Silvia Modamio-Høybjør

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.

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 …

In vivo and in vitro effects of two novel gamma actin (ACTG1) mutations that cause DFNA20/26 hearing impairment

Here we report the functional assessment of two novel deafness-associated gamma-actin mutants, K118N and E241K, in a spectrum of different situations with increasing biological complexity by combining biochemical and cell biological analysis in yeast and mammalian cells. Our in vivo experiments showed that while the K118N had a very mild effect on yeast behaviour, the phenotype caused by the E241K mutation was very severe and characterized by a highly compromised ability to grow on glycerol as a carbon source, an aberrant multi-vacuolar pattern and the deposition of thick F-actin bundles randomly in the cell. The latter feature is consistent with the highly unusual spontaneous tendency of the E241K mutant to form bundles in vitro, although this propensity to bundle was neutralized by tropomyosin and the E241K filament bundles were hypersensitive to severing in the presence of cofilin. In transiently transfected NIH3T3 cells both mutant actins were normally incorporated into cytoskeleton structures, although cytoplasmic aggregates were also observed indicating an element of abnormality caused by the mutations in vivo. Interestingly, gene-gun mediated expression of these mutants in cochlear hair cells results in no gross alteration in cytoskeletal structures or the morphology of stereocilia. Our results provide a more complete picture of the biological consequences of deafness-associated gamma-actin mutants and support the hypothesis that the post-lingual and progressive nature of the DFNA20/26 hearing loss is the result of a progressive deterioration of the hair cell cytoskeleton over time.

DFNA8/12 caused by TECTA mutations is the most identified subtype of nonsyndromic autosomal dominant hearing loss.

The prevalence of DFNA8/DFNA12 (DFNA8/12), a type of autosomal dominant nonsyndromic hearing loss (ADNSHL), is unknown as comprehensive population‐based genetic screening has not been conducted. We therefore completed unbiased screening for TECTA mutations in a Spanish cohort of 372 probands from ADNSHL families. Three additional families (Spanish, Belgian, and English) known to be linked to DFNA8/12 were also included in the screening. In an additional cohort of 835 American ADNSHL families, we preselected 73 probands for TECTA screening based on audiometric data. In aggregate, we identified 23 TECTA mutations in this process. Remarkably, 20 of these mutations are novel, more than doubling the number of reported TECTA ADNSHL mutations from 13 to 33. Mutations lie in all domains of the α‐tectorin protein, including those for the first time identified in the entactin domain, as well as the vWFD1, vWFD2, and vWFD3 repeats, and the D1–D2 and TIL2 connectors. Although the majority are private mutations, four of them—p.Cys1036Tyr, p.Cys1837Gly, p.Thr1866Met, and p.Arg1890Cys—were observed in more than one unrelated family. For two of these mutations founder effects were also confirmed. Our data validate previously observed genotype–phenotype correlations in DFNA8/12 and introduce new correlations. Specifically, mutations in the N‐terminal region of α‐tectorin (entactin domain, vWFD1, and vWFD2) lead to mid‐frequency NSHL, a phenotype previously associated only with mutations in the ZP domain. Collectively, our results indicate that DFNA8/12 hearing loss is a frequent type of ADNSHL. Hum Mutat 32:1–10, 2011.

Differential Biological Role of CD3 Chains Revealed by Human Immunodeficiencies

The biological role in vivo of the homologous CD3γ and δ invariant chains within the human TCR/CD3 complex is a matter of debate, as murine models do not recapitulate human immunodeficiencies. We have characterized, in a Turkish family, two new patients with complete CD3γ deficiency and SCID symptoms and compared them with three CD3γ-deficient individuals belonging to two families from Turkey and Spain. All tested patients shared similar immunological features such as a partial TCR/CD3 expression defect, mild αβ and γδ T lymphocytopenia, poor in vitro proliferative responses to Ags and mitogens at diagnosis, and very low TCR rearrangement excision circles and CD45RA+ αβ T cells. However, intrafamilial and interfamilial clinical variability was observed in patients carrying the same CD3G mutations. Two reached the second or third decade in healthy conditions, whereas the other three showed lethal SCID features with enteropathy early in life. In contrast, all reported human complete CD3δ (or CD3ε) deficiencies are in infants with life-threatening SCID and very severe αβ and γδ T lymphocytopenia. Thus, the peripheral T lymphocyte pool was comparatively well preserved in human CD3γ deficiencies despite poor thymus output or clinical outcome. We propose a CD3δ ≫ CD3γ hierarchy for the relative impact of their absence on the signaling for T cell production in humans.

A novel KCNQ4 pore-region mutation (p.G296S) causes deafness by impairing cell-surface channel expression

Mutations in the potassium channel gene KCNQ4 underlie DFNA2, a subtype of autosomal dominant progressive, high-frequency hearing loss. Based on a phenotype-guided mutational screening we have identified a novel mutation c.886G>A, leading to the p.G296S substitution in the pore region of KCNQ4 channel. The possible impact of this mutation on total KCNQ4 protein expression, relative surface expression and channel function was investigated. When the G296S mutant was expressed in Xenopus oocytes, electrophysiological recordings did not show voltage-activated K+ currents. The p.G296S mutation impaired KCNQ4 channel activity in two manners. It greatly reduced surface expression and, secondarily, abolished channel function. The deficient expression at the cell surface membrane was further confirmed in non-permeabilized NIH-3T3 cells transfected with the mutant KCNQ4 tagged with the hemagglutinin epitope in the extracellular S1–S2 linker. Co-expression of mutant and wild type KCNQ4 in oocytes was performed to mimic the heterozygous condition of the p.G296S mutation in the patients. The results showed that the G296S mutant exerts a strong dominant-negative effect on potassium currents by reducing the wild type KCNQ4 channel expression at the cell surface. This is the first study to identify a trafficking-dependent dominant mechanism for the loss of KCNQ4 channel function in DFNA2.

A mutation in CCDC50, a gene encoding an effector of epidermal growth factor-mediated cell signaling, causes progressive hearing loss

We previously mapped a novel autosomal dominant deafness locus, DFNA44, by studying a family with postlingual, progressive, nonsyndromic hearing loss. We report here on the identification of a mutation in CCDC50 as the cause of hearing loss in the family. CCDC50 encodes Ymer, an effector of epidermal growth factor (EGF)-mediated cell signaling that is ubiquitously expressed in different organs and has been suggested to inhibit down-regulation of the EGF receptor. We have examined its expression pattern in mouse inner ear. Western blotting and cell transfection results indicate that Ymer is a soluble, cytoplasmic protein, and immunostaining shows that Ymer is expressed in a complex spatiotemporal pattern during inner ear development. In adult inner ear, the expression of Ymer is restricted to the pillar cells of the cochlea, the stria vascularis, and the vestibular sensory epithelia, where it shows spatial overlap with the microtubule-based cytoskeleton. In dividing cells, Ymer colocalizes with microtubules of the mitotic apparatus. We suggest that DFNA44 hearing loss may result from a time-dependent disorganization of the microtubule-based cytoskeleton in the pillar cells and stria vascularis of the adult auditory system.

A novel locus for autosomal dominant nonsyndromic hearing loss, DFNA50, maps to chromosome 7q32 between the DFNB17 and DFNB13 deafness loci

Progressive hearing loss is a significant problem in all ageing populations. By the age of 80 years, nearly 50% of individuals have hearing loss that impairs their ability to communicate easily, leading to increasing social isolation.1 Progressive hearing loss in middle and late adulthood is considered multifactorial, with involvement of both genetic and environmental factors.2 In contrast, childhood or adolescent hearing loss is often inherited as an autosomal dominant Mendelian trait, representing about 20% of all cases of hereditary nonsyndromic sensorineural hearing impairment (NSSHI). Postlingual inherited deafness is usually moderate to severe and progressive, and it often affects a particular range of frequencies.3 Hereditary deafness has proved extremely heterogeneous genetically; more than 40 loci have been mapped for autosomal dominant (AD) NSSHI, and 17 deafness genes from these loci have been identified to date.4 Linkage analysis using large pedigrees is a useful tool for mapping and identifying novel deafness genes, a key step for improving our current understanding of auditory function. Here we describe the localisation of a novel DFNA locus on chromosome 7q32, involving the study of a Spanish family with postlingual and progressive hearing loss affecting all frequencies. ### Family data A five generation family (S403) with a history of ADNSSHI was identified through the Hospital Universitario de Valladolid, Spain. The pedigree consisted of 74 members, 37 of whom were affected (fig 1). Appropriate informed consent was obtained from all participants of the study and from parents of subjects younger than 18 years. Clinical evaluation was carried out, blood samples were collected from 55 family members, and DNA was extracted by standard techniques. Environmental factors were excluded as causes of hearing impairment. No syndromic features were present. Tympanometry with acoustic reflex testing indicated proper functioning of the middle ear, and pure tone audiometry was performed to test …

Three deaf mice: mouse models for TECTA-based human hereditary deafness reveal domain-specific structural phenotypes in the tectorial membrane

Tecta is a modular, non-collagenous protein of the tectorial membrane (TM), an extracellular matrix of the cochlea essential for normal hearing. Missense mutations in Tecta cause dominant forms of non-syndromic deafness and a genotype–phenotype correlation has been reported in humans, with mutations in different Tecta domains causing mid- or high-frequency hearing impairments that are either stable or progressive. Three mutant mice were created as models for human Tecta mutations; the TectaL1820F,G1824D/+ mouse for zona pellucida (ZP) domain mutations causing stable mid-frequency hearing loss in a Belgian family, the TectaC1837G/+ mouse for a ZP-domain mutation underlying progressive mid-frequency hearing loss in a Spanish family and the TectaC1619S/+ mouse for a zonadhesin-like (ZA) domain mutation responsible for progressive, high-frequency hearing loss in a French family. Mutations in the ZP and ZA domains generate distinctly different changes in the structure of the TM. Auditory brainstem response thresholds in the 8–40 kHz range are elevated by 30–40 dB in the ZP-domain mutants, whilst those in the ZA-domain mutant are elevated by 20–30 dB. The phenotypes are stable and no evidence has been found for a progressive deterioration in TM structure or auditory function. Despite elevated auditory thresholds, the Tecta mutant mice all exhibit an enhanced tendency to have audiogenic seizures in response to white noise stimuli at low sound pressure levels (≤84 dB SPL), revealing a previously unrecognised consequence of Tecta mutations. These results, together with those from previous studies, establish an allelic series for Tecta unequivocally demonstrating an association between genotype and phenotype.

Characterization of a Spontaneous, Recessive, Missense Mutation Arising in the Tecta Gene

The TECTA gene encodes alpha-tectorin (TECTA), a major noncollagenous component of the tectorial membrane (TM). In humans, mutations in TECTA lead to either dominant (DFNA8/A12) or recessive (DFNB21) forms of nonsyndromic hearing loss. All missense mutations in TECTA that have been reported thus far are associated with the dominant subtype, whereas those leading to recessive deafness are all inactivating mutations. In this paper, we characterize a spontaneous missense mutation (c.1046C > A, p.A349D) arising in the mouse Tecta gene that is, unlike all previously reported missense mutations in TECTA, recessive. The morphological phenotype of the TectaA349D/A349D mouse resembles but is not identical to that previously described for the