Miguel A. Moreno-Pelayo

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.

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.

An ENU-induced mutation of miR-96 associated with progressive hearing loss in mice

Progressive hearing loss is common in the human population, but little is known about the molecular basis. We report a new N-ethyl-N-nitrosurea (ENU)-induced mouse mutant, diminuendo, with a single base change in the seed region of Mirn96. Heterozygotes show progressive loss of hearing and hair cell anomalies, whereas homozygotes have no cochlear responses. Most microRNAs are believed to downregulate target genes by binding to specific sites on their mRNAs, so mutation of the seed should lead to target gene upregulation. Microarray analysis revealed 96 transcripts with significantly altered expression in homozygotes; notably, Slc26a5, Ocm, Gfi1, Ptprq and Pitpnm1 were downregulated. Hypergeometric P-value analysis showed that hundreds of genes were upregulated in mutants. Different genes, with target sites complementary to the mutant seed, were downregulated. This is the first microRNA found associated with deafness, and diminuendo represents a model for understanding and potentially moderating progressive hair cell degeneration in hearing loss more generally.

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 …

A multicenter study on the prevalence and spectrum of mutations in the otoferlin gene (OTOF) in subjects with nonsyndromic hearing impairment and auditory neuropathy

Autosomal recessive nonsyndromic hearing impairment (NSHI) is a heterogeneous condition, for which 53 genetic loci have been reported, and 29 genes have been identified to date. One of these, OTOF, encodes otoferlin, a membrane‐anchored calcium‐binding protein that plays a role in the exocytosis of synaptic vesicles at the auditory inner hair cell ribbon synapse. We have investigated the prevalence and spectrum of deafness‐causing mutations in the OTOF gene. Cohorts of 708 Spanish, 83 Colombian, and 30 Argentinean unrelated subjects with autosomal recessive NSHI were screened for the common p.Gln829X mutation. In compound heterozygotes, the second mutant allele was identified by DNA sequencing. In total, 23 Spanish, two Colombian and two Argentinean subjects were shown to carry two mutant alleles of OTOF. Of these, one Colombian and 13 Spanish subjects presented with auditory neuropathy. In addition, a cohort of 20 unrelated subjects with a diagnosis of auditory neuropathy, from several countries, was screened for mutations in OTOF by DNA sequencing. A total of 11 of these subjects were shown to carry two mutant alleles of OTOF. In total, 18 pathogenic and four neutral novel alleles of the OTOF gene were identified. Haplotype analysis for markers close to OTOF suggests a common founder for the novel c.2905_2923delinsCTCCGAGCGCA mutation, frequently found in Argentina. Our results confirm that mutation of the OTOF gene correlates with a phenotype of prelingual, profound NSHI, and indicate that OTOF mutations are a major cause of inherited auditory neuropathy. Hum Mutat 29(6), 823–831, 2008.

Q829X, a novel mutation in the gene encoding otoferlin (OTOF), is frequently found in Spanish patients with prelingual non-syndromic hearing loss

Inherited hearing impairment is a highly heterogeneous group of disorders with an overall incidence of about 1 in 2000 newborns.1 Among them, prelingual, severe hearing loss with no other associated clinical feature (non-syndromic) is by far the most frequent.1 It represents a serious handicap for speech acquisition, and therefore early detection is essential for the application of palliative treatment and special education. Hence genetic diagnosis and counselling are being increasingly demanded. Non-syndromic prelingual deafness is mainly inherited as an autosomal recessive trait. To date, 28 different loci for autosomal recessive non-syndromic hearing loss have been reported and 10 genes have been identified.2 Mutations in the gene encoding connexin-26 ( GJB2 , DFNB1 locus) are responsible for up to 50% of all cases of autosomal recessive deafness, with a frequent mutation (35delG) accounting for up to 86% of the GJB2 mutant alleles in several populations.3–10 Other mutations, 235delC and 167delT, account for the majority of GJB2 mutant alleles among the Japanese11 and Ashkenazi Jewish populations,12 respectively. However, little is known about the individual contribution of other genes and their mutations to the remaining uncharacterised cases. Two factors explain this lack of knowledge. First, most of the deafness genes identified so far are large, with many exons and no mutational hotspots, a problem that hampers routine molecular diagnosis. Second, the recent impressive progress in the investigation of genetic deafness has been the result of a research strategy based on the study of large pedigrees with many affected subjects.13 As a consequence, for most of the genes identified so far, genetic linkage has been reported only for a few families, and a small number of mutations have been published.14–23 In contrast, most of the families asking for a genetic diagnosis are small, with only …

High prevalence of the W24X mutation in the gene encoding connexin-26 (GJB2) in spanish romani (gypsies) with autosomal recessive non-syndromic hearing loss

Molecular testing for mutations in the gene encoding connexin‐26 (GJB2) at the DFNB1 locus has become the standard of care for genetic diagnosis and counseling of autosomal recessive non‐syndromic hearing impairment (ARNSHI). The spectrum of mutations in GJB2 varies considerably among the populations, different alleles predominating in different ethnic groups. A cohort of 34 families of Spanish Romani (gypsies) with ARNSHI was screened for mutations in GJB2. We found that DFNB1 deafness accounts for 50% of all ARNSHI in Spanish gypsies. The predominating allele is W24X (79% of the DFNB1 alleles), and 35delG is the second most common allele (17%). An allele‐specific PCR test was developed for the detection of the W24X mutation. By using this test, carrier frequencies were determined in two sample groups of gypsies from different Spanish regions (Andalusia and Catalonia), being 4% and 0%, respectively. Haplotype analysis for microsatellite markers closely flanking the GJB2 gene revealed five different haplotypes associated with the W24X mutation, all sharing the same allele from marker D13S141, suggesting that a founder effect for this mutation is responsible for its high prevalence among Spanish gypsies.

KCNQ4 K⁺ channels tune mechanoreceptors for normal touch sensation in mouse and man

Mutations inactivating the potassium channel KCNQ4 (Kv7.4) lead to deafness in humans and mice. In addition to its expression in mechanosensitive hair cells of the inner ear, KCNQ4 is found in the auditory pathway and in trigeminal nuclei that convey somatosensory information. We have now detected KCNQ4 in the peripheral nerve endings of cutaneous rapidly adapting hair follicle and Meissner corpuscle mechanoreceptors from mice and humans. Electrophysiological recordings from single afferents from Kcnq4−/− mice and mice carrying a KCNQ4 mutation found in DFNA2-type monogenic dominant human hearing loss showed elevated mechanosensitivity and altered frequency response of rapidly adapting, but not of slowly adapting nor of D-hair, mechanoreceptor neurons. Human subjects from independent DFNA2 pedigrees outperformed age-matched control subjects when tested for vibrotactile acuity at low frequencies. This work describes a gene mutation that modulates touch sensitivity in mice and humans and establishes KCNQ4 as a specific molecular marker for rapidly adapting Meissner and a subset of hair follicle afferents.

Molecular and clinical characterisation of three Spanish families with maternally inherited non-syndromic hearing loss caused by the 1494C→T mutation in the mitochondrial 12S rRNA gene

Mutations in the 12S rRNA gene of the mitochondrial genome are responsible for maternally inherited non-syndromic hearing loss (NSHL), and for increased susceptibility to the ototoxicity of aminoglycoside antibiotics. Among these mutations, 1555A→G is the most prevalent in all populations tested so far. Recently, the 1494C→T mutation was reported in two large Chinese pedigrees with maternally inherited NSHL. In this study, sequencing of the 12S rRNA gene in a Spanish family with maternally inherited NSHL showed the presence of the 1494C→T mutation. An additional screening of 1339 unrelated Spanish patients with NSHL allowed the authors to find two other families with the mutation. Audiological data were obtained from 17 confirmed 1494C→T carriers, which showed that the hearing loss was sensorineural, bilateral and symmetrical, with a remarkable variability in age of onset and severity. Three carriers were asymptomatic. Three affected carriers had a history of treatment with aminoglycoside antibiotics. The mitochondrial genome of one affected person from each of these three families was entirely sequenced, and it was established that they belong to different mitochondrial haplogroups (H, U5b, U6a). The study results further support the pathogenic role of 1494C→T on hearing, and show that this mutation can be found in different Caucasian mitochondrial DNA backgrounds.