Search

Hearing loss is highly heterogeneous but one common form involves a failure to maintain the local ionic environment of the sensory hair cells reflected in a reduced endocochlear potential. We used a genetic approach to ask if this type of pathology can be reversed, using theSpns2tm1amouse mutant known to show this defect. By activatingSpns2gene transcription at different ages after the onset of hearing loss we found that an existing auditory impairment can be reversed to give close to normal thresholds for an Auditory Brainstem Response (ABR), at least at low to mid stimulus frequencies. Delaying the activation ofSpns2led to less effective recovery of ABR thresholds suggesting there is a critical period for intervention. Early activation ofSpns2not only led to improvement in auditory function but also to protection of sensory hair cells from secondary degeneration. The genetic approach we have used to establish that this type of hearing loss is in principle reversible could be extended to many other diseases using available mouse resources.Significance statementNeurological diseases are often thought to be irreversible, including hearing loss. In this study we found that one type of hearing loss can be reversed as long as the treatment is delivered within a critical period early in disease progression. This result is a proof-of-concept that hearing loss not only can be avoided but also may be reversed. This genetic approach can be used for a wide range of diseases using existing mouse resources.

Adult-onset progressive hearing loss is a common, complex disease with a strong genetic component. Although to date over 150 genes have been identified as contributing to human hearing loss, many more remain to be discovered, as does most of the underlying genetic diversity. Many different variants have been found to underlie adult-onset hearing loss, but they tend to be rare variants with a high impact upon the gene product. It is likely that combinations of more common, lower impact variants also play a role in the prevalence of the disease.Here we present our exome study of hearing loss in a cohort of 532 older adult volunteers with extensive phenotypic data, including 99 older adults with normal hearing, an important control set. Firstly, we carried out an outlier analysis to identify genes with a high variant load in older adults with hearing loss compared to those with normal hearing. Secondly, we used audiometric threshold data to identify individual variants which appear to contribute to different threshold values. We followed up these analyses in a second cohort. Using these approaches, we identified genes and variants linked to better hearing as well as those linked to worse hearing.These analyses identified some known deafness genes, demonstrating proof of principle of our approach. However, most of the candidate genes are novel associations with hearing loss. While the results support the suggestion that genes responsible for severe deafness may also be involved in milder hearing loss, they also suggest that there are many more genes involved in hearing which remain to be identified. Our candidate gene lists may provide useful starting points for improved diagnosis and drug development.

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in MINAR2 , encoding membrane integral NOTCH2-associated receptor 2, in four families underlying autosomal recessive nonsyndromic deafness. Neurologic evaluation of affected individuals at ages ranging from 4 to 80 y old does not show additional abnormalities. MINAR2 is a recently annotated gene with limited functional understanding. We detected three MINAR2 variants, c.144G > A (p.Trp48*), c.412_419delCGGTTTTG (p.Arg138Valfs*10), and c.393G > T, in 13 individuals with congenital- or prelingual-onset severe-to-profound sensorineural hearing loss (HL). The c.393G > T variant is shown to disrupt a splice donor site. We show that Minar2 is expressed in the mouse inner ear, with the protein localizing mainly in the hair cells, spiral ganglia, the spiral limbus, and the stria vascularis. Mice with loss of function of the Minar2 protein ( Minar2 tm1b/tm1b ) present with rapidly progressive sensorineural HL associated with a reduction in outer hair cell stereocilia in the shortest row and degeneration of hair cells at a later age. We conclude that MINAR2 is essential for hearing in humans and mice and its disruption leads to sensorineural HL. Progressive HL observed in mice and in some affected individuals and as well as relative preservation of hair cells provides an opportunity to interfere with HL using genetic therapies.

Discovery of deafness genes and elucidating their functions have substantially contributed to our understanding of hearing physiology and its pathologies. Here we report on DNA variants in

Mice carrying targeted mutations are important for investigating gene function and the role of genes in disease, but the process of culturing embryonic stem cells during the making of a targeted allele offers opportunities for spontaneous mutations to arise. Identifying spontaneous mutations relies on the detection of phenotypes segregating independently of targeted alleles, and many phenotypes are easy to miss if not specifically looked for. Here we present data from a large, targeted knockout programme in which mice were analysed through a phenotyping pipeline. Twenty-five lines out of 1311 displayed different deafness phenotypes that did not segregate with the targeted allele. We have identified 8 different mutations causing deafness in 16 of these 25 lines and characterised the resulting phenotypes. Our data show that spontaneous mutations with observable effects on phenotype are a common side effect of intensive breeding programmes, including those underlying targeted mutation programmes.

Cell cycle associated protein 1 (Caprin1) is an RNA-binding protein that can regulate the cellular post-transcriptional response to stress. It is a component of both stress granules and neuronal RNA granules and is implicated in neurodegenerative disease, synaptic plasticity and long-term memory formation. Our previous work suggested that Caprin1 also plays a role in the response of the cochlea to stress. Here, targeted inner ear-deletion of Caprin1 in mice leads to an early onset, progressive hearing loss. Auditory brainstem responses from Caprin1-deficient mice show reduced thresholds, with a significant reduction in wave-I amplitudes compared to wildtype. Whilst hair cell structure and numbers were normal, the inner hair cell-spiral ganglion neuron (IHC-SGN) synapse revealed abnormally large post-synaptic GluA2 receptor puncta, a defect consistent with the observed wave-I reduction. Unlike wildtype mice, mild-noise-induced hearing threshold shifts in Caprin1-deficient mice did not recover. Oxidative stress triggered TIA-1/HuR-positive stress granule formation in ex-vivo cochlear explants from Caprin1-deficient mice, showing that stress granules could still be induced. Taken together, these findings suggest that Caprin1 plays a key role in maintenance of auditory function, where it regulates the normal status of the IHC-SGN synapse.

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency hearing impairment in two mutant mouse lines, carrying different mutant alleles of theKlhl18gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Auditory brainstem response (ABR) thresholds (a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window ofKlhl18lowfmutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed thatKlhl18lowfmutant mice had abnormally tapering inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.Klhl18mutant mice exhibit an uncommon low frequency hearing impairment with physiological features consistent with Auditory Neuropathy Spectrum Disorder (ANSD).SIGNIFICANCE STATEMENTWe describe a novel progressive hearing loss inKlhl18mutant mice that affects the lower frequencies of its’ hearing range. Investigation of two mutant alleles of this gene revealed primary inner hair cell defects affecting the neural output of the cochlea while outer hair cell function appeared normal. The tallest stereocilia of inner hair cells showed an abnormal tapering shape, especially notable in the apical half of the cochlear duct corresponding to the low frequency hearing loss. Our finding of a primary inner hair cell defect associated with raised thresholds for auditory brainstem responses combined with normal outer hair cell function suggests that Klhl18 deficiency and inner hair cell pathology may contribute to Auditory Neuropathy Spectrum Disorder in humans.

Norrie disease is caused by mutation of the NDP gene, presenting as congenital blindness followed by later onset of hearing loss. Protecting patients from hearing loss is critical for maintaining their quality of life. This study aimed to understand the onset of pathology in cochlear structure and function. By investigating patients and juvenile Ndp-mutant mice, we elucidated the sequence of onset of physiological changes (in auditory brainstem responses, distortion product otoacoustic emissions, endocochlear potential, blood-labyrinth barrier integrity) and determined the cellular, histological, and ultrastructural events leading to hearing loss. We found that cochlear vascular pathology occurs earlier than previously reported and precedes sensorineural hearing loss. The work defines a disease mechanism whereby early malformation of the cochlear microvasculature precedes loss of vessel integrity and decline of endocochlear potential, leading to hearing loss and hair cell death while sparing spiral ganglion cells. This provides essential information on events defining the optimal therapeutic window and indicates that early intervention is needed. In an era of advancing gene therapy and small-molecule technologies, this study establishes Ndp-mutant mice as a platform to test such interventions and has important implications for understanding the progression of hearing loss in Norrie disease., JCI Insight, 7 (3), ISSN:2379-3708

Age-related hearing loss in humans (presbycusis) typically involves impairment of high frequency sensitivity before becoming progressively more severe at lower frequencies. Pathologies initially affecting lower frequency regions of hearing are less common. Here we describe a progressive, predominantly low-frequency recessive hearing impairment in two mutant mouse lines carrying different mutant alleles of the Klhl18 gene: a spontaneous missense mutation (Klhl18lowf) and a targeted mutation (Klhl18tm1a(KOMP)Wtsi). Both males and females were studied, and the two mutant lines showed similar phenotypes. Threshold for auditory brainstem responses (ABR; a measure of auditory nerve and brainstem neural activity) were normal at 3 weeks old but showed progressive increases from 4 weeks onwards. In contrast, distortion product otoacoustic emission (DPOAE) sensitivity and amplitudes (a reflection of cochlear outer hair cell function) remained normal in mutants. Electrophysiological recordings from the round window of Klhl18lowf mutants at 6 weeks old revealed 1) raised compound action potential thresholds that were similar to ABR thresholds, 2) cochlear microphonic potentials that were normal compared with wildtype and heterozygous control mice and 3) summating potentials that were reduced in amplitude compared to control mice. Scanning electron microscopy showed that Klhl18lowf mutant mice had abnormally tapering of the tips of inner hair cell stereocilia in the apical half of the cochlea while their synapses appeared normal. These results suggest that Klhl18 is necessary to maintain inner hair cell stereocilia and normal inner hair cell function at low frequencies.

Tasmanian devil (tde) mice are deaf and exhibit circling behaviour. Sensory hair cells of mutants show disorganised hair bundles with abnormally thin stereocilia. The origin of this mutation is the insertion of a transgene which disrupts expression of the Grxcr1 (glutaredoxin cysteine rich 1) gene. We report here that Grxcr1 exons and transcript sequences are not affected by the transgene insertion in tde homozygous (tde/tde) mice. Furthermore, 5’RACE PCR experiments showed the presence of two different transcripts of the Grxcr1 gene, expressed in both tde/tde and in wild-type controls. However, quantitative analysis of Grxcr1 transcripts revealed a significantly decreased mRNA level in tde/tde mice. The key stereociliary proteins ESPN, MYO7A, EPS8 and PTPRQ were distributed in hair bundles of homozygous tde mutants in a similar pattern compared with control mice. We found that the abnormal morphology of the stereociliary bundle was associated with a reduction in the size and Ca2+-sensitivity of the mechanoelectrical transducer (MET) current. We propose that GRXCR1 is key for the normal growth of the stereociliary bundle prior to the onset of hearing, and in its absence hair cells are unable to mature into fully functional sensory receptors.

The peripheral deafness gene Mir96 is expressed in both the cochlea and central auditory circuits. To investigate whether it plays a role in the auditory system beyond the cochlea, we characterized homozygous Dmdo/Dmdo mice with a point mutation in miR-96. Anatomical analysis demonstrated a significant decrease in volume of auditory nuclei in Dmdo/Dmdo mice. This decrease resulted from decreased cell size. Non-auditory structures in the brainstem of Dmdo/Dmdo mice or auditory nuclei of the congenital deaf Cldn14-/- mice revealed no such differences. Electrophysiological analysis in the medial nucleus of the trapezoid body (MNTB) showed that principal neurons fired preferentially multiple action potentials upon depolarization, in contrast to the single firing pattern prevalent in controls and Cldn14-/- mice. Immunohistochemistry identified significantly reduced expression of two predicted targets of the mutated miR-96, Kv1.6 and BK channel proteins, possibly contributing to the electrophysiological phenotype. Microscopic analysis of the Dmdo/Dmdo calyx of Held revealed a largely absent compartmentalized morphology, as judged by SV2-labeling. Furthermore, MNTB neurons from Dmdo/Dmdo mice displayed larger synaptic short-term depression, slower AMPA-receptor decay kinetics and a larger NMDA-receptor component, reflecting a less matured stage. Again, these synaptic differences were not present between controls and Cldn14-/- mice. Thus, deafness genes differentially affect the auditory brainstem. Furthermore, our study identifies miR-96 as an essential gene regulatory network element of the auditory system which is required for functional maturation in the peripheral and central auditory system alike.

The microRNA miR-96 is important for hearing, as point mutations in humans and mice result in dominant progressive hearing loss.Mir96is expressed in sensory cells along withMir182andMir183, but the roles of these closely-linked microRNAs are as yet unknown. Here we analyse mice carrying null alleles ofMir182, and ofMir183andMir96together to investigate their roles in hearing. We found thatMir183/96heterozygous mice had normal hearing and homozygotes were completely deaf with abnormal hair cell stereocilia bundles and reduced numbers of inner hair cell synapses at four weeks old.Mir182knockout mice developed normal hearing then exhibited progressive hearing loss. Our transcriptional analyses revealed significant changes in a range of other genes, but surprisingly there were fewer genes with altered expression in the organ of Corti ofMir183/96null mice compared with our previous findings inMir96Dmdomutants, which have a point mutation in the miR-96 seed region. This suggests the more severe phenotype ofMir96Dmdomutants compared withMir183/96mutants, including progressive hearing loss inMir96Dmdoheterozygotes, is likely to be mediated by the gain of novel target genes in addition to the loss of its normal targets. We propose three mechanisms of action of mutant miRNAs; loss of targets that are normally completely repressed, loss of targets whose transcription is normally buffered by the miRNA, and gain of novel targets. Any of these mechanisms could lead to a partial loss of a robust cellular identity and consequent dysfunction.

Highlights • Presbyacusis presents with metabolic, neural, and/or sensory phenotypes. • Reduced endocochlear potential is likely to be a key contributor to presbyacusis. • Genetic factors contribute to presbyacusis, including single-gene variants. • There appear to be downstream effects of presbyacusis on brain function and structure., There are multiple etiologies and phenotypes of age-related hearing loss or presbyacusis. In this review we summarize findings from animal and human studies of presbyacusis, including those that provide the theoretical framework for distinct metabolic, sensory, and neural presbyacusis phenotypes. A key finding in quiet-aged animals is a decline in the endocochlear potential (EP) that results in elevated pure-tone thresholds across frequencies with greater losses at higher frequencies. In contrast, sensory presbyacusis appears to derive, in part, from acute and cumulative effects on hair cells of a lifetime of environmental exposures (e.g., noise), which often result in pronounced high frequency hearing loss. These patterns of hearing loss in animals are recognizable in the human audiogram and can be classified into metabolic and sensory presbyacusis phenotypes, as well as a mixed metabolic+sensory phenotype. However, the audiogram does not fully characterize age-related changes in auditory function. Along with the effects of peripheral auditory system declines on the auditory nerve, primary degeneration in the spiral ganglion also appears to contribute to central auditory system aging. These inner ear alterations often correlate with structural and functional changes throughout the central nervous system and may explain suprathreshold speech communication difficulties in older adults with hearing loss. Throughout this review we highlight potential methods and research directions, with the goal of advancing our understanding, prevention, diagnosis, and treatment of presbyacusis.

The underlying causes of age-related hearing loss (ARHL) are not well understood, but it is clear from heritability estimates that genetics plays a role in addition to environmental factors. Genome-wide association studies (GWAS) in human populations can point to candidate genes that may be involved in ARHL, but follow-up analysis is needed to assess the role of these genes in the disease process. Some genetic variants may contribute a small amount to a disease, while other variants may have a large effect size, but the genetic architecture of ARHL is not yet well-defined. In this study, we asked if a set of 17 candidate genes highlighted by early GWAS reports of ARHL have detectable effects on hearing by knocking down expression levels of each gene in the mouse and analysing auditory function. We found two of the genes have an impact on hearing. Mutation of Dclk1 led to late-onset progressive increase in ABR thresholds and the A430005L14Rik (C1orf174) mutants showed worse recovery from noise-induced damage than controls. We did not detect any abnormal responses in the remaining 15 mutant lines either in thresholds or from our battery of suprathreshold ABR tests, and we discuss the possible reasons for this., Highlights • Dclk1 may play a role in progressive age-related hearing loss. • A430005L14Rik may play a role in the susceptibility of the auditory system to noise-damage. • Fifteen other candidate genes (from GWAS) knocked out or knocked down in mutant mice do not lead to changes in ABRs.

Adult-onset hearing loss is very common, but we know little about the underlying molecular pathogenesis impeding the development of therapies. We took a genetic approach to identify new molecules involved in hearing loss by screening a large cohort of newly generated mouse mutants using a sensitive electrophysiological test, the auditory brainstem response (ABR). We review here the findings from this screen. Thirty-eight unexpected genes associated with raised thresholds were detected from our unbiased sample of 1,211 genes tested, suggesting extreme genetic heterogeneity. A wide range of auditory pathophysiologies was found, and some mutant lines showed normal development followed by deterioration of responses, revealing new molecular pathways involved in progressive hearing loss. Several of the genes were associated with the range of hearing thresholds in the human population and one, SPNS2, was involved in childhood deafness. The new pathways required for maintenance of hearing discovered by this screen present new therapeutic opportunities.

The role of sex in biomedical studies has often been overlooked, despite evidence of sexually dimorphic effects in some biological studies. Here, we used high-throughput phenotype data from 14,250 wildtype and 40,192 mutant mice (representing 2,186 knockout lines), analysed for up to 234 traits, and found a large proportion of mammalian traits both in wildtype and mutants are influenced by sex. This result has implications for interpreting disease phenotypes in animal models and humans., Systemic dissection of sexually dimorphic phenotypes in mice is lacking. Here, Karp and the International Mouse Phenotype Consortium show that approximately 10% of qualitative traits and 56% of quantitative traits in mice as measured in laboratory setting are sexually dimorphic.

In the mammalian inner ear, bicellular and tricellular tight junctions (tTJs) seal the paracellular space between epithelial cells. Tricellulin and immunoglobulin-like (Ig-like) domain containing receptor 1 (ILDR1, also referred to as angulin-2) localize to tTJs of the sensory and non-sensory epithelia in the organ of Corti and vestibular end organs. Recessive mutations of TRIC (DFNB49) encoding tricellulin and ILDR1 (DFNB42) cause human nonsyndromic deafness. However, the pathophysiology of DFNB42 deafness remains unknown. ILDR1 was recently reported to be a lipoprotein receptor mediating the secretion of the fat-stimulated cholecystokinin (CCK) hormone in the small intestine, while ILDR1 in EpH4 mouse mammary epithelial cells in vitro was shown to recruit tricellulin to tTJs. Here we show that two different mouse Ildr1 mutant alleles have early-onset severe deafness associated with a rapid degeneration of cochlear hair cells (HCs) but have a normal endocochlear potential. ILDR1 is not required for recruitment of tricellulin to tTJs in the cochlea in vivo; however, tricellulin becomes mislocalized in the inner ear sensory epithelia of ILDR1 null mice after the first postnatal week. As revealed by freeze-fracture electron microscopy, ILDR1 contributes to the ultrastructure of inner ear tTJs. Taken together, our data provide insight into the pathophysiology of human DFNB42 deafness and demonstrate that ILDR1 is crucial for normal hearing by maintaining the structural and functional integrity of tTJs, which are critical for the survival of auditory neurosensory HCs.

miR-96 is a microRNA, a non-coding RNA gene which regulates a wide array of downstream genes. The miR-96 mouse mutant diminuendo exhibits deafness and arrested hair cell functional and morphological differentiation. We have previously shown that several genes are markedly downregulated in the diminuendo organ of Corti; one of these is Ptprq, a gene known to be important for maturation and maintenance of hair cells. In order to study the contribution that downregulation of Ptprq makes to the diminuendo phenotype, we carried out microarrays, scanning electron microscopy and single hair cell electrophysiology to compare diminuendo mutants (heterozygous and homozygous) with mice homozygous for a functional null allele of Ptprq. In terms of both morphology and electrophysiology, the auditory phenotype of mice lacking Ptprq resembles that of diminuendo heterozygotes, while diminuendo homozygotes are more severely affected. A comparison of transcriptomes indicates there is a broad similarity between diminuendo homozygotes and Ptprq-null mice. The reduction in Ptprq observed in diminuendo mice appears to be a major contributor to the morphological, transcriptional and electrophysiological phenotype, but does not account for the complete diminuendo phenotype.

Recent findings suggest that the manipulation of the EphA/ephrinA system can improve hearing threshold sensitivity in the auditory system (Yates et al., 2014). These results appear to open-up the possibility that pharmacological manipulation of this system could lead to the development of treatments to cure some types of hearing loss. As a first step towards this goal, we have performed a further series of auditory brainstem evoked potential recordings on ephrinA2 homozygous knockout mice and their wildtype littermates in order to replicate the previously reported findings. However, we found that ephrinA2 knockout mice had auditory threshold sensitivity for click and 3–42 kHz tone pip frequencies comparable to that of their wildtype littermates. Evoked potential wave amplitudes, latencies and inter-peak intervals were also comparable between ephrinA2 knockout mice and wild type control littermates. Thus in our experiments we could not replicate the findings of Yates et al. (2014). Whilst the EphA/ephrinA system may therefore play a role in the development of innervation of the cochlea and neural circuitry of the auditory brainstem, there appears to be a functional redundancy between members of this family such that loss of ephrinA2 function alone is insufficient to alter auditory function in the mouse.

Highlights • We studied the expression of Pitpnm1 in the developing mouse inner ear. • We covered several ages between E14.5 and P5, and also looked at adults. • Pitpnm1 is expressed in the inner hair cells from before birth to adulthood. • Pitpnm1 is expressed transiently in the outer hair cells at early postnatal stages. • Mice lacking Pitpnm1 display no obvious auditory defects., Deafness is a genetically complex disorder with many contributing genes still unknown. Here we describe the expression of Pitpnm1 in the inner ear. It is expressed in the inner hair cells of the organ of Corti from late embryonic stages until adulthood, and transiently in the outer hair cells during early postnatal stages. Despite this specific expression, Pitpnm1 null mice showed no hearing defects, possibly due to redundancy with the paralogous genes Pitpnm2 and Pitpnm3.

SummaryMutations in the ATP6V0A4 gene lead to autosomal recessive distal renal tubular acidosis in patients, who often show sensorineural hearing impairment. A first Atp6v0a4 knockout mouse model that recapitulates the loss of H+-ATPase function seen in humans has been generated and recently reported (Norgett et al., 2012). Here, we present the first detailed analysis of the structure and function of the auditory system in Atp6v0a4-/- knockout mice. Measurements of the auditory brainstem response (ABR) showed significantly elevated thresholds in homozygous mutant mice, which indicate severe hearing impairment. Heterozygote thresholds were normal. Analysis of paint-filled inner ears and sections from E16.5 embryos revealed a marked expansion of cochlear and endolymphatic ducts in Atp6v0a4-/- mice. A regulatory link between Atp6v0a4, Foxi1 and Pds has been reported and we found that the endolymphatic sac of Atp6v0a4-/- mice expresses both Foxi1 and Pds, which suggests a downstream position of Atp6v0a4. These mutants also showed a lack of endocochlear potential, suggesting a functional defect of the stria vascularis on the lateral wall of the cochlear duct. However, the main K+ channels involved in the generation of endocochlear potential, Kcnj10 and Kcnq1, are strongly expressed in Atp6v0a4-/- mice. Our results lead to a better understanding of the role of this proton pump in hearing function.

Summary Mutations in whole organisms are powerful ways of interrogating gene function in a realistic context. We describe a program, the Sanger Institute Mouse Genetics Project, that provides a step toward the aim of knocking out all genes and screening each line for a broad range of traits. We found that hitherto unpublished genes were as likely to reveal phenotypes as known genes, suggesting that novel genes represent a rich resource for investigating the molecular basis of disease. We found many unexpected phenotypes detected only because we screened for them, emphasizing the value of screening all mutants for a wide range of traits. Haploinsufficiency and pleiotropy were both surprisingly common. Forty-two percent of genes were essential for viability, and these were less likely to have a paralog and more likely to contribute to a protein complex than other genes. Phenotypic data and more than 900 mutants are openly available for further analysis. PaperClip, Graphical Abstract, Highlights • Large openly available resource of targeted mouse mutants and phenotypic data • Screen for broad range of disease features and traits • Many novel phenotypes suggest functions for both studied and unstudied genes • Haploinsufficiency and pleiotropy are common, More than 900 new mutant mice lines and a multifaceted phenotypic screening platform reveal unanticipated pleiotropies, widespread effects of haploinsufficiency, potential disease models, and functions for unstudied genes.

Progressive hearing loss is very common in the population but we still know little about the underlying pathology. A new spontaneous mouse mutation (stonedeaf, stdf ) leading to recessive, early-onset progressive hearing loss was detected and exome sequencing revealed a Thr289Arg substitution in Sphingosine-1-Phosphate Receptor-2 (S1pr2). Mutants aged 2 weeks had normal hearing sensitivity, but at 4 weeks most showed variable degrees of hearing impairment, which became severe or profound in all mutants by 14 weeks. Endocochlear potential (EP) was normal at 2 weeks old but was reduced by 4 and 8 weeks old in mutants, and the stria vascularis, which generates the EP, showed degenerative changes. Three independent mouse knockout alleles of S1pr2 have been described previously, but this is the first time that a reduced EP has been reported. Genomic markers close to the human S1PR2 gene were significantly associated with auditory thresholds in the 1958 British Birth Cohort (n = 6099), suggesting involvement of S1P signalling in human hearing loss. The finding of early onset loss of EP gives new mechanistic insight into the disease process and suggests that therapies for humans with hearing loss due to S1P signalling defects need to target strial function.

WBP2 encodes the WW domain‐binding protein 2 that acts as a transcriptional coactivator for estrogen receptor α (ESR1) and progesterone receptor (PGR). We reported that the loss of Wbp2 expression leads to progressive high‐frequency hearing loss in mouse, as well as in two deaf children, each carrying two different variants in the WBP2 gene. The earliest abnormality we detect in Wbp2‐deficient mice is a primary defect at inner hair cell afferent synapses. This study defines a new gene involved in the molecular pathway linking hearing impairment to hormonal signalling and provides new therapeutic targets.

Key points The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano‐electrical transducer (MET) channels.The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli.In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles.We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop.We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role. Abstract Mutations in Myo6, the gene encoding the (F‐actin) minus end‐directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano‐electrical transduction. We report that Ca2+‐dependent adaptation of the mechano‐electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca2+‐dependent adaptation., Key points The transduction of sound into electrical signals occurs at the hair bundles atop sensory hair cells in the cochlea, by means of mechanosensitive ion channels, the mechano‐electrical transducer (MET) channels.The MET currents decline during steady stimuli; this is termed adaptation and ensures they always work within the most sensitive part of their operating range, responding best to rapidly changing (sound) stimuli.In this study we used a mouse model (Snell's waltzer) for hereditary deafness in humans that has a mutation in the gene encoding an unconventional myosin, myosin VI, which is present in the hair bundles.We found that in the absence of myosin VI the MET current fails to acquire its characteristic adaptation as the hair bundles develop.We propose that myosin VI supports the acquisition of adaptation by removing key molecules from the hair bundle that serve a temporary, developmental role.

Large-scale N-ethyl-N-nitrosourea (ENU) mutagenesis has provided many rodent models for human disease. Here we describe the initial characterization and mapping of a recessive mutation that leads to degeneration of the incisors, failure of molars to erupt, a grey coat colour, and mild osteopetrosis. We mapped the omi mutation to chromosome 10 between D10Mit214 and D10Mit194. The Ostm1 gene is a likely candidate gene in this region and the grey-lethal allele, Ostm1gl, and omi mutations fail to complement each other. We show that om/om mice have reduced levels of Ostm1 protein. To date we have not been able to identify the causative mutation. We propose that omi is a novel hypomorphic mutation affecting Ostm1 expression, potentially in a regulatory element. Electronic supplementary material The online version of this article (doi:10.1007/s00335-012-9438-7) contains supplementary material, which is available to authorized users.

Objective: To explore the future potential of genetic screening to detect newborns at risk of childhood-onset hearing loss. Design: An expert led discussion of current and future developments in genetic technology and the knowledge base of genetic hearing loss to determine the viability of genetic screening and the implications for screening policy. Results and Discussion: Despite increasing pressure to adopt genetic technologies, a major barrier for genetic screening in hearing loss is the uncertain clinical significance of the identified mutations and their interactions. Only when a reliable estimate of the future risk of hearing loss can be made at a reasonable cost, will genetic screening become viable. Given the speed of technological advancement this may be within the next 10 years. Decision-makers should start to consider how genetic screening could augment current screening programmes as well as the associated data processing and storage requirements. Conclusion: In the interim, we suggest that decision makers consider the benefits of (1) genetically testing all newborns and children with hearing loss, to determine aetiology and to increase knowledge of the genetic causes of hearing loss, and (2) consider screening pregnant women for the m.1555A> G mutation to reduce the risk of aminoglycoside antibiotic-associated hearing loss.

Sphingosine-1-phosphate (S1P) is lipid messenger involved in the regulation of embryonic development, immune system functions, and many other physiological processes. However, the mechanisms of S1P transport across cellular membranes remain poorly understood, with several ATP-binding cassette family members and the spinster 2 (Spns2) member of the major facilitator superfamily known to mediate S1P transport in cell culture. Spns2 was also shown to control S1P activities in zebrafish in vivo and to play a critical role in zebrafish cardiovascular development. However, the in vivo roles of Spns2 in mammals and its involvement in the different S1P-dependent physiological processes have not been investigated. In this study, we characterized Spns2-null mouse line carrying the Spns2tm1a(KOMP)Wtsi allele (Spns2tm1a). The Spns2tm1a/tm1a animals were viable, indicating a divergence in Spns2 function from its zebrafish ortholog. However, the immunological phenotype of the Spns2tm1a/tm1a mice closely mimicked the phenotypes of partial S1P deficiency and impaired S1P-dependent lymphocyte trafficking, with a depletion of lymphocytes in circulation, an increase in mature single-positive T cells in the thymus, and a selective reduction in mature B cells in the spleen and bone marrow. Spns2 activity in the nonhematopoietic cells was critical for normal lymphocyte development and localization. Overall, Spns2tm1a/tm1a resulted in impaired humoral immune responses to immunization. This study thus demonstrated a physiological role for Spns2 in mammalian immune system functions but not in cardiovascular development. Other components of the S1P signaling network are investigated as drug targets for immunosuppressive therapy, but the selective action of Spns2 may present an advantage in this regard.

To determine the molecular cause of sector retinitis pigmentosa and hearing loss in two affected siblings.Direct DNA sequencing of the USH1C gene was performed in two affected siblings. Putative pathogenic sequence changes were assayed in their parent's chromosomes and in control chromosomes. Clinical examination included visual acuity measurement, visual field measurement, electrophysiologic assessment, and fine matrix mapping. Retinal imaging with fundus photography, scanning laser ophthalmoscope (fundus autofluorescence), and optical coherence tomography was performed. Hearing and vestibular function was also assessed.The siblings were aged 42 years and 40 years, and both were compound heterozygotes for the p.R103H missense change and the novel splice site change c.2227-1GA in the USH1C gene. Both alleles were found to be in trans. Neither allele was identified in a panel of 866 control chromosomes, and both were considered pathogenic. Both siblings had sector retinitis pigmentosa restricted to the inferior and nasal retina. Fundus autofluorescence imaging showed a clear demarcation between normal and abnormal areas of retina, which corresponded to areas of reduced sensitivity on fine matrix mapping and loss of visual field. Both siblings had severe hearing loss but were able to develop language.We report a novel molecular cause of sector retinitis pigmentosa associated with hearing loss representing a new phenotype associated with mutations in the USH1C gene.

Measurements of auditory evoked potentials can be used to determine reliably an audiometric representation of hearing sensitivity in mice. In a high-throughput phenotyping screen of mice carrying targeted mutations of single genes, the auditory brainstem response (ABR) is used to gain an estimate of hearing threshold for broadband click stimuli and pure tone frequencies ranging from 6 to 30 kHz. Comparison of thresholds obtained in mutant and wild-type mice give a means to determine mild, moderate, and severe hearing impairment. This gives a clear advantage over using a “clickbox” test to assess hearing by observations of the Preyer reflex. The ABR screen has identified several mutant lines with mild and moderate hearing loss, which appear to demonstrate normal Preyer responses. The ABR technique also allows frequency-selective hearing loss to be identified. Curr. Protoc. Mouse Biol. 1:279-287 © 2011 by John Wiley & Sons, Inc. Keywords: hearing; evoked potential; electrophysiology; high-throughput screening

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30(T5M/T5M) mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca(2+) signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.