Felipe Moreno

Mutations in the seed region of human miR-96 are responsible for nonsyndromic progressive hearing loss

MicroRNAs (miRNAs) bind to complementary sites in their target mRNAs to mediate post-transcriptional repression, with the specificity of target recognition being crucially dependent on the miRNA seed region. Impaired miRNA target binding resulting from SNPs within mRNA target sites has been shown to lead to pathologies associated with dysregulated gene expression. However, no pathogenic mutations within the mature sequence of a miRNA have been reported so far. Here we show that point mutations in the seed region of miR-96, a miRNA expressed in hair cells of the inner ear, result in autosomal dominant, progressive hearing loss. This is the first study implicating a miRNA in a mendelian disorder. The identified mutations have a strong impact on miR-96 biogenesis and result in a significant reduction of mRNA targeting. We propose that these mutations alter the regulatory role of miR-96 in maintaining gene expression profiles in hair cells required for their normal function.

GJB2 Mutations and Degree of Hearing Loss: A Multicenter Study

Hearing impairment (HI) affects 1 in 650 newborns, which makes it the most common congenital sensory impairment. Despite extraordinary genetic heterogeneity, mutations in one gene, GJB2, which encodes the connexin 26 protein and is involved in inner ear homeostasis, are found in up to 50% of patients with autosomal recessive nonsyndromic hearing loss. Because of the high frequency of GJB2 mutations, mutation analysis of this gene is widely available as a diagnostic test. In this study, we assessed the association between genotype and degree of hearing loss in persons with HI and biallelic GJB2 mutations. We performed cross-sectional analyses of GJB2 genotype and audiometric data from 1,531 persons, from 16 different countries, with autosomal recessive, mild-to-profound nonsyndromic HI. The median age of all participants was 8 years; 90% of persons were within the age range of 0-26 years. Of the 83 different mutations identified, 47 were classified as nontruncating, and 36 as truncating. A total of 153 different genotypes were found, of which 56 were homozygous truncating (T/T), 30 were homozygous nontruncating (NT/NT), and 67 were compound heterozygous truncating/nontruncating (T/NT). The degree of HI associated with biallelic truncating mutations was significantly more severe than the HI associated with biallelic nontruncating mutations (P<.0001). The HI of 48 different genotypes was less severe than that of 35delG homozygotes. Several common mutations (M34T, V37I, and L90P) were associated with mild-to-moderate HI (median 25-40 dB). Two genotypes--35delG/R143W (median 105 dB) and 35delG/dela(GJB6-D13S1830) (median 108 dB)--had significantly more-severe HI than that of 35delG homozygotes.

A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment

Hearing impairment is a common and highly heterogeneous sensory disorder. Genetic causes are thought to be responsible for more than 60% of the cases in developed countries.1 In the majority of cases, non-syndromic hearing impairment is inherited in an autosomal recessive pattern.2 Thirty eight different loci and 20 genes for autosomal recessive non-syndromic hearing impairment (ARNSHI) have been identified to date.3 In many populations, up to 50% of all cases of ARNSHI are caused by mutations in the DFNB1 locus (MIM 220290) on 13q12.4 This locus contains the GJB2 gene (MIM 121011), encoding connexin-26 (Cx26),5 which belongs to a family of transmembrane proteins with about 20 members in humans. Hexamers of connexins (connexons) are displayed in the plasma membrane. Docking of connexons on the surfaces of two adjacent cells results in the formation of intercellular gap junction channels.6 Several different connexins, including Cx26, have been shown to participate in the complex gap junction networks of the cochlea.7,8 It has been postulated that these networks play a key role in potassium homeostasis, which is essential for the sound transduction mechanism.9 Given the high prevalence of DFNB1 deafness, molecular testing for GJB2 mutations has become the standard of care for the diagnosis of patients with non-syndromic hearing impairment of unknown cause.10 However, the finding of a large number of affected subjects with only one GJB2 mutant allele complicates the molecular diagnosis of DFNB1 deafness. In different studies, these have accounted for 10–50% of deaf subjects with GJB2 mutations.4 It was hypothesised that there could be other mutations in the DFNB1 locus but outside the GJB2 gene. This hypothesis gained support by the finding of a deletion in the DFNB1 locus outside GJB2 but truncating the neighbouring GJB6 gene (MIM 604418), which …

Prevalence and Evolutionary Origins of the del(GJB6-D13S1830) Mutation in the DFNB1 Locus in Hearing-Impaired Subjects: A Multicenter Study

Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%-50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in approximately 50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%-20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.

A genotype-phenotype correlation for GJB2 (connexin 26) deafness

Introduction: Mutations in GJB2 are the most common cause of non-syndromic autosomal recessive hearing impairment, ranging from mild to profound. Mutation analysis of this gene is widely available as a genetic diagnostic test. Objective: To assess a possible genotype-phenotype correlation for GJB2. Design: Retrospective analysis of audiometric data from people with hearing impairment, segregating two GJB2 mutations. Subjects: Two hundred and seventy seven unrelated patients with hearing impairment who were seen at the ENT departments of local and university hospitals from Italy, Belgium, Spain, and the United States, and who harboured bi-allelic GJB2 mutations. Results: We found that 35delG homozygotes have significantly more hearing impairment, compared with 35delG/non-35delG compound heterozygotes. People with two non-35delG mutations have even less hearing impairment. We observed a similar gradient of hearing impairment when we categorised mutations as inactivating (that is, stop mutations or frame shifts) or non-inactivating (that is, missense mutations). We demonstrated that certain mutation combinations (including the combination of 35delG with the missense mutations L90P, V37I, or the splice-site mutation IVS1+1G>A, and the V37I/V37I genotype) are associated with significantly less hearing impairment compared with 35delG homozygous genotypes. Conclusions: This study is the first large systematic analysis indicating that the GJB2 genotype has a major impact on the degree of hearing impairment, and identifying mild genotypes. Furthermore, this study shows that it will be possible to refine this correlation and extend it to additional genotypes. These data will be useful in evaluating habilitation options for people with GJB2 related deafness.

The export of the DNA replication inhibitor microcin B17 provides immunity for the host cell

Microcin B17 (MccB17) is a peptide antibiotic which inhibits DNA replication in Enterobacteriaceae. Microcin‐producing strains are immune to the action of the microcin. Physical and genetic studies showed that immunity is mediated by three genes: mcbE, mcbF and mcbG. We sequenced these genes and identified polypeptide products for mcbF and mcbG. By studying the contribution of each gene to the expression of immunity we found that immunity is determined by two different mechanisms. One of these, encoded by mcbE and mcbF, is also involved in the production of extracellular MccB17. To reconcile these observations we propose that McbE and McbF serve as a ‘pump’ for the export of active MccB17 from the cytoplasm. This model is supported by the predicted properties of the McbE and McbF proteins, which are thought to be, respectively, an integral membrane protein and an ATP‐binding protein with homology to other transport proteins.

The peptide antibiotic microcin B17 induces double-strand cleavage of DNA mediated by E. coli DNA gyrase.

Microcin B17 (MccB17) is a bactericidal peptide antibiotic which inhibits DNA replication. Two Escherichia coli MccB17 resistant mutants were isolated and the mutations were shown to map to 83 min of the genetic map. Cloning of the mutations and Tn5 insertional analysis demonstrated that they were located inside gyrB. The approximate location of the mutations within gyrB was determined by constructing hybrid genes, as a previous step to sequencing. Both mutations were shown to consist of a single AT‐‐‐‐GC transition at position 2251 of the gene, which produces a Trp751‐‐‐‐Arg substitution in the amino acid sequence of the GyrB polypeptide. The inhibitory effect of MccB17 on replicative cell‐free extracts was assayed. In this in vitro system, interaction of MccB17 with a component of the extracts induced double‐strand cleavage of plasmid DNA. In vivo treatment with MccB17 also induced a well‐defined cleavage pattern on chromosomal DNA. These effects were not observed with a MccB17‐resistant, gyrB mutant. Altogether, our results indicate that MccB17 blocks DNA gyrase by trapping an enzyme‐DNA cleavable complex. Thus, the mode of action of this peptide antibiotic resembles that of quinolones and a variety of antitumour drugs currently used in cancer chemotherapy. MccB17 is the first peptide shown to inhibit a type II DNA topoisomerase.

An E. coli promoter induced by the cessation of growth

The production of the bacterial DNA replication inhibitor Microcin B17 is induced as cultures enter stationary phase. Using SI nuclease protection assays we have shown that this induction is the result of increased levels of transcription initiation from a promoter located upstream from mcbA, the structural gene for Microcin B17. Upstream from the start site of transcription there is a rather typical ‐35 region. However, there is no good homology to the consensus –10 region. While most of the cells transcription is shut off as a result of the cessation of growth, transcription from the mcbA promoter continues for several hours in stationary phase. A single‐copy gene fusion between mcbA and lacZ was used to monitor the response of the promoter to different nutritional conditions and in different host backgrounds altered in metabolic regulatory loci. Starvation for nitrogen, phosphate or carbon sources all induced transcription from the promoter. Levels of transcription were reduced in ompR backgrounds, in contrast, mutations in other global regulatory loci, fnr, relA and cya had little or no effect.

Heteroplasmy for the 1555A>G mutation in the mitochondrial 12S rRNA gene in six Spanish families with non-syndromic hearing loss

Hearing impairment is the most prevalent sensory disorder and genetic causes are thought to be responsible for over 60% of the cases in developed countries.1 Inherited hearing impairment is highly heterogeneous from both the clinical and genetic points of view.1,2 It varies in age of onset, severity, and audiological characteristics, and it can be associated or not with other clinical features (syndromic or non-syndromic hearing impairment). Genetic transmission includes autosomal (dominant and recessive), X linked, and maternal inheritance patterns. This unparalleled heterogeneity is well illustrated by the fact that over 70 loci in the nuclear genome have been reported to be involved in non-syndromic hearing impairment, and about 30 genes have been isolated from their critical intervals.3 Furthermore, a number of different mutations in several genes of the mitochondrial genome are responsible for syndromic and non-syndromic forms of hearing loss.4,5 Mutations responsible for maternally inherited non-syndromic hearing loss are so far confined to only two genes in the mitochondrial genome. These include mutations 7510T>C6 and 7511T>C7 in the tRNASer(UCN) gene, and 1095T>C8 and 1555A>G9 in the gene for the 12S rRNA. This last mutation is responsible for a dual phenotype, since it also confers increased susceptibility to the ototoxic action of aminoglycoside antibiotics.9 Most of these mutations have been reported in a small number of families from several countries, with the exception of 1555A>G, which seems to be more frequent than the others,10–13 although its real prevalence remains to be determined in most populations. Remarkably, in Spain it accounts for about 15–20% of all familial cases of non-syndromic hearing loss, irrespective of their mode of inheritance and age of onset14,15 (our unpublished results). In a majority of these patients, the hearing loss is not …

Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus.

Hearing impairment affects about 1 in 1,000 children at birth. Approximately 70 loci implicated in non-syndromic forms of deafness have been reported in humans and 24 causative genes have been identified (see also http://www.uia.ac.be/dnalab/hhh). We report a mouse transcript, isolated by a candidate deafness gene approach, that is expressed almost exclusively in the inner ear. Genomic analysis shows that the human ortholog STRC (so called owing to the name we have given its protein—stereocilin), which is located on chromosome 15q15, contains 29 exons encompassing approximately 19 kb. STRC is tandemly duplicated, with the coding sequence of the second copy interrupted by a stop codon in exon 20. We have identified two frameshift mutations and a large deletion in the copy containing 29 coding exons in two families affected by autosomal recessive non-syndromal sensorineural deafness linked to the DFNB16 locus. Stereocilin is made up of 1,809 amino acids, and contains a putative signal petide and several hydrophobic segments. Using immunohistolabeling, we demonstrate that, in the mouse inner ear, stereocilin is expressed only in the sensory hair cells and is associated with the stereocilia, the stiff microvilli forming the structure for mechanoreception of sound stimulation.