocytes inducing a significant majority of kinetochore-negative MN.

Phenotypic variability of patients homozygous for the GJB2 mutation 35delG cannot be explained by the influence of one major modifier gene

Hereditary hearing loss (HL) is a very heterogeneous trait, with 46 gene identifications for non-syndromic HL. Mutations in GJB2 cause up to half of all cases of severe-to-profound congenital autosomal recessive non-syndromic HL, with 35delG being the most frequent mutation in Caucasians. Although a genotype–phenotype correlation has been established for most GJB2 genotypes, the HL of 35delG homozygous patients is mild to profound. We hypothesise that this phenotypic variability is at least partly caused by the influence of modifier genes. By performing a whole-genome association (WGA) study on 35delG homozygotes, we sought to identify modifier genes. The association study was performed by comparing the genotypes of mild/moderate cases and profound cases. The first analysis included a pooling-based WGA study of a first set of 255 samples by using both the Illumina 550K and Affymetrix 500K chips. This analysis resulted in a ranking of all analysed single-nucleotide polymorphisms (SNPs) according to their P-values. The top 250 most significantly associated SNPs were genotyped individually in the same sample set. All 192 SNPs that still had significant P-values were genotyped in a second independent set of 297 samples for replication. The significant P-values were replicated in nine SNPs, with combined P-values between 3 × 10−3 and 1 × 10−4. This study suggests that the phenotypic variability in 35delG homozygous patients cannot be explained by the effect of one major modifier gene. Significantly associated SNPs may reflect a small modifying effect on the phenotype. Increasing the power of the study will be of greatest importance to confirm these results.

A novel hearing loss-related mutation occurring in the GJB2 basal promoter

Mutations in the GJB2 gene are a major cause of non-syndromic recessive hearing loss in many countries. In a significant fraction of patients, only monoallelic GJB2 mutations known to be either recessive or of unclear pathogenicity are identified. This paper reports a novel GJB2 mutation, −3438C→T, found in the basal promoter of the gene, in trans with V84M, in a patient with profound hearing impairment. This novel mutation can abolish the basal promoter activity of GJB2. These results highlight the importance of extending the mutational screening to regions outside the coding region of GJB2.

A novel M163L mutation in connexin 26 causing cell death and associated with autosomal dominant hearing loss

Mutations in GJB2 gene (encoding connexin 26) are the most common cause of hereditary non-syndromic sensorineural hearing loss (NSSHL) in different populations. The majority of GJB2 mutations are recessive, but a few dominant mutations have been associated with hearing loss either isolated or associated with skin disease. We describe a novel dominant pathogenic GJB2 mutation, identified in a Portuguese family affected with bilateral mild/moderate high-frequency NSSHL. In vitro functional studies demonstrate that the mutant protein (p.M163L) has defective trafficking to the plasma membrane and is associated with increased cell death.

Spectrum and frequency of GJB2 mutations in a cohort of 264 Portuguese nonsyndromic sensorineural hearing loss patients

Abstract Objective: To assess the spectrum and prevalence of mutations in the GJB2 gene in Portuguese nonsyndromic sensorineural hearing loss (NSSHL) patients. Design: Sequencing of the coding region, basal promoter, exon 1, and donor splice site of the GJB2 gene; screening for the presence of the two common GJB6 deletions. Study sample: A cohort of 264 Portuguese NSSHL patients. Results: At least one out of 21 different GJB2 variants was identified in 80 (30.2%) of the 264 patients analysed. Two mutant alleles were found in 53 (20%) of these probands, of which 83% (44/53) harboured at least one c.35delG allele. Twenty-seven (10.2%) of the probands harboured only one mutant allele. Subsequent analysis revealed that the GJB6 deletion del(GJB6-D13S1854) was present in at least 7.4% (2/27) of the patients carrying only one mutant GJB2 allele. Overall, one in five (55/264) of the patients were diagnosed as having DFNB1-related NSSHL, of which the vast majority (53/55) harboured only GJB2 mutations. Conclusions: This study provides clear demonstration that mutations in the GJB2 gene are an important cause of NSSHL in Portugal, thus representing a valuable indicator as regards therapeutical and rehabilitation options, as well as genetic counseling of these patients and their families.

The Controversial p.Arg127His Mutation in GJB2: Report on Three Portuguese Hearing Loss Family Cases

Mutations in the GJB2 gene account for up to 50% of hereditary nonsyndromic hearing loss in several populations. Over 200 mutations are already described in this gene, and three of them, c.35delG, c.167delT, and c.235delC, are the most frequent in Caucasians, Ashkenazi Jews, and Asians, respectively. Most of GJB2 hearing loss-related mutations are recessive, but a few dominant alleles have also been described. Apart from the clearly pathogenic mutations, there are some other variants whose pathogenicity is still controversial, such as p.Met34Thr, p.Val37Ile, p.Arg127His, and p.Val153Ile. The p.Arg127His allele has been found in some mono- and biallelic hearing-impaired patients from several countries. In this article we report on some Portuguese patients harboring this mutation. Taking into consideration the analysis of these Portuguese cases as well as the genetic and functional data regarding p.Arg127His available in the literature, we conclude that this variant may be a cause of hearing loss depending on environmental factors and/or genetic background.

The role of poly(ADP-ribose)polymerase in the induction of sister chromatid exchanges and micronuclei by mitomycin C in Down's syndrome cells as compared to euploid cells

Inhibitors of poly(ADP-ribose)polymerase (PARP; EC 2.4.2.30), such as 3-aminobenzamide (3-AB), can be used to assess the role of the enzyme in the induction of DNA lesions in euploid cells as compared to cells of genetic conditions known to exhibit increased susceptibility to chemical or physical mutagens, such as Downs syndrome (DS) lymphocytes. We report in this work on the effect of PARP inhibition by 3-AB in the induction of sister chromatid exchanges (SCE) and micronuclei (MN) in DS lymphocytes as compared to lymphocytes from normal controls exposed in vitro to a gradient of mitomycin C (MMC). For both types of cells, DS and normal lymphocytes, MMC induces a significant increase in frequencies of SCE and MN in the absence and in the presence of 3-AB. In the presence of 3-AB the yield of SCE and MN induced by MMC was significantly higher in normal lymphocytes as compared to lymphocytes from DS patients. The molecular mechanisms by which 3-AB affects the yield of SCE and MN remains to be fully elucidated; however, it seems clear that DS patients display a different behavior in what concerns poly(ADP-ribosyl)ation as compared to normal individuals.

Preferential sensitivity of acrocentric chromosomes to the aneugenic effect of colchicine

In order to evaluate the predisposition to the aneuploidy-inducing agent colchicine (Col) on lymphocytes from trisomic 21 patients compared with their parents and with a control group of subjects without trisomic children, we performed the micronucleus (MN) assay associated with C-banding, CREST staining, and nucleolar organizing region (NOR)-banding. According to our results Col behaves as an aneugenic agent independently of the population studied for CREST and C-banding. The Col-induced MN exhibited a clear majority (> 80%) of positive NOR-MN, meaning that they contain a NOR region transcriptionally active or inactive. The same data were observed in trisomic 21 individuals, their parents, and the control group, without significant differences between them. These results seem to suggest a preferential effect of the aneugen Col on acrocentric chromosomes in all of the three groups studied.

Pathophysiology, Diagnosis and Treatment of Somatosensory Tinnitus: A Scoping Review

Somatosensory tinnitus is a generally agreed subtype of tinnitus that is associated with activation of the somatosensory, somatomotor, and visual-motor systems. A key characteristic of somatosensory tinnitus is that is modulated by physical contact or movement. Although it seems common, its pathophysiology, assessment and treatment are not well defined. We present a scoping review on the pathophysiology, diagnosis, and treatment of somatosensory tinnitus, and identify priority directions for further research. Methods: Literature searches were conducted in Google Scholar, PubMed, and EMBASE databases. Additional broad hand searches were conducted with the additional terms etiology, diagnose, treatment. Results: Most evidence on the pathophysiology of somatosensory tinnitus suggests that somatic modulations are the result of altered or cross-modal synaptic activity within the dorsal cochlear nucleus or between the auditory nervous system and other sensory subsystems of central nervous system (e.g., visual or tactile). Presentations of somatosensory tinnitus are varied and evidence for the various approaches to treatment promising but limited. Discussion and Conclusions: Despite the apparent prevalence of somatosensory tinnitus its underlying neural processes are still not well understood. Necessary involvement of multidisciplinary teams in its diagnosis and treatment has led to a large heterogeneity of approaches whereby tinnitus improvement is often only a secondary effect. Hence there are no evidence-based clinical guidelines, and patient care is empirical rather than research-evidence-based. Somatic testing should receive further attention considering the breath of evidence on the ability of patients to modulate their tinnitus through manouvers. Specific questions for further research and review are indicated.

Novel splice-site mutation c.1615-2A>G (IVS14-2A>G) in the SLC26A4 gene causing Pendred syndrome in a consanguineous Portuguese family.

Novel Splice-Site Mutation c.1615-2A>G (IVS14-2A>G) in the SLC26A4 Gene Causing Pendred Syndrome in a Consanguineous Portuguese Family Helena Sim~oes-Teixeira, Tiago D. Matos, Marta Canas Marques, Óscar Dias, M ario Andrea, Eduardo Barreiros, Lu ıs Barreiros, Felipe Moreno, Graça Fialho, Helena Caria, and Ignacio del Castillo* Faculty of Science, Centre for Biodiversity, Functional and Integrative Genomics (BioFIG), University of Lisbon, Lisbon, Portugal Unidad de Gen etica Molecular, Hospital Universitario Ram on y Cajal, IRYCIS, Madrid, Spain ORL Service, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, EPE, Lisbon, Portugal Endocrinology Department, Hospital de Santa Maria, Centro Hospitalar Lisboa Norte, EPE, Lisbon, Portugal Centro de Investigaci on Biom edica en Red de Enfermedades Raras (CIBERER), Madrid, Spain Higher School of Health, Polytechnic Institute of Set ubal, Set ubal, Portugal