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Pendred syndrome (PS) is the second most common type of autosomal recessive syndromic hearing loss (HL). It is characterised by sensorineural HL and goiter with occasional hypothyroidism. These features are generally accompanied by malformations of the inner ear, as enlarged vestibular aqueduct (EVA). In about 50% of probands, mutations in the SLC26A4 gene are the cause of the disease. Here we report the case of a Portuguese female, aged 47, presenting with severe to profound HL and hypothyroidism. Her mother and sister, both deceased, had suffered from HL and goiter. By MRI and CT, an enlarged vestibular aqueduct and endolymphatic sac were observed. Molecular study of the patient included screening for GJB2 coding mutations and GJB6 common deletions followed by screening of all SLC26A4 exons, as well as intronic regions 8 and 14. Mutation c.918+2T>C was found for the first time in homozygosity in the intronic region 7 of the SLC26A4 gene. Whilst sequencing the control samples, a novel mutation c.821C>G was found in heterozygosity in the exon 7 of SLC26A4 gene and was predicted to be damaging. This study thus led to the finding of two novel SLC26A4 genotypes and provides new insight on the phenotypic features associated with PS.

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Low-frequency sensorineural hearing loss (LFSNHL) is an unusual type of HL in which frequencies at 2,000 Hz and below are predominantly affected. Most of the families with LFSNHL carry missense mutations in WFS1 gene, coding for wolframin. A Portuguese patient aged 49, reporting HL since her third decade of life, and also referring tinnitus, was shown to display bilateral moderate LFSNHL after audiological evaluation. Molecular analysis led to the identification of a novel mutation, c.511G>A (p.Asp171Asn), found in heterozygosity in the exon 5 of the WFS1 gene, and changing the aspartic acid at position 171 to an asparagine, in the extracellular N-terminus domain of the wolframin protein. This novel mutation wasn't present either in 200 control chromosomes analyzed or in the hearing proband's half-brother, and it had not been reported in 1000 Genomes, Exome Variant Server, HGMD or dbSNP databases. No mutations were found in GJB2 and GJB6 genes. Multi-alignment of 27 wolframin sequences from mammalian species, against the human wolframin sequence in ConSurf, indicated a conservation score corresponding to 7 in a 1-9 color scale where 9 is conserved and 1 is variable. In addition, the mutation p.Asp171Asn was predicted to be damaging and possibly damaging by SIFT and Polyphen-2, respectively. The auditory phenotype of this patient could thus be due to the novel mutation p.Asp171Asn. Further functional characterization might enable to elucidate in which way the change in the residue 171, as other changes introduced by LFSNHL-associated mutations previously described, leads to this type of HL.

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Involvement of GJB2 noncoding regions in hearing loss (HL) has not been extensively investigated. However, three noncoding mutations, c.-259C>T, c.-23G>T, and c.-23+1G>A, were reported. Also, c.-684_-675del, of uncertain pathogenicity, was found upstream of the basal promoter. We performed a detailed analysis of GJB2 noncoding regions in Portuguese HL patients (previously screened for GJB2 coding mutations and the common GJB6 deletions) and in control subjects, by sequencing the basal promoter and flanking upstream region, exon 1, and 3'UTR. All individuals were genotyped for c.-684_-675del and 14 SNPs. Novel variants (c.-731C>T, c.-26G>T, c.*45G>A, and c.*985A>T) were found in controls. A hearing individual homozygous for c.-684_-675del was for the first time identified, supporting the nonpathogenicity of this deletion. Our data indicate linkage disequilibrium (LD) between SNPs rs55704559 (c.*168A>G) and rs5030700 (c.*931C>T) and suggest the association of c.[*168G;*931T] allele with HL. The c.*168A>G change, predicted to alter mRNA folding, might be involved in HL.

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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.

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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.

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Mitochondrial DNA (mtDNA) A7445G point mutation has been shown to be responsible for familial nonepidermolytic palmoplantar keratoderma (NEPPK) associated with deafness without any additional features. To date, only a few cases have been described. We report a Portuguese pedigree presenting an inherited combination of NEPPK and sensorineural deafness compatible with maternal transmission. Clinical expression and age of onset of NEPPK and deafness were variable. Normal expression patterns of epidermal keratins and filaggrin, intercellular junction proteins including connexin 26, loricrin and cornified envelope proteins, were observed. Molecular analysis revealed that all the affected members, previously screened for Cx26 mutations with negative results, presented the mtDNA A7445G point mutation in the homoplasmic form. To our knowledge, this is the fifth family in whom inherited NEPPK and hearing loss are related to this mitochondrial mutation.

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A possible predisposition to aneuploidy in trisomic 21 individuals, their parents, and a control group was evaluated. Peripheral blood lymphocytes from those three groups were used to study the induction of micronuclei (MN) by mitomycin C, cyclophosphamide, and quercetin. Induced MN were further analysed by C-banding and CREST antibody. Trisomic 21 individuals have spontaneous frequencies of MN significantly higher than their parents and the control group. Quercetin without metabolic activation induces MN in trisomic 21 and their parents at a significantly higher level than in control group. The group of the parents of trisomic 21 individuals exhibits higher frequencies of induced MN by mitomycin C and cyclophosphamide than controls. Mitomycin C significantly induced CREST-positive-MN in ten of the sixteen parents evaluated. The results obtained seem to suggest a unique behaviour for the parents of trisomic 21 patients consisting in an increased susceptibility to chromosome loss in the presence of clastogenic genotoxicants, suggesting a higher predisposition to aneuploidy.

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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 Down's 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.

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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.

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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.

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