Cingulin regulates hair cell cuticular plate morphology and is required for hearing in human and mouse

Abstract Cingulin (CGN) is a cytoskeleton‐associated protein localized at the apical junctions of epithelial cells. CGN interacts with major cytoskeletal filaments and regulates RhoA activity. However, physiological roles of CGN in development and human diseases are currently unknown. Here, we report a multi‐generation family presenting with autosomal dominant non‐syndromic hearing loss (ADNSHL) that co‐segregates with a CGN heterozygous truncating variant, c.3330delG (p.Leu1110Leufs*17). CGN is normally expressed at the apical cell junctions of the organ of Corti, with enriched localization at hair cell cuticular plates and circumferential belts. In mice, the putative disease‐causing mutation results in reduced expression and abnormal subcellular localization of the CGN protein, abolishes its actin polymerization activity, and impairs the normal morphology of hair cell cuticular plates and hair bundles. Hair cell‐specific Cgn knockout leads to high‐frequency hearing loss. Importantly, Cgn mutation knockin mice display noise‐sensitive, progressive hearing loss and outer hair cell degeneration. In summary, we identify CGN c.3330delG as a pathogenic variant for ADNSHL and reveal essential roles of CGN in the maintenance of cochlear hair cell structures and auditory function.


Introduction
Based on a recent report from the World Health Organization, more than 430 million people worldwide suffer from disabling hearing loss (Chadha et al, 2021).Genetic defects, excessive noise exposure, aging, and ototoxic drugs are all associated with hearing loss (Wang & Puel, 2018).Roughly one out of every 1,000 newborns have hearing impairment, and approximately 50% of these cases are associated with genetic factors (Wrobel et al, 2021).Non-syndromic hearing loss (NSHL) is the most common hereditary sensory defect (Kochhar et al, 2007).Eighty percent of genetic NSHL cases are autosomal recessive (ARNSHL), whereas 18% are autosomal dominant (ADNSHL) and the remaining 2% exhibit either X-linked or mitochondrial inheritance.ARNSHL is mainly characterized by congenital prelingual deafness that is generally not progressive, while ADNSHL is usually postlingual and progressive.
Currently, over 170 deafness loci have been mapped and mutations in more than 130 genes have been identified in individuals with hereditary hearing loss (Hereditary hearing loss homepage http://hereditaryhearingloss.org)(Petit et al, 2023).However, identification of ADNSHL deafness genes remains a difficult task due to substantial genetic and phenotypic heterogeneity.Indeed, some of the identified mutations have not been rigorously verified in vitro or in vivo.Therefore, identifying and validating novel mutations in genes associated with ADNSHL, and clarifying their pathogenic mechanisms, would not only help evaluate the efficacy of cochlear implant surgery but also provide tools for personalized treatment of hereditary deafness cases.
Tight junctions (TJs) are specialized intercellular junctions that form a continuous, belt-like seal between adjacent cells in epithelial and endothelial tissues (Sawada et al, 2003).TJs in the inner ear are critical for the maintenance of the unique fluid environment in the cochlea and of the electrochemical gradient that drives mechanotransduction of sound (Gao et al, 2023).Defects in multiple tight junction-related proteins, such as ZO-2 (TJP2), claudin-9 (CLDN9), claudin-14 (CLDN14), and claudin-11 (CLDN11) are associated with hearing loss in humans and mice (Wilcox et al, 2001;Gow et al, 2004;Walsh et al, 2010;Sineni et al, 2019).In addition, mutations in genes encoding the tricellular TJ components, including tricellulin (TRIC) and angulin-2 (ILDR1), also result in human deafness (Riazuddin et al, 2006;Borck et al, 2011), which can be recapitulated in knockout mouse models (Nayak et al, 2013;Morozko et al, 2015).These reports indicate that the integrity of cochlear TJs is pivotal to auditory function.
Cingulin (CGN, OMIM: 609437) is a 140-kDa TJ-related protein localized in the apical circumferential belts of epithelial cells (Citi et al, 2012).CGN was discovered by its copurification with myosin II from intestinal epithelial cells.Studies have shown that CGN can regulate myosin phosphorylation through RhoA and Rac1 (Aijaz et al, 2005), tether the nonmuscle myosin-2 to the junction (Rouaud et al, 2023), regulate the activity of Rho family GTPases, and thereby regulate the formation and membrane tortuosity of epithelial tight junctions (Gonzalez-Mariscal et al, 2014).CGN also interacts with TJ proteins ZO-1 and ZO-2, thereby linking TJs with the actin cytoskeleton (Citi et al, 2000(Citi et al, , 2012)).Interestingly, a recent report demonstrated that inhibition of Cingulin b expression leads to abnormal development of the zebrafish lateral line (Lu et al, 2022).However, the physiological function of CGN in the auditory system is still unclear, and no CGN mutations have been associated with human diseases, including deafness.
Using linkage analysis and exome sequencing, here we report an ADNSHL family in which all affected individuals carry a deletion mutation in exon 20 of the CGN gene (c.3330delG, p.Leu1110Leufs*17).We demonstrate that CGN is preferentially localized at the hair cell cuticular plate and circumferential belt in normal control mice.Studies in vitro and in a knockin mouse line indicate that the p.Leu1110Leufs*17 mutation results in reduced expression and abnormal subcellular localization of CGN.Mice with either CGN conditional knockout or p.Leu1110Leufs*17 knockin display increased sensitivity to noise damage, progressive hearing loss, and hair cell degeneration.Our findings support CGN as a novel deafness gene and indicate that CGN is required for maintenance of the normal apical structure of sensory hair cells in the cochlea and plays a critical role in auditory function.

ADNSHL is diagnosed in 10 individuals from a Chinese pedigree
We ascertained a pedigree of Chinese Han origin, with bilateral, postlingual ADNSHL, without tinnitus or vestibular symptoms.In affected individuals, hearing loss started at middle to high frequencies by 6-7 years of age, and subsequently progressed to all frequencies by the first or second decade (Figs 1A and EV1).For example, during our 6 years of clinical follow-up, the pure tone threshold of the proband (IV:2) was elevated by about 30 dB on average (Fig 1B).Otoscopic examinations revealed a normal external auditory canal and tympanic membranes with normal tympanometry.Auditory steady-state response (ASSR), temporal bone high-resolution computed tomography (HRCT), and magnetic resonance imaging (MRI) for the proband (at 13 years of age) were normal, excluding auditory neuropathy spectrum disorders and inner ear malformations as potential causes of hearing loss.Excessive noise exposure, ototoxic medication history, or complaints of other systemic abnormalities were absent from the clinical reports of the affected individuals.A summary of the auditory phenotype is shown in Table 1.

A CGN variant co-segregates with the ADNSHL
To identify the pathogenic variant, exome sequencing (ES) was carried out on seven affected and five unaffected individuals of the family.Genome-wide linkage analysis of the ES results identified two candidate regions on chromosome 1 and one candidate region on chromosome 6, all with a maximum logarithm of the odds (LOD) score of 2.1 (Appendix Fig S1A and B).Further analysis revealed variants in a total of seven candidate genes (ITGA10, CGN, IL6R, MST1L, SERPINB6, SSR1, and TUBB2B/PSMG4) that co-segregated with the disease phenotype (Appendix Fig S1C).However, except for the CGN variant, all other variants were common singlenucleotide polymorphisms (SNPs) and, therefore, excluded from further analyses.
Sanger sequencing on the seven affected and five unaffected individuals, confirmed that the CGN variant co-segregating with hearing loss was c.3330delG located in exon 20 of the human gene (NM_020770) (Fig 1C and D).The variant resulted in a frameshift and premature truncation of the normal C-terminal tail of the CGN protein at amino acid Leu1110 (p.Leu1110Leufs*17), which is highly conserved among the mammalian species (Fig 1E and F).The variant was predicted to be deleterious by the Mutation Taster, PolyPhen-2, and SIFT computational tools and was not present in the dbSNP, 1000 Genomes, ESP6500, nci60, GnomAD databases or in 10,588 ethically matched control individuals (Cao et al, 2020).This variant also was not detected in 150 additional sporadic cases of postlingual NSHL patients.
We then enrolled in the study a selection of 96 probands of independent familiar cases with hereditary hearing loss compatible with an autosomal dominant inheritance pattern.These samples have been previously subjected to genetic screening using the custom gene panel, OTO-NGS, version 1 and 2, designed in our laboratory (Morin et al, 2020), with no causative deafness mutations being identified.CGN exon screening in this Spanish cohort only revealed benign or likely benign genetic variations, all missense in nature  with some detected in two probands of independent familial cases (Appendix Table S1).These results indicate that the CGN-associated hearing loss is likely one of the rarer forms of genetic deafness, similar to those linked to HOMER2 or CCDC50 genes (Modamio-Hoybjor et al, 2007;Lachgar et al, 2021).

CGN is expressed in the mouse cochlea
To explore potential roles of CGN, we first examined its expression pattern in different mouse tissues, including the cochlea by RT-qPCR analysis.Cgn mRNA is widely expressed in mouse tissues, including cochlea, kidney, and liver, with higher expression levels in lung, gonad, and intestine (Fig 2A We then assessed the auditory function in 2-month-old Cgn-cKO mice by measuring distortion product otoacoustic emissions (DPOAE) which reflect the activity of OHCs, and auditory brainstem responses (ABR), which represent the sound-evoked neural activity in the auditory nerve and brainstem.Compared to wild-type littermates, Cgn-cKO mice had significant elevations in DPOAE and ABR thresholds at 32 kHz (Fig 4A and B).In addition, ABR peak 1 (P1) amplitudes, the summed activity of the cochlear nerve, were also reduced at 8-32 kHz (Fig 4C -I).Histological analyses demonstrated significant loss of OHCs in Cgn-cKO mice at the 32 kHz cochlear region (Fig 4J and K), consistent with observed ABR and DPOAE threshold elevations.These data indicate that Cgn deletion in cochlear hair cells leads to highfrequency OHC loss and hearing impairment.

Cgn delG knockin mice display dose-dependent progressive hearing loss
To determine the pathophysiological roles of the CGN variant (c.3330delG) identified in the affected individuals, we generated      and K).Together, these data show that the reduced expression of CGN in the Cgn delG/delG mice results in hearing impairment and loss of OHCs.However, OHCs of the heterozygous knockin mice were not lost, suggesting that other hair cell pathologies may also contribute to the high-frequency hearing loss in Cgn delG mice.Interestingly, CGN is also highly expressed in the apical junctions of the utricular sensory epithelia of wild-type mice (Fig EV5A and B) but reduced in Cgn delG mice (Fig EV5C).However, balance function behavior assessed by rotarod tests is normal in the mutant mice (Fig EV5D).These results suggest that although CGN is widely expressed in sensory epithelia of cochlea and utricle, it is essential for normal auditory function but dispensable for vestibular function, consistent with a lack of overt vestibular symptoms in human patients.

Cgn delG knockin mice have increased sensitivity to noise exposure
To evaluate whether carrying the Cgn delG mutation alters the sensitivity to environmental insults, 2-month-old mice were exposed to broadband noise at 100 dB SPL for 2 h (Fig 6A).This noise exposure results in temporary shifts of auditory thresholds, which typically recover to baseline after 1 week in wild-type mice (Cgn +/+ ).In contrast, DPOAE thresholds, ABR thresholds, and ABR P1 amplitudes (32 kHz) of the Cgn delG/delG mice exhibited larger impairment after noise exposure (Fig 6B -D).Remarkably, the heterozygous Cgn delG/+ mice also showed significant hearing impairment after noise exposure in all three auditory measures (Fig 6B -D).Consistently, OHCs were lost at high frequency (32 kHz) in Cgn delG/+ and Cgn delG/delG mice after noise exposure (Fig 6E and F).These data indicate that both Cgn delG/+ and Cgn delG/delG mice are more sensitive to moderate noise insults than wild types, and that noise exposure exacerbates overall hearing impairment in the heterozygous Cgn delG mice.
Cgn delG mice show normal synaptic density, tight junction, and microtubule structures Since the reduced ABR P1 amplitudes that we observed in Cgn delG mice are often associated with loss of synapses formed between IHCs and SGN fibers, a.k.a.cochlear synaptopathy (Kohrman et al, 2020), we compared the number of ribbon synapses in wildtype and Cgn mutants (Appendix Fig S4A).The synaptic ribbon density in Cgn delG mice was comparable to that in wildtype mice (Appendix Fig S4B), indicating that the hearing impairment of the Cgn delG mice may not be due to synaptic loss.
Cingulin is specifically localized at the apical junctions of cells (Citi et al, 2012) (Fig 3E) and the circumferential belts in the organ of Corti (Fig 2F and G).It has been suggested that CGN might stabilize tight junction structures by regulating the activity of Rho family GTPases (Gonzalez-Mariscal et al, 2014).Therefore, we speculated that hearing impairment in Cgn mutant mice might be related to the disruption of its regulatory function in tight junctions.Therefore, we evaluated the expression of several markers associated with tight junction formation in MDCK cells overexpressing wildtype and mutant human CGN plasmids.Overexpression of the mutant human CGN had no significant effect on mRNA and protein expression of tight junction markers, Occludin and ZO-1 (Appendix Fig S5A and B). mRNA expression levels of other tight junction-related markers (Claudin-1, Claudin-3, Claudin-5, ZO-2, ZO-3, GEF-H1, RhoA, and ZONAB) were also unaffected (Guillemot & Citi, 2006) (Appendix Fig S5B).We next used shRNAs reported in a previous study to knock down (KD) endogenous Cgn in MDCK cells (Guillemot & Citi, 2006)     Cingulin has been reported to regulate the localization of microtubules at tight junctions (Yano et al, 2018) and microtubules are widely expressed in the organ of Corti (Steyger et al, 1989).During inner ear development, each hair cell has a single kinocilium, which is a microtubule-based organelle.Kinocilia spontaneously degenerate postnatally and are thought to play an important role in the proper orientation of the actin filamentbased stereocilia bundles in mature hair cells (Bieniussa et al, 2023).In mature hair cells, microtubules are mainly found in the apical and lateral membrane regions of the cell (Steyger et al, 1989).We examined microtubule expression in mature cochlea from 2-month-old Cgn-cKO and 7-month-old Cgn delG mice.However, both types of Cgn mutant mice exhibited normal microtubule structures (Appendix Fig S6A and B).We also examined kinocilium position in immature cochlea of P0 Cgn delG mice and found no significant difference between the wild-type and Cgn delG mice (Appendix Fig S6C).These data indicate that abnormal expression of CGN had no obvious effects on microtubule expression patterns, and that the hearing impairment in Cgn mutant mice thus may not be related to microtubule alterations in the cochlea.

Cgn delG mice display abnormal morphology of the hair cell cuticular plates
Cingulin is considered a classical actin-binding protein and its N-terminal globular head domain has high binding affinity for actin filaments, thus serving as an important contributor to epithelial structural formation (Cordenonsi et al, 1999;Yano et al, 2018).
Therefore, we examined the effects of wild-type and mutant CGN on actin dynamics using the SRF-RE luciferase reporter system (Miralles et al, 2003)  We also used fluorescent labeled-Phalloidin to visualize F-actin structures present in the cuticular plates and hair cell stereocilia in wild-type and mutant mice.Interestingly, in addition to the loss of the OHCs (visualized as loss of F-actin signals), the cuticular plates of the surviving OHCs appeared abnormal in the Cgn delG cochleae (Figs 5J and EV4B).To further interrogate this morphological feature, we immunostained the organ of Corti for two cuticular platespecific markers, LMO7 and Spectrin b II (Du et al, 2019;Liu et al, 2019).The areas of both cuticular plates and circumferential belts from three rows of OHCs were also quantified (Fig 7D).Bases on the LMO7 immunostaining, the morphology of the cuticular plates in Cgn delG/delG mice was significantly altered compared to the wild-type mice at both basal (32 kHz, Fig 7E and F ◀ Figure 6.Exacerbated hearing loss and OHC degeneration in the Cgn delG mice after noise exposure.

A
Low-magnification SEM images of hair bundles from 2-month-old Cgn +/+ and Cgn delG/delG mice.B High-magnification SEM images of IHC hair bundles from 2-month-old Cgn +/+ and Cgn delG/delG mice.C High-magnification SEM images of OHC hair bundles from 2-month-old Cgn +/+ and Cgn delG/delG mice.Shown are hair bundles of the second-row OHCs (OHC2).D Width index of each hair bundle is calculated as the bundle width (w) divided by the height of the bundle arc (h).E-G Width indices of OHC hair bundles at (E) 8 kHz, (F) 16 kHz, and (G) 32 kHz cochlear regions were calculated from SEM images.n = 28-37 from 6 mice (Cgn +/+ ) or 3 mice (Cgn delG/delG ).
Data information: Data are presented as mean AE SEM; unpaired Student's t-test was used in (E-G).****P < 0.0001.Source data are available online for this figure.
not been reported.Here, we identify a novel CGN variant (CGN delG) co-segregating with affected individuals in an ADNSHL family.CGN is normally enriched at the apical cuticular plates and circumferential belts of cochlear hair cells and mediates actin polymerization.The CGN delG disease variant abolishes the expression and localization of CGN protein, altering the morphology of cuticular plates and stereocilia bundles in hair cells, and resulting in progressive and noise-sensitive OHC loss in mutant mouse models.These data, together with our human genetic and sequencing data, strongly supports the CGN delG variant as the causative mutation underlying hearing loss in this Chinese pedigree.
In the sensory epithelium of the mammalian inner ear, the apical surface of hair cells has an organelle rich in a dense actin filament network (named "cuticular plate"), where the roots of the stereocilia bundles are inserted (Liberman, 1987;DeRosier & Tilney, 1989;Pollock & McDermott, 2015).A structurally separate group of actin filaments form the circumferential belts and encircle the lateral edges of the cuticular plates (Hirokawa et al, 1982;Jaeger et al, 1994).Between the cuticular plate and the circumferential belt, there is a region without F-actin, known as the pericuticular necklace (Raphael et al, 1994).The morphology of these apical structures is dynamically remodeled during postnatal development in mouse, in which an immature non-convex apical circumference is molded to the mature V-shape of the overlying hair bundle in cochlear OHCs (Etournay et al, 2010).Interestingly and consistent with our findings, this remodeling is significantly impaired in mouse mutants with defective hair bundles (such as Cdh23 À/À , Ush1c À/À , Myo7a SB/ SB , Whrn wi/wi , and Myo15 ash2/sh2 ), suggesting potential interplay between morphogenesis of hair bundles and the underlying cuticular plates (Etournay et al, 2010).Actomyosin cytoskeleton tension is thought to play a pivotal role in these processes, although the regulatory mechanisms are largely unknown (Etournay et al, 2010).Our study thus provides an important piece to the puzzle on how the delicate morphology of cuticular plates on the apical hair cells may be maintained.Whether CGN also regulates the remodeling of hair cell circumferential belts and cuticular plates during development remains to be addressed.
Our observation that CGN regulates actin polymerization is consistent with previous in vitro studies (Cordenonsi et al, 1999;D'Atri & Citi, 2001;D'Atri et al, 2002;Mangan et al, 2016;Yano et al, 2018).Two main types of actin molecules, b-actin and c-actin, are expressed in hair cells (Drummond et al, 2012).b-actin is mainly distributed in stereocilia, while c-actin is the main component of cuticular plates (Drummond et al, 2012).c-actin-KO mice show progressive high-frequency hearing loss at 16 weeks of age (Belyantseva et al, 2009), while b-actin hair cell cKO mice show similar high-frequency hearing loss, but subsequent progression is slower (Perrin et al, 2010).Interestingly, loss of CGN expression has recently been shown to affect the localization of c-actin in MDCK cells at cell junctions (Rouaud et al, 2023).Based on our findings, we speculate that the restricted expression of CGN at the apical regions of hair cells is responsible for the morphology and tension of the cuticular plates by regulating c-actin dynamics.
It is worth to note that auditory phenotypes of the Cgn mutant mice are milder than those observed in human patients.Such differences are not uncommon in monogenic disorders in which the disease symptoms and severities are modifiable by environmental factors (Rossetti & Harris, 2007;Singer, 2015;Sacco & Milner, 2019).
We previously generated a Pou4f3 knockin mouse model for DFNA15, another human ADNSHL locus (Vahava et al, 1998), in which the progressive loss of hearing and OHCs is significantly modified by aging, noise exposure, and genetic background (Zhu et al, 2020).The Cgn delG mice in this study also display agedepedentn progressive hearing loss and are more susceptible to noise exposures, reminiscent of the DFNA15 mouse model.In addition, hearing loss in DFNB59 (PVJK) and Foxo3-KO mouse models is also greatly influenced by prior moderate noise exposure (Delmaghani et al, 2015;Gilels et al, 2017).Lastly, P2rx2-null mice, which model human DFNA41 (P2RX2), only show mild hearing loss at 17 months of age but are susceptible to noise exposure at 3 months of age (Yan et al, 2013).Although potential functional differences of CGN in mice and human cochleae cannot completely be excluded, these studies highlight the importance of gene-environment interactions in the pathogenesis and severity of progressive genetic hearing loss.
In summary, this study reports human CGN as a novel deafness gene in ADNSHL and provides both in vitro and in vivo evidence on the mechanisms of the CGN variant causing hearing loss in human and mouse.Furthermore, the findings highlight the important physiological roles of CGN in maintaining the cochlear hair cell cuticular plate morphology and auditory function.

Subjects and clinical evaluations
We recruited a three-generation Chinese Han family with postlingual autosomal-dominant non-syndromic hearing loss.Twelve members (seven affected and five unaffected) participated in the present study, informed consent was signed by all participating subjects.The experiments conformed to the principles set out in the WMA Declaration of Helsinki and the Department of Health and Human Services Belmont Report.This study was approved by the ethics committee of Nanjing Drum Tower Hospital, the Affiliated Hospital of Nanjing University Medical School (2019-170 and 2021-122-02).
Hearing levels of all participating members were measured by pure tone audiometry.For affected members, a complete medical history and physical examination were performed to exclude the possibility of environmental causes or syndromic hearing loss.For the proband, additional auditory evaluations were performed including distortion product otoacoustic emission (DPOAE), auditory steady-state response (ASSR), auditory brainstem response (ABR), temporal bone high-resolution computed tomography scanning (HRCT), and internal acoustic meatus magnetic resonance imaging (MRI).

Exome sequencing (ES) and linkage analysis of the Chinese family
All the 12 participating members were included in the ES study.Genomic DNA samples were extracted from whole blood samples of the participating members.
The exomes and flanking intronic regions from whole blood DNA samples were captured by Agilent SureSelect Human All Exon Kit (Agilent Technologies).The captured DNA was sequenced on

EMBO Molecular Medicine
Guang-Jie Zhu et al Illumina HiSeq 4000 sequencing platform (Illumina).Bioinformatics were aligned to NCBI build37/hg19 assembly using the BWA (0.7.12) software.Each sample was covered to an average sequencing depth of at least 100x.SNPs and indels were identified using GATK HaplotypeCaller software.Candidate pathogenic variants were defined as nonsense, missense, splice-site, and indel variants with allele frequencies of 0.001 or less in public variant databases dbSNP, 1000 Genomes, ESP6500, nci60, GnomAD and in disease database of COSMIC, Clinvar, OMIM, GWAS.Genotypes distributed in every 0.3 cM of genomic region were chosen for calculation of the logarithm of odds (LOD) scores using the Merlin v. 1.1.2parametric linkage analysis package.

ES of selected Spanish subjects
Exome sequencing was performed on the 96 Spanish index cases using the Agilent SureSelect V6 exome capture-based method in a NovaSeq6000 platform (Illumina, 150 PE, 18Gb/sample).The sequence data were mapped against the human genome sequence (build GRCh37/hg19), and data analysis was performed using the DNANEXUS' software that enables the single nucleotide variations (SNVs) and the copy number variation (CNV) analysis of the targeted exonic sequences.The analysis of the variant calling files (VCF) was restricted to the cingulin gene (CGN) and variant prioritization was carried out using a custom filtering strategy (Morin et al, 2020).

Sanger sequencing
Possible pathogenic variants from the ES analysis were evaluated by computational tools, including Mutation Taster, PolyPhen-2, and SIFT.Candidate pathogenic variants were further genotyped for the 12 family members by Sanger sequencing.

Mouse models and genotyping
Cgn flox mice, named Cgn fl , were generated by CRISPR-Cas9 genome-editing technology on C57BL/6J background, by inserting LoxP fragments at both ends of the exon 6 and exon 21 of mouse CGN (ENSMUSG00000068876) (Gempharmatech Inc, China).Genotypes of Cgn fl mice were identified by a primer pair targeting the LoxP insert (forward primer: 5 0 -CTG GGC TGG GCT CTT CTA TGT AG-3 0 , reverse primer: 5 0 -CTA GGG ATT GAA CCC AGA ACT TGA C-3 0 ).Cgn disease mutation knockin mice, named Cgn delG , were generated by CRISPR-Cas9 genome-editing technology on C57BL/6J background.A repair DNA harboring the G-base deleted in the identified human patients was used as donor template.The mutant allele was amplified using a specific forward primer (5 0 -ACT CTG CTT AGC TAA CAT TAC GT-3 0 ) paired with the reverse primer (5 0 -GCA GGG CTG GCT GGG ATC CT-3 0 ).The wildtype-allele was genotyped in a similar manner using the same reverse primer combined with a specific forward primer for the wildtype-allele (5 0 -ACT CTG CTT AGC TAA CCC TGA GG-3 0 ).
C57BL/6J were purchased from Gempharmatech Inc, China.Both male and female mice were used in this study.Animals were assigned to control or experimental groups based on their genotypes.For noise exposure experiments, animals of the same genotype were randomly assigned to control or noise exposure groups.Genotypes of the animals were blinded to the experimenters.All animal procedures were approved by the Institutional Animal Care and Use Committee of Model Animal Research Center of Nanjing University, China with protocol approval number #WGQ04.

Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analyses
Cultured cells or mouse tissues were homogenized and the total RNA extracted by RNAiso Plus kit (9109, Takara).The quantity and purity of samples were examined by determining absorbance at 260/280 nm by Nanodrop (Thermo Scientific).Reverse transcription reactions were performed by HiScript III RT SuperMix for qPCR (+gDNA wiper) (R323-01, Vazyme).QPCR was performed with AceQ qPCR SYBR Green Master Mix (Q111-02, Vazyme) on a Light-Cyclerâ 96 Instrument (Roche).QPCR reactions were performed with the primers listed in Appendix Table S2.Expression of target genes was normalized to the internal reference genes (GAPDH for human and mouse cells/tissues, Actb/b-actin for the canine MDCK cells).Fold changes of expression were normalized to control treatments or wildtype CGN control samples.

Cgn shRNA lentiviral vectors and lentivirus packaging
For Cgn gene silencing, previously described target sequences for silencing canine CGN (Guillemot & Citi, 2006) were cloned into pLKO1, a lentiviral vector with puromycin resistance screening label, using EcoRI (R3101V, New England Biolabs) and AgeI (R3552S, New England Biolabs) sites.The lentiviral particles were produced by co-transfecting each pLKO1 vector with other two lentiviral packaging helper plasmids (pSPAX2 package and pMD2.G envelope plasmids) in HEK293T cell.The supernatant was collected and concentrated by ultracentrifugation.The Cgn shRNA lentivirus were used to infect MDCK cells and puromycin was used to select infected cells to obtain stable shRNA-expressing cell lines.Cgn knockdown efficiency in MDCK cell was determined by RT-qPCR, Western blot and immunofluorescence.

SRF-RE dual-luciferase reporter assays
The pGL4.34[luc2P/SRF-RE/Hygro] vector contains a serum response factor (SRF) response element (SRF-RE) that drives transcription of the luciferase reporter gene luc2P in response to the activation of serum response factors through multiple signaling pathways, including RhoA GTPase activation.HEK293T cells in 24well plates were transfected with plasmids overnight as follows: pGL4.34[luc2P/SRF-RE/Hygro](Promega), pRL-TK, and CGN (WT or mutant), with or without co-transfecting the constitutively active RhoA.Transfected cells were serum starved for 6 at 18 h after transfection.Starved cells were lysed and the supernatants collected.The dual-luciferase assay kit (RG089M, Beyotime) was used to detect SRF activity, an indirectly representation of actin polymerization levels.

Immunofluorescence
Isolated mouse cochleae were fixed in 4% paraformaldehyde (PFA) in PBS for 2 h at RT.Samples were then decalcified with 5% (w/v) EDTA for 4 days at RT. Decalcified cochleae can be stored in PBS at 4°C.After micro-dissection of the cochleae, samples were permeabilized by freeze-thawing in 30% sucrose and blocked in 5% normal horse serum (NHS) (008-000-121, Jackson ImmunoResearch) with 0.3% Triton X-100 in PBS for 1 h.Then, the samples were incubated with primary antibodies (diluted in 1% NHS with 0.3% Triton X-100 in PBS) overnight at 4°C.Next day, samples were incubated with corresponding secondary antibodies for 2 h at RT and mounted to cover slides.
Transfected cells were fixed on ice in 2% PFA for 15 min and then blocked in 5% NHS with 0.3% Triton X-100 in PBS for 1 h at RT.Then, samples were incubated with primary antibodies (diluted in 1% NHS with 0.3% Triton X-100 in PBS) overnight at 4°C.Next day, samples were incubated with corresponding secondary antibodies for 2 h at RT.

Scanning electron microscopy (SEM)
Temporal bones of 2-month-old mice were fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer at 4°C overnight.The cochleae were dissected out and postfixed with 1% osmium tetroxide in 0.1 M phosphate buffer at 4°C for 2 h.The samples were then dehydrated in ethanol, followed by critically point drying using a Leica EM CPD300 (Leica, Germany).After sputter coating with platinum to a thickness of 15 nm using a Cressington 108 sputter coater (Cressington, United Kingdom), the samples were imaged with a Quanta250 field-emission scanning electron microscope (FEI, The Netherlands).The bundle width is defined as the distance between the two ends of each hair bundle.The height of the bundle arc is defined as the distance from the vertex to the line connecting the two ends of each hair bundle.Width index of each hair bundle is calculated as the bundle width divided by the height of the bundle arc.

DPOAE and ABR measurements
Mice were anesthetized by intraperitoneal injections of 375 mg/kg Avertin (Sigma) before ABR and DPOAE tests.
For ABR measurements, three needle electrodes, A, B, and G, were placed under the skin: A at the dorsal midline between the two ear flaps, B behind the right pinna, and G at the base of the tail.ABR potentials were evoked with 5-ms tone pips (0.5-ms rise-fall, with a cos2 envelope, at 33/s) delivered to the eardrum at log-spaced frequencies at 5.6, 8, 11.3, 16, 22.6, and 32 kHz.The responses were amplified (10,000×) and filtered (0.3-3 kHz) with an analog-to-digital board in a PC-based data-acquisition system.Sound pressure levels (SPLs) were raised at 5 dB-step from 10 to 80 decibels (dB).At each SPL, 1,024 responses were averaged (with alternating polarity) after "artifact rejection".DPOAEs were measured immediately following ABR tests.The DPOAE signal in response to primary and secondary tones with frequencies f1 and f2 respectively was recorded at the third frequency (2 × f1)Àf2, with f2/f1 = 1.2, and the f2 level 10 dB lower than the f1 level.SPLs at the ear canal was amplified and digitally sampled at 4-ms intervals.DPOAE thresholds were defined as the f1 level required to evoke a response at À10 dB SPL.Both DPOAE and ABR recordings were carried out using EPL cochlear function test suite software (Mass Eye and Ear, Boston, MA, USA).ABR peak 1 amplitudes were analyzed with ABR peak analysis software (Mass Eye and Ear).

Noise exposure
Both Cgn wild-type and mutant mice were subjected to noise exposure at 2 months of age.Individual mouse was housed within small cells in a subdivided cage on a rotation station, which is suspended in a reverberant chamber.Noise exposure experiment was carried out using LabState software (AniLab Software & Instruments, China) to produce 2-20 kHz broadband noise at 100 dB for 2 h.

Rotarod tests
Vestibular function of 2-month-old wildtype and mutant mice was performed using a rotarod system (ZB-200, Chengdu Techman Software, China).Mice were trained each day for 3 consecutive days at 15 round per minute (rpm) for 240 s prior to the experiment.Mice were subjected to three different rotarod test protocols: (i) 12 rpm fixed speed for 240 s; (ii) 20 rpm fixed speed for 240 s; and (iii) 0-44 rpm accelerating speed with 1 rpm/s and maintained at 44 rpm from 44 to 60 s.All three test protocols were performed for three times with 30-60 m resting time in between.The latencies to fall from the rotarod were then recorded and presented as averages of the three tests for each test protocol.

Statistical analysis
Statistical tests were performed using GraphPad Prism 9. Specific statistical tests used in each experiment and replicates were described in figure legends.Distribution of the data was assumed to be normal, but this was not formally tested.No statistical methods were used predetermine sample sizes, but our sample sizes are similar to those reported in our previous publications (Wan et al, 2014;Wan & Corfas, 2017;Zhu et al, 2020).The genotypes of the control and experimental animals were blinded to the investigators until the completion of data acquisition and analysis.For noise exposure experiments, mice with the same genotype were randomly assigned to control or noise exposure conditions.

The paper explained Problem
Non-syndromic hearing loss (NSHL) is the most common hereditary sensory impairment.Half of the congenital deaf patients are associated with genetic defects.While more than 100 deafness genes have been identified, the etiologies and pathological mechanisms in a great number of deaf patients are still unknown.Cingulin (CGN) is a cytoskeleton-associated protein and an important component of the tight junction in vertebrate epithelial cells; however, associations of CGN mutations with human diseases have not been reported.

Results
We report a novel CGN variant (c.3330delG, p.L1110Lfs*17) that cosegregates with the deaf patients in an autosomal dominant NSHL family.CGN is preferentially enriched at the apical cuticular plates and circumferential belts of the hair cells.The CGN mutation abolishes the expression and subcellular localization of CGN protein and fails to promote actin polymerization.A knockin mouse model carrying the disease mutation shows altered morphology of the actinenriched cuticular plates and hair bundles of sensory hair cells.Importantly, the Cgn mutation results in progressive and noise-sensitive hearing impairment and hair cell degeneration in the knockin mouse model.

Impact
Identifying novel deafness genes and the underlying pathogenic mechanisms are essential to personalized treatment for genetic hearing loss.Combining human genetics, cell culture, and animal model studies, we demonstrate that CGN is a novel deafness gene and plays an important role in the maintenance of hair cell cuticular plate and auditory function.This work not only provides mechanistic insights into development and maintenance of hair cell cuticular plates but also affords an experimental basis for genetic counseling and personalized therapeutics to deaf patients carrying CGN mutations.

Figure 1 .
Figure 1.A heterozygous CGN variant identified in affected individuals of an autosomal dominant non-syndromic hearing loss family.A Pedigree of the CGN c.3330delG variant within the family.Circles and squares represent females and males, respectively.Filled symbols denote individuals with hearing loss and non-filled symbols show individuals with normal hearing.B Audiograms of the proband (IV:2) over 6 years of follow-up.C Sanger sequencing of the CGN c.3330delG variant present in affected proband (IV:2) but not in his unaffected father (III:3).D Exon organization of the human CGN gene with position of the variant (c.3330delG).E Domain structure of the CGN protein and position of the variant (p.Leu1110Leufs*17).F Conservation of the Leu1110 residues of CGN in different mammalian species.Protein products with the CGN mutation (mut) in various species were also displayed.Source data are available online for this figure.

▸Figure 2 .
Figure 2. Expression pattern of CGN in mouse cochlea and other organs.A, B RT-qPCR (n = 3-12 biological replicates) (A) and Western blot (B) analyses of CGN expression in different mouse organs at P21. C, D RT-qPCR (n = 6 cochleae) (C) and Western blot (D) analyses of CGN expression in postnatal mouse cochlea.E Expression levels of Cgn in different cochlear cell types of P28-35 mouse (n = 4-6 biological replicates).Data were extracted from RNA-seq results published by Liu et al (2018a) and Liu et al (Data ref: Liu et al, 2018b).Unit: RPKM, Reads Per Kilo base per Million mapped reads; IHC, inner hair cell; OHC, outer hair cell; DC, Deiters cell; PC, Pillar cell.F, G Whole mount immunofluorescence of CGN expression in P3, P14, and P21 mouse cochlea coimmunostained with hair cell marker Parvalbumin (F) or cuticular plate marker LMO7 (G).CGN expression levels at the cuticular plates were normalized to LMO7 immunofluorescent signals (n = 130-193 hair cells from 3 to 4 cochleae).Data information: Data are presented as mean AE SEM; unpaired Student's t-test was used in (C), one-way ANOVA was used in (E and G).*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001.Source data are available online for this figure.

Cgn
knockin mice (Cgn delG mice) harboring the exact G-base deletion using CRISPR-Cas9 technology (Fig EV3E-G).Cgn mRNA expression in cochlea of homozygous Cgn knockin (CGN delG/delG ) mice was significantly decreased compared with wild-type (Cgn +/+ ) and heterozygous Cgn knock in (Cgn delG/+ ) mice (Fig 5A), likely due to nonsense mediated mRNA decay (NMD) of the frameshifted mutant Cgn mRNA.Western blot results confirmed that CGN protein was expressed as a truncated form and that expression was significantly reduced in cochlea and lung tissues from both heterozygous and homozygous Cgn delG mice (Figs 5B and EV4A).

Figure 3 .
Figure 3. Abnormal expression pattern of the mutant human CGN in MDCK cells.A A schematic diagram of C-terminal Flag-tagged CGN WT and p.Leu1110Leufs*17 (p.L1110Lfs*17) constructs used in this study.B MDCK cells expressing WT or mutant CGN were immunolabeled with CGN or Flag antibodies.C Western blot analysis of whole cell lysates from MDCK cells transfected with WT or mutant CGN.Exogenous CGN was immunoblotted with Flag antibody.D RT-qPCR of CGN expression in transfected MDCK cells (n = 3 biological replicates).E High magnification immunofluorescent images showing subcellular localizations of WT and mutant CGN (white arrows).Data information: Data are presented as mean AE SEM; unpaired Student's t-test was used in (D).ns, not significant, P > 0.05.Source data are available online for this figure.
deficiency would affect the formation of tight junctions (Appendix Fig S5D and E).Again, Cgn KD in MDCK cells did not affect expression of the tight junction markers (Appendix Fig S5F).Lastly, we examined the expression of ZO-1, a marker of tight junction formation and maintenance, in cochleae of the Cgn delG and Cgn-cKO mice.The expression patterns of ZO-1 in cochleae of the two mutant mice
(Fig 7A).Expression of wild-type CGN significantly enhanced actin polymerization.Moreover, co-expression of wild-type CGN and RhoA, a member of Rho GTPase family, synergistically promote actin polymerization (Fig7B).In contrast, the mutant CGN (p.L1110Lfs*17) showed significantly reduced actin polymerization activity(Fig 7C), likely due to its reduced expression levels and abnormal subcellular localization.
) and middle (16 kHz, Appendix Fig S7A) cochlear regions.Quantitative results showed that the cuticular plate areas of Cgn delG/delG mice decreased (Fig 7G and Appendix Fig S7B), while the circumferential belt areas increased (Fig 7H and Appendix Fig S7C) relative to those of wildtype mice.Importantly, the relative areas of the cuticular plates were significantly reduced (Fig 7I and Appendix Fig S7D).Similar results were obtained with the Spectrin b II antibody (Fig 7J-N and Appendix Fig S7E-H).Together, these data suggest that the Cgn mutant reduces actin polymerization and alters the morphology of the actin-rich cuticular plates of the OHCs.
E Whole mount immunofluorescence of the organ of Corti from the 2-month-old Cgn delG mice 1 week after noise exposure.White arrows indicate lost OHCs labeled with F-actin (Phalloidin, magenta) or Myosin 7a (green).F Percentage of OHC loss in noise exposed Cgn delG mice (n = 5-9 cochleae).Data information: Data are presented as mean AE SEM; two-way ANOVA was used in (B-D), one-way ANOVA was used in (F).**P < 0.01, ***P < 0.001, and ****P < 0.0001.Source data are available online for this figure.

▸Figure 7 .
Figure 7. Effects of the CGN mutation on actin polymerization and hair cell cuticular plate morphology.A Schematic diagram of actin polymerization activity detected by the SRF-RE luciferase reporter system.B Effects of CGN and RhoA expression on actin polymerization (n = 3 biological replicates).C Effect of WT and mutant CGN on actin polymerization (n = 3 biological replicates).D Experimental approach to quantify areas of cuticular plates, circumferential belts, and relative cuticular plate areas.The image shows apical surface of an OHC from a wild-type mouse.Phalloidin (magenta), LMO7 (green).E-I Whole mount immunofluorescence with LMO7 antibody on 2-month-old Cgn delG mice at 32 kHz cochlear region (E, F), areas of the OHC cuticular plates (G), areas of the OHC circumferential belts (H) and relative areas of the OHC cuticular plates (I) (n = 159-187).J-N Whole mount immunofluorescence with Spectrin b II antibody on 2-month-old Cgn delG mice at 32 kHz cochlear region (J, K), areas of the OHC cuticular plates (L), areas of the OHC circumferential belts (M), and relative areas of the OHC cuticular plates (N) (n = 56-62).Data information: Data are presented as mean AE SEM; unpaired Student's t-test was used in (B, C, G-I and L-N).**P < 0.01, ***P < 0.001, and ****P < 0.0001.Source data are available online for this figure.

Figure 8 .
Figure 8. Effects of the CGN mutation on hair bundle morphology.

Table 1 .
Audiological phenotypes of the affected individuals.
a Age at last examination.