Clarin‐2 is essential for hearing by maintaining stereocilia integrity and function
2019; Springer Nature; Volume: 11; Issue: 9 Linguagem: Inglês
10.15252/emmm.201910288
ISSN1757-4684
AutoresLucy A Dunbar, Pranav Patni, Carlos Aguilar, Philomena Mburu, Laura F. Corns, Helena RR. Wells, Sedigheh Delmaghani, Andrew Parker, Stuart L. Johnson, Debbie Williams, Christopher T. Esapa, Michelle M. Simon, Lauren Chessum, Sherylanne Newton, Joanne Dorning, Prashanthini Jeyarajan, Susan Morse, Andrea Lelli, Gemma Codner, Thibault Peineau, Suhasini R. Gopal, Kumar N. Alagramam, Ronna Hertzano, Didier Dulon, Sara Wells, Frances M. K. Williams, Christine Petit, Sally J. Dawson, Steve D. M. Brown, Walter Marcotti, A. Amraoui, Michael R. Bowl,
Tópico(s)Bat Biology and Ecology Studies
ResumoArticle26 August 2019Open Access Transparent process Clarin-2 is essential for hearing by maintaining stereocilia integrity and function Lucy A Dunbar Lucy A Dunbar Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Pranav Patni Pranav Patni Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France Search for more papers by this author Carlos Aguilar Carlos Aguilar Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Philomena Mburu Philomena Mburu Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Laura Corns Laura Corns Department of Biomedical Science, University of Sheffield, Sheffield, UK Search for more papers by this author Helena RR Wells Helena RR Wells Department of Twin Research & Genetic Epidemiology, King's College London, London, UK Search for more papers by this author Sedigheh Delmaghani Sedigheh Delmaghani Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France Search for more papers by this author Andrew Parker Andrew Parker Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Stuart Johnson Stuart Johnson Department of Biomedical Science, University of Sheffield, Sheffield, UK Search for more papers by this author Debbie Williams Debbie Williams Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Christopher T Esapa Christopher T Esapa Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Michelle M Simon Michelle M Simon Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Lauren Chessum Lauren Chessum Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Sherylanne Newton Sherylanne Newton Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Joanne Dorning Joanne Dorning Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Prashanthini Jeyarajan Prashanthini Jeyarajan Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Susan Morse Susan Morse Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Andrea Lelli Andrea Lelli Génétique et Physiologie de l'Audition, Institut Pasteur, INSERM UMR-S 1120, Collège de France, Sorbonne Universités, Paris, France Search for more papers by this author Gemma F Codner Gemma F Codner Mary Lyon Centre, MRC Harwell Institute, Harwell, UK Search for more papers by this author Thibault Peineau Thibault Peineau Laboratoire de Neurophysiologie de la Synapse Auditive, Université de Bordeaux, Bordeaux, France Search for more papers by this author Suhasini R Gopal Suhasini R Gopal Department of Otolaryngology – Head and Neck Surgery, University Hospitals Cleveland Medical Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Kumar N Alagramam Kumar N Alagramam Department of Otolaryngology – Head and Neck Surgery, University Hospitals Cleveland Medical Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Ronna Hertzano Ronna Hertzano Department of Otorhinolaryngology Head and Neck Surgery, Anatomy and Neurobiology and Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Didier Dulon Didier Dulon Laboratoire de Neurophysiologie de la Synapse Auditive, Université de Bordeaux, Bordeaux, France Search for more papers by this author Sara Wells Sara Wells Mary Lyon Centre, MRC Harwell Institute, Harwell, UK Search for more papers by this author Frances M Williams Frances M Williams Department of Twin Research & Genetic Epidemiology, King's College London, London, UK Search for more papers by this author Christine Petit Christine Petit Génétique et Physiologie de l'Audition, Institut Pasteur, INSERM UMR-S 1120, Collège de France, Sorbonne Universités, Paris, France Search for more papers by this author Sally J Dawson Sally J Dawson UCL Ear Institute, University College London, London, UK Search for more papers by this author Steve DM Brown Steve DM Brown Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Walter Marcotti Walter Marcotti orcid.org/0000-0002-8770-7628 Department of Biomedical Science, University of Sheffield, Sheffield, UK Search for more papers by this author Aziz El-Amraoui Corresponding Author Aziz El-Amraoui [email protected] orcid.org/0000-0003-2692-4984 Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France Search for more papers by this author Michael R Bowl Corresponding Author Michael R Bowl [email protected] orcid.org/0000-0001-7971-445X Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Lucy A Dunbar Lucy A Dunbar Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Pranav Patni Pranav Patni Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France Search for more papers by this author Carlos Aguilar Carlos Aguilar Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Philomena Mburu Philomena Mburu Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Laura Corns Laura Corns Department of Biomedical Science, University of Sheffield, Sheffield, UK Search for more papers by this author Helena RR Wells Helena RR Wells Department of Twin Research & Genetic Epidemiology, King's College London, London, UK Search for more papers by this author Sedigheh Delmaghani Sedigheh Delmaghani Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France Search for more papers by this author Andrew Parker Andrew Parker Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Stuart Johnson Stuart Johnson Department of Biomedical Science, University of Sheffield, Sheffield, UK Search for more papers by this author Debbie Williams Debbie Williams Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Christopher T Esapa Christopher T Esapa Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Michelle M Simon Michelle M Simon Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Lauren Chessum Lauren Chessum Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Sherylanne Newton Sherylanne Newton Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Joanne Dorning Joanne Dorning Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Prashanthini Jeyarajan Prashanthini Jeyarajan Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Susan Morse Susan Morse Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Andrea Lelli Andrea Lelli Génétique et Physiologie de l'Audition, Institut Pasteur, INSERM UMR-S 1120, Collège de France, Sorbonne Universités, Paris, France Search for more papers by this author Gemma F Codner Gemma F Codner Mary Lyon Centre, MRC Harwell Institute, Harwell, UK Search for more papers by this author Thibault Peineau Thibault Peineau Laboratoire de Neurophysiologie de la Synapse Auditive, Université de Bordeaux, Bordeaux, France Search for more papers by this author Suhasini R Gopal Suhasini R Gopal Department of Otolaryngology – Head and Neck Surgery, University Hospitals Cleveland Medical Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Kumar N Alagramam Kumar N Alagramam Department of Otolaryngology – Head and Neck Surgery, University Hospitals Cleveland Medical Center, Case Western Reserve University, Cleveland, OH, USA Search for more papers by this author Ronna Hertzano Ronna Hertzano Department of Otorhinolaryngology Head and Neck Surgery, Anatomy and Neurobiology and Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA Search for more papers by this author Didier Dulon Didier Dulon Laboratoire de Neurophysiologie de la Synapse Auditive, Université de Bordeaux, Bordeaux, France Search for more papers by this author Sara Wells Sara Wells Mary Lyon Centre, MRC Harwell Institute, Harwell, UK Search for more papers by this author Frances M Williams Frances M Williams Department of Twin Research & Genetic Epidemiology, King's College London, London, UK Search for more papers by this author Christine Petit Christine Petit Génétique et Physiologie de l'Audition, Institut Pasteur, INSERM UMR-S 1120, Collège de France, Sorbonne Universités, Paris, France Search for more papers by this author Sally J Dawson Sally J Dawson UCL Ear Institute, University College London, London, UK Search for more papers by this author Steve DM Brown Steve DM Brown Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Walter Marcotti Walter Marcotti orcid.org/0000-0002-8770-7628 Department of Biomedical Science, University of Sheffield, Sheffield, UK Search for more papers by this author Aziz El-Amraoui Corresponding Author Aziz El-Amraoui [email protected] orcid.org/0000-0003-2692-4984 Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France Search for more papers by this author Michael R Bowl Corresponding Author Michael R Bowl [email protected] orcid.org/0000-0001-7971-445X Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK Search for more papers by this author Author Information Lucy A Dunbar1, Pranav Patni2, Carlos Aguilar1, Philomena Mburu1, Laura Corns3, Helena RR Wells4, Sedigheh Delmaghani2, Andrew Parker1, Stuart Johnson3, Debbie Williams1, Christopher T Esapa1, Michelle M Simon1, Lauren Chessum1, Sherylanne Newton1, Joanne Dorning1, Prashanthini Jeyarajan1, Susan Morse1, Andrea Lelli5, Gemma F Codner6, Thibault Peineau7, Suhasini R Gopal8, Kumar N Alagramam8, Ronna Hertzano9, Didier Dulon7, Sara Wells6, Frances M Williams4, Christine Petit5, Sally J Dawson10, Steve DM Brown1, Walter Marcotti3, Aziz El-Amraoui *,2,‡ and Michael R Bowl *,1,‡ 1Mammalian Genetics Unit, MRC Harwell Institute, Harwell, UK 2Déficits Sensoriels Progressifs, Institut Pasteur, INSERM UMR-S 1120, Sorbonne Universités, Paris, France 3Department of Biomedical Science, University of Sheffield, Sheffield, UK 4Department of Twin Research & Genetic Epidemiology, King's College London, London, UK 5Génétique et Physiologie de l'Audition, Institut Pasteur, INSERM UMR-S 1120, Collège de France, Sorbonne Universités, Paris, France 6Mary Lyon Centre, MRC Harwell Institute, Harwell, UK 7Laboratoire de Neurophysiologie de la Synapse Auditive, Université de Bordeaux, Bordeaux, France 8Department of Otolaryngology – Head and Neck Surgery, University Hospitals Cleveland Medical Center, Case Western Reserve University, Cleveland, OH, USA 9Department of Otorhinolaryngology Head and Neck Surgery, Anatomy and Neurobiology and Institute for Genome Sciences, University of Maryland School of Medicine, Baltimore, MD, USA 10UCL Ear Institute, University College London, London, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +33 1 45 68 88 92; E-mail: [email protected] *Corresponding author. Tel: +44 1235 841161; E-mail: [email protected] EMBO Mol Med (2019)11:e10288https://doi.org/10.15252/emmm.201910288 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Hearing relies on mechanically gated ion channels present in the actin-rich stereocilia bundles at the apical surface of cochlear hair cells. Our knowledge of the mechanisms underlying the formation and maintenance of the sound-receptive structure is limited. Utilizing a large-scale forward genetic screen in mice, genome mapping and gene complementation tests, we identified Clrn2 as a new deafness gene. The Clrn2clarinet/clarinet mice (p.Trp4* mutation) exhibit a progressive, early-onset hearing loss, with no overt retinal deficits. Utilizing data from the UK Biobank study, we could show that CLRN2 is involved in human non-syndromic progressive hearing loss. Our in-depth morphological, molecular and functional investigations establish that while it is not required for initial formation of cochlear sensory hair cell stereocilia bundles, clarin-2 is critical for maintaining normal bundle integrity and functioning. In the differentiating hair bundles, lack of clarin-2 leads to loss of mechano-electrical transduction, followed by selective progressive loss of the transducing stereocilia. Together, our findings demonstrate a key role for clarin-2 in mammalian hearing, providing insights into the interplay between mechano-electrical transduction and stereocilia maintenance. Synopsis The study identifies CLRN2 as a new deafness gene required for maintenance of transducing stereocilia in the sensory cochlear hair cells. Lack of clarin-2 leads to an early-onset hearing loss in mice. CLRN2 is associated with human non-syndromic progressive hearing loss. Utilizing an unbiased forward genetic screen in mice, a Clrn2 gene nonsense mutation (p.Trp4*) was identified as the cause of deafness in the clarinet mouse mutant (Clrn2clarinet). Analysis of data from the UK Biobank study identifies CLRN2 as a novel candidate gene for human non-syndromic progressive age-related hearing loss. While clarin-2 is non-essential for stereocilia bundle patterning, it is indispensable for their maintenance, with loss of clarin-2 leading to stereocilia resorption, decreased mechano-electrical transduction, and progressive hearing impairment. Introduction The process of hearing requires the transduction of sound wave-induced mechanical energy into neuronal signals. This process is achieved by the mechanosensitive inner ear hair cells located in the cochlea. These specialized sensory cells, named inner hair cells (IHCs) and outer hair cells (OHCs), have an array of actin-filled stereocilia protruding from their apical surface. Each hair cell stereocilia bundle is arranged as 3–4 rows in a highly ordered "staircase-like" structure, which is essential for function. Each taller stereocilium is connected to a shorter neighbour, in an adjacent row, by an extracellular tip link (Kazmierczak et al, 2007), with the upper end of the tip link extending from the side of a taller-row stereocilium to the tip of a shorter-row stereocilium, where it is tethered to the transduction channel complex. In response to sound-induced fluid movement within the inner ear, hair cell bundles are deflected towards the tallest stereocilia causing tension in the tip links, which opens the mechanically gated transduction channels, allowing the influx of K+ and Ca2+ ions into the hair cell, leading to depolarization and release of neurotransmitter (Corey & Hudspeth, 1983; Schwander et al, 2010). Components of the elusive transduction channel complex include LHFPL tetraspan subfamily member 5 (LHFPL5), transmembrane inner ear (TMIE) and transmembrane channel-like 1 (TMC1) and TMC2 (Kawashima et al, 2011; Kurima et al, 2015; Corns et al, 2016, 2017; Fettiplace, 2016; Beurg et al, 2018). All these proteins are reported to interact with protocadherin-15 (PCDH15), a component of the tip link, anchoring it to the stereocilia membrane (Xiong et al, 2012; Maeda et al, 2014; Zhao et al, 2014). The development and maintenance of the "staircase" stereocilia bundle, and the inter-stereociliary tip links, are therefore critical for auditory transduction and essential for hearing. Currently, our knowledge of the mechanisms underlying stereocilia bundle formation and maintenance is limited, and the precise molecular composition of the transduction channel complex remains elusive. The Clarin (CLRN) proteins belong to a superfamily of small integral proteins with four alpha-helical transmembrane domains, which also includes Tetraspanins, Connexins, Claudins, Occludins and calcium channel gamma subunit-like proteins (Adato et al, 2002; Aarnisalo et al, 2007). In humans, the CLRN family comprises three proteins encoded by the paralogous genes CLRN1, CLRN2 and CLRN3, which contain no known functional domains apart from their four transmembrane domains and a C-terminal class-II PDZ-binding motif (PBM type II) (Fig 1A). In humans, CLRN1 mutations have been found to cause Usher syndrome type 3A (USH3A), which is characterized by post-lingual, progressive hearing loss, variable vestibular dysfunction and onset of retinitis pigmentosa leading to vision loss (Adato et al, 2002; Bonnet & El-Amraoui, 2012). Similarly, Clrn1 knockout (Clrn1−/−) mice are reported to show early-onset profound hearing loss, and consistent with this, these mice exhibit disrupted stereocilia bundles in the early postnatal period (Geng et al, 2009, 2012). However, to date Clrn2 has not been associated with any disease and has never been the focus of a scientific paper. Figure 1. Clrn2 is essential for mammalian hearing A. Identification of the ENU-induced hearing loss pedigree MPC169, subsequently named clarinet. ABR phenotyping of pedigree Muta-Ped-C3PDE-169 at 3 months of age identified 8 mice with elevated hearing thresholds (red triangles) compared to their normal hearing colony mates (n = 61, black triangles). Indeed, all eight affected mice were found to not respond to the highest intensity stimulus (90 dB SPL) at the three frequencies tested, or the click stimulus, and so their thresholds are shown as 95 dB SPL. B–D. The genomic structure of mouse Clrn2 (ENSMUST00000053250), and domains of the encoded tetraspan-like glycoprotein (232 amino acids). Black-filled boxes represent untranslated region of Clrn2. The positions of the transmembrane (TM) domains (blue) and the structure of Clrn2clarinet (C) and Clrn2del629 (D) alleles are indicated. The clarinet mutation, Clrn2clarinet (c.12G > A) (red asterisk), is predicted to lead to a premature stop codon at position 4 (p.Trp4*) (C), whereas the Clrn2del629 allele consists of a CRISPR/Cas9-mediated 629 nucleotide deletion encompassing exon 2, leading to splicing of exon 1 to exon 3, which if translated would produce a protein lacking 2 (TM2 and TM3) of the 4 transmembrane domains (D). E. Averaged ABR thresholds for Clrn2clarinet/del629 compound heterozygotes at P21, showing significantly elevated thresholds compared to Clrn2+/+, Clrn2clarinet/+ and Clrn2del629/+ control colony mates. All five Clrn2clarinet/del629 mice were found to not respond at the highest intensity stimulus (90 dB SPL) for at least one frequency/click stimulus. Data shown are mean ± SD ***P < 0.001, one-way ANOVA (Please see Appendix Table S1 for exact P-values). F. Regional plot of P-values for SNP association with hearing difficulty around the CLRN2 gene locus. The genes within the region are annotated, and the direction of the transcripts is shown by arrows. Colouring is based on linkage disequilibrium (LD) across the region with the most associated SNP, rs35414371, shown in purple. Download figure Download PowerPoint Utilizing an unbiased forward genetic screen, we have identified an ENU-induced Clrn2 mutation as the cause of deafness in the clarinet mouse mutant (Clrn2clarinet). Moreover, we have employed Clrn2clarinet mice and a second CRISPR/Cas9-induced mutant (Clrn2del629) to investigate the requirement of clarin-2 in the auditory, vestibular and visual systems. While clarin-2 appears to have a nonessential role in the retina and vestibular apparatus, its absence leads to an early-onset progressive hearing loss. In addition, we identify that genetic variation at the human CLRN2 locus is highly associated with adult hearing difficulty in the UK Biobank Cohort. Expression of tagged clarin-2 in cochlear cultures shows enrichment of the protein in hair cell stereocilia. We demonstrate that clarin-2 is not required for the initial patterning, or formation, of the "staircase" stereocilia bundle, but instead is essential for the process of maintenance of the stereocilia bundle and mechano-electrical transduction. This study establishes a critical role for the tetraspan protein clarin-2 in the function of the mammalian auditory system. Results Clarin-2, a novel protein essential for mammalian hearing During a recent phenotype-driven ENU-mutagenesis screen undertaken at the MRC Harwell Institute, pedigree MPC169 was identified as containing mice with hearing impairment (Potter et al, 2016). In a G3 cohort of 69 mice, 8 were found to have severely elevated auditory brainstem response (ABR) thresholds at 3 months of age (Fig 1A). A genome scan and subsequent single nucleotide polymorphism (SNP) mapping of affected (deaf) and unaffected (hearing) G3 mice demonstrated linkage to a ~12-Mb region on Chromosome 5 (Fig EV1A). Whole-genome sequencing of an affected mouse identified a homozygous mutation within the critical interval consisting of a non-synonymous G-to-A transition at nucleotide 12 of the Clrn2 gene (ENSMUST00000053250). The Clrn2 mutation, confirmed using Sanger sequencing (Fig EV1B), leads to a tryptophan-to-stop (p.Trp4*) nonsense mutation in the encoded clarin-2 protein, a tetraspan-like glycoprotein with a class-II PDZ-binding motif (Fig 1B and C). We subsequently named this mutant clarinet and backcrossed the Clrn2clarinet allele to C57BL/6J for ten generations. Click here to expand this figure. Figure EV1. Clrn2 is essential for mammalian hearing A. The clarinet mutation mapped to a ˜12 Mb region on Chromosome 5 between SNPs rs6341620 and rs6192958 (Chr5:37101560-49346495, GRCm38), containing 110 genes. B. DNA sequencing identified a nucleotide transition (c.12G > A) in the Clrn2 gene at codon 4, thus altering the wild-type (WT) sequence TGG, encoding a tryptophan (Trp), to the mutant (M) sequence TGA, encoding a premature stop codon (p.Trp4*). Electropherograms derived from a clarinet mutant mouse (Clrn2clarinet/clarinet) and a wild-type colony mate (Clrn2+/+) control showing the sequence surrounding Clrn2 nucleotide 12 (indicated by an arrow). C. A second Clrn2 mutant allele (Clrn2del629) was generated employing CRIPSR/Cas9 genome editing, deleting the second coding exon of the Clrn2 gene. Schematic representations of the genomic structure of the wild-type (Clrn2+) and mutant (Clrn2del629) alleles are shown. Mouse Clrn2 consists of 3 exons, which are all in-frame to each other, spanning 10.4 kb of genomic DNA. Wild-type clarin-2 is a 232 amino acid protein, containing 4 transmembrane (TM) domains (dark grey bars). TM1 is encoded by exon 1, TM2 and part of TM3 are encoded by exon 2, and TM4 is encoded by exon 3. The ATG (translation start) and the TGA (Stop) sites are in exons 1 and 3, respectively, and the 5′ and 3′ untranslated regions are shown as black. RT–PCR of RNA extracted from cochleae of Clrn2+/+, Clrn2+/del629 and Clrn2del629/del629 mice, using oligonucleotide primers designed to exon 1 (forward primer) and exon 3 (reverse primer) of the Clrn2 gene, confirms deletion of exon 2 in the mutant mice and identifies aberrant splicing of exon 1 to exon 3, which are in-frame. As such, the Clrn2del629/del629 transcript has the potential to generate a shorter clarin-2 isoform, but this would be missing two of the four transmembrane domains that define the tetraspan clarin-2 protein. D. Averaged ABR click waveforms for Clrn2+/+, Clrn2clarinet/+, Clrn2del629/+, Clrn2clarinet/del629 and Clrn2clarinet/clarinet mice at P21. Arrows indicate the sound intensity at which the auditory threshold was called. Download figure Download PowerPoint To confirm Clrn2clarinet is the causal mutation underlying the auditory dysfunction observed in clarinet mice, we first used a CRISPR/Cas9 approach to engineer a second Clrn2 mutant mouse model, named Clrn2del629. This allele consists of a 629 nucleotide deletion that encompasses exon 2 (ENSMUSE00000401986) of the Clrn2 gene, which encodes the second, and part of the third, transmembrane domains of clarin-2. As such, while the remaining exons 1 and 3 splice together and are in-frame, any translated protein is predicted to have reduced, or absent, function (Figs 1D and EV1C). Next, we undertook a complementation test crossing together these two Clrn2 mutant lines (Figs 1E and EV1D). ABR measurements, recorded in postnatal day 28 (P28) mice in response to click and tone-burst stimuli, showed that compound heterozygous (Clrn2clarinet/del629) mice display very elevated thresholds (> 80 decibel sound pressure level (dB SPL)) at all frequencies tested: 8, 16 and 32 kHz, whereas Clrn2clarinet/+ and Clrn2del629/+ mice exhibit thresholds comparable with those of wild-type (Clrn2+/+) littermates (< 40 dB SPL) (Figs 1E and EV1D), demonstrating the absence of a heterozygous auditory phenotype. Failure of complementation in Clrn2clarinet/del629 mice confirms the gene Clrn2 is essential for hearing. Utilizing the UK Biobank Cohort (Sudlow et al, 2015), a multi-phenotype study of 500,000 people aged between 40 and 69 years, we also sought whether genetic variation at the CLRN2 locus is related to self-reported human hearing difficulty. The association was performed using a case–control design (n = 250,389) based on answers to questions regarding participants' self-assessed hearing ability and self-reported hearing difficulty in the presence of background noise. An association was tested between all 484 UK Biobank genotyped and imputed SNPs within 100 kb of the CLRN2 gene. Within this region, 36 SNPs were significantly associated with the hearing difficulty phenotype, including a cluster of five highly associated SNPs that lie within or very close to the CLRN2 gene (Fig 1F). Within the 20 most highly associated SNPs, the majority are either intronic or intergenic (Table EV1). The rs35414371 SNP with the highest association has a P-value of 1.60E-11 and lies just 2 kb downstream of the CLRN2 gene. The second most associated SNP, rs13147559 (P = 1.70E-11), is in exon 2 of the CLRN2 gene at coding nucleotide position 337 (c.337, ENST00000511148.2). Presence of the ancestral allele (cytosine, c.337C) encodes for leucine (p.113Leu), whereas presence of the minor allele (guanine, c.337G) encodes for valine (p.113Val). As such, this SNP (c.337C > G) represents a missense variant (p.Leu113Val) within the predicted transmembrane domain 2 of the clarin-2 protein (NP_001073296). In silico studies show that the leucine at position 113 is evolutionarily conserved across species. Furthermore, two prediction tools, PolyPhen-2 and MutationAssessor, suggest that substitution of a valine at this position might be detrimental to clarin-2 function returning scores of "possibly damaging" and "medium", respectively. Together, our findings indicate that clarin-2 is key to hearing in both mice and humans. Clarin-2 is essential for hearing function Clarin-2 displays 56% amino acid similarity with clarin-1, the USH3A protein. Indeed, CLRN1 loss of function has been shown to cause progressive hearing loss, variable vestibular dysfunction and progressive retinitis pigmentosa, which prompted us to seek whether Clrn2 is a candidate Usher gene. RT–PCR analyses from wild-type P30 mice revealed the presence of Clrn2 transcripts in the inner ear (notably in the auditory hair cells) and the eye, but not in brain or muscle (Fig 2A and B). Thus, functional measurements were performed to characterize hearing, vestibular and visual phenotypes in clarinet mice. Figure 2. Clarin-2 is required for hearing function A. RT–PCR analysis in P30 mice showing the presence of Clrn2 transcripts in the inner ear and eye, but not in brain or muscle. β-actin was used as a positive control. B. Clrn2 transcripts could be detected in both inner (IHCs) and outer (OHCs) hair cells of P15 wild-type mice. Otoferlin (Otof) and oncomodulin (Ocm) transcripts were used as positive controls for IHCs and OHCs, respectively. Ocm transcripts were only present in the OHC lysate, demonstrating that the IHC sample had not been contaminated with OHCs. C–F. Auditory phenotyping of clarinet mice at P16 (C), P21 (D), P28 (E) and P42 (F). ABR threshold measurements show that Clrn2clarinet/clarinet mice (red) exhibit a severe-to-profound hearing loss affecting all frequencies tested. At 16 kHz in Clrn2clarinet/clarinet mice, the mean ABR hearing thresholds vary from 55–65 dB SPL at P16, 60–90 dB SPL at P21 and to 80–100 dB SPL at P28 and P42. Age-matched Clrn2+/+ (black) and Clrn2clarinet/+ (grey) controls display thresholds within the expected range (15–45 dB SPL) at all frequencies and timepoints tested. At P16, all eight Clrn2clarinet/clarinet mice exhibited recordable ABR responses for each frequency tested and the click stimulus. For the longitudinal ABR study, at P21 and P28 three of the seven Clrn2clarinet/clarinet mice were found to not respond at the highest intensity stimulus (90 dB SPL) for a
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