
==== Front
Hum Mol Genet
Hum Mol Genet
hmg
Human Molecular Genetics
0964-6906
1460-2083
Oxford University Press

38981620
10.1093/hmg/ddae095
ddae095
Original Article
AcademicSubjects/SCI01140
A novel copy number variant in the murine Cdh23 gene gives rise to profound deafness and vestibular dysfunction
https://orcid.org/0000-0002-9544-6800
Boehler Nicholas A Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada

Seheult Shane D I Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

Wahid Muhammad Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada

Hase Kazuma Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

D’Amico Sierra F Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

Saini Shakshi Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

Mascarenhas Brittany Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada

Bergman Matthew E Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada

Phillips Michael A Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada

Faure Paul A Department of Psychology, Neuroscience & Behaviour, McMaster University, 1280 Main Street West, Hamilton, ON L8S 4K1, Canada

https://orcid.org/0000-0002-9292-7406
Cheng Hai-Ying Mary Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON L5L 1C6, Canada
Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON M5S 3G5, Canada

Corresponding author. Department of Biology, University of Toronto Mississauga, 3359 Mississauga Road, Mississauga, ON, L5L 1C6, Canada. E-mail: haiying.cheng@utoronto.ca
01 10 2024
09 7 2024
09 7 2024
33 19 16481659
07 2 2024
10 4 2024
30 5 2024
© The Author(s) 2024. Published by Oxford University Press.
2024
https://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

Abstract

Hearing loss is the most common congenital sensory deficit worldwide and exhibits high genetic heterogeneity, making molecular diagnoses elusive for most individuals. Detecting novel mutations that contribute to hearing loss is crucial to providing accurate personalized diagnoses, tailored interventions, and improving prognosis. Copy number variants (CNVs) are structural mutations that are understudied, potential contributors to hearing loss. Here, we present the Abnormal Wobbly Gait (AWG) mouse, the first documented mutant exhibiting waltzer-like locomotor dysfunction, hyperactivity, circling behaviour, and profound deafness caused by a spontaneous CNV deletion in cadherin 23 (Cdh23). We were unable to identify the causative mutation through a conventional whole-genome sequencing (WGS) and variant detection pipeline, but instead found a linked variant in hexokinase 1 (Hk1) that was insufficient to recapitulate the AWG phenotype when introduced into C57BL/6J mice using CRISPR-Cas9. Investigating nearby deafness-associated genes revealed a pronounced downregulation of Cdh23 mRNA and a complete absence of full-length CDH23 protein, which is critical for the development and maintenance of inner ear hair cells, in whole head extracts from AWG neonates. Manual inspection of WGS read depth plots of the Cdh23 locus revealed a putative 10.4 kb genomic deletion of exons 11 and 12 that was validated by PCR and Sanger sequencing. This study underscores the imperative to refine variant detection strategies to permit identification of pathogenic CNVs easily missed by conventional variant calling to enhance diagnostic precision and ultimately improve clinical outcomes for individuals with genetically heterogenous disorders such as hearing loss.

hearing loss
copy number variation
cadherin 23
spontaneous mouse mutant
auditory brainstem response
Canadian Institutes of Health Research 10.13039/501100000024 PJT-166046 PJT-183946 Natural Sciences and Engineering Research Council of Canada 10.13039/501100000038 RGPIN-2016-05563 RGPIN-2020-06906 John Evans Leadership Fund Canadian Foundation for Innovation 36131
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pmcIntroduction

There are over 7000 documented genetic disorders, and it is estimated that 1 in 100 people are affected by a single-gene disorder [1]. However, more than 20% of these documented genetic disorders have no known molecular basis [2]. It is crucial to elucidate the molecular mechanisms by which these disorders manifest to permit the downstream development of preventative, diagnostic, or therapeutic strategies. Using animal models to study human disease is well-established and effective in characterizing abnormal gene function [3–5].

Congenital hearing loss is the complete or partial loss of hearing present at birth and is the most common sensory impairment, with an estimated prevalence ranging from between 1.3 to 3.4 per 1000 live births [6–8]. It is estimated that 50% to 60% of newborn hearing loss is genetic, with over 100 genes in humans known to contribute to the development of hearing loss [9–11]. However, due to the genetic heterogeneity of congenital hearing loss, conventional clinical testing fails to identify an underlying genetic cause for hearing loss in most patients [12].

Mouse models have served an essential role in the study of hearing loss over the last century, with over 200 deafness-associated genes characterized in mice [13]. These findings have rapidly accelerated hearing research in humans since mice have short generation times, have cochlea that are strikingly similar to humans, and are genetically tractable with an ever-growing toolkit for molecular and bioinformatic analyses [14, 15]. Numerous spontaneous mouse models of hearing loss due to single nucleotide polymorphisms (SNPs) or small indels have been well-documented in the literature [16–22]. However, deletions and/or insertions larger than 50 bp, referred to as copy number variants (CNVs), have received little attention in their role of hearing loss onset in mice, despite recent evidence suggesting that CNVs contribute significantly to the development of non-syndromic hearing loss in humans [23].

Hyperactivity and circling behaviours in several mouse mutants have been linked to morphological inner ear defects that give rise to profound deafness and vestibular dysfunction [5, 24, 25]. Recently, a spontaneous mutant mouse line exhibiting severe locomotor dysfunction, hyperactivity, head-tossing, and circling behaviour was discovered in our colony, which we hereafter refer to as abnormal wobbly gait (AWG) mice. AWG mice appear indistinguishable from phenotypically wild-type (pWT) littermates at birth but are easily identifiable by weaning age (postnatal day 21). Here, we perform behavioural and physiological investigations to characterize the AWG phenotype. Bioinformatic and molecular analyses uncover a novel copy number variant (CNV) deletion of exons 11 and 12 in cadherin 23 (Cdh23) that result in vestibular dysfunction and profound deafness, giving rise to the AWG phenotype.

Results

Inheritance of the AWG phenotype is autosomal recessive and independent of the miR-132/212 KO genetic background

In the course of maintaining an in-bred colony of miR-132/212 KO mice, we observed a breeding pair that produced mice of both sexes exhibiting the AWG phenotype, which is characterized by circling and head-tossing behaviour, hyperactivity, and erratic behaviour during typical handling [Supplementary Movie 1]. To determine whether the AWG phenotype is dominant or recessive, we backcrossed AWG males and females to C57BL/6J mice. Both sexes were fertile, although pups from AWG females did not survive past birth, presumably as a consequence of the dam’s hyperactivity interfering with its ability to nurse. However, F1 pups sired from AWG males did not exhibit the AWG phenotype. Sibling matings of these unaffected F1 progeny resulted in the re-emergence of the AWG phenotype in approximately 1 in 4 animals of the F2 generation (Fig. 1A). Thereafter, the AWG strain was maintained in-house over successive generations by backcrossing AWG males to C57BL/6J females followed by sibling matings. Tracking offspring from the 4th and 5th generations of backcrossed breeders revealed that approximately 20% of F2 mice manifested the AWG phenotype irrespective of sex (Table 1) and independent of the miR-132/212 null allele. Collectively, these results strongly suggest that the AWG trait is inherited in an autosomal recessive fashion and is likely caused by a single gene mutation.

Figure 1 Breeding schematic for maintaining the spontaneous AWG mutant mouse line that exhibits reduced weight gain. (A) AWG mice were backcrossed to C57BL/6J mice prior to sibling mating of F1 mice. F2 AWG mice were then backcrossed to C57BL/6J mice repeatedly for a total of five generations. (B) Weekly weight tracking of a total of 526 mice (184 pWT♂, 180 pWT♀, 75 AWG♂, 87 AWG♀) shows a significant reduction in weight gain for both male and female AWG mice. For the later age points, body weight data are missing from animals that were removed for other experiments.

Graphical representation showing the breeding strategy and inheritance of the AWG mutation. Graph showing the body weights of AWG mice as a function of age.

Table 1 The AWG phenotype follows an approximately autosomal recessive mode of inheritance pattern.

F2 Offspring	Phenotype	
	pWT	AWG	
Females	185	44	
Males	198	50	
Total	383	94	
Ratio	0.80	0.20	
Affected offspring from the 4th and 5th generation AWG mice backcrossed against the C57BL/6J background. All breeding pairs were F1 heterozygotes in brother x sister mating schemes. Phenotype was recorded at time of weaning (postnatal day 21), by which point the AWG phenotype was easily observable in all affected mice.

The AWG phenotype is characterized by reduced weight gain, hyperactivity, circling and head tossing behaviours, and deafness

Next, we quantitatively assessed the AWG phenotype by comparing AWG mice and pWT littermates in a series of behavioural and physiological experiments. AWG mice weighed significantly less than same-sex pWT controls from 4 to ~40 weeks of age (Fig. 1B). We did not observe any increase in mortality of AWG mice compared to pWT littermate controls from the time of weaning to the end of weight tracking at 40 weeks of age. In the Open Field Maze (OFM) assay, AWG mice engaged in a dramatically increased number of circling events (Fig. 2A) and tended to travel greater distances (Fig. 2B). Motor coordination was measured during rotarod tests: AWG mice fell at reduced latencies during the constant rotarod test (Fig. S1A) and at lower speeds (Fig. S1B) compared to pWT controls.

Figure 2 AWG mice demonstrate elevated hyperactivity and circling behaviour. Behavioural changes in AWG mice compared to pWT littermate controls (n = 8) as measured by (A) number of circling events, and (B) total distance travelled during a 10 min open field maze (OFM) task. Asterisks represent the results of Welch’s t-test (* = P < 0.05, **P < 0.01, ***P < 0.001). Open circles represent females and crosses represent males. (C) Data generated by ToxTrac motion detection software during an OFM task.

Graphs and data on the performance of AWG mice and phenotypically wild-type littermate controls in the open field maze task. Number of circling events and distance traveled are quantified.

To evaluate hearing, we performed acoustic startle response (ASR) experiments on AWG (awg/awg) and pWT littermates. The pWT mice were later separated into WT (+/+) and heterozygous (awg/+) groups once the causative gene had been identified. In the ASR assay, mice are exposed to a sudden loud sound and reflexive muscular activity indicative of an ASR is assessed from video recordings. Across 19 raters, 92% were confident or somewhat confident that an ASR was absent in all recordings of AWG mice (Fig. 3A). In contrast, 64% of raters were confident or somewhat confident that an ASR was present in all +/+ mice and unrelated C57BL/6J mice (C57), the latter of which served as positive controls (Fig. 3A). Interestingly, raters were markedly less confident that an ASR was present for awg/+ littermates compared to WT littermates and C57 mice. The ASR scores were consistent across raters (ICC = 0.732; F119, 1042 = 58.6, P < 0.01) (Fig. S2).

Figure 3 AWG mice exhibit profound deafness as measured by acoustic startle response (ASR) and auditory brainstem response (ABR). (A) ASR rating using a 4-point Likert scale (1 = ASR definitely not present, 2 = ASR likely not present, 3 = ASR likely present, 4 = ASR definitely present) for AWG (awg/awg; n = 12), C57BL/6J (C57; n = 10), heterozygous (awg/+; n = 8), and WT (+/+, n = 10) mice show an absence of ASR in AWG mice in response to a pulse of loud broadband noise. (B-E) ABR tests for awg/awg (n = 15), C57 (n = 11), awg/+, (n = 11), and +/+ (n = 9) mice show that AWG mice exhibit an absence or significantly impaired response to clicks and tones up to 85 dB SPL. Asterisks represent the results of Welch’s t-test (* = P < 0.05, **P < 0.01, ***P < 0.001).

Graphs and data comparing AWG mice to heterozygous and wild-type littermates and C57BL/6J mice in the acoustic startle response (ASR) and auditory brainstem response (ABR). ASR is measured on a Likert scale that rates the confidence of the observer to the presence or absence of the startle response. ABR measures the presence or absence of response to clicks and tones up to 85 dB SPL.

Although behavioural measures provide reliable and accurate estimates of hearing capability, we also sought to measure hearing thresholds using an electrophysiological approach of auditory brainstem response (ABR) recordings. We determined click- and tone-evoked hearing thresholds across the different mouse types (awg/awg, awg/+, +/+, and C57) through visual inspection and through an automated thresholding algorithm (Table S1) [26]. Visual inspection of hearing thresholds was completed by two raters (SS and KH) using stacked plot displays\, which permits observers to scan the amplitude and latency of each component of the evoked response across all SPLs and integrate this information to inform a threshold estimate. The visually determined hearing thresholds were highly correlated (r2 = 0.896; Fig. S3A and D). By comparing the Spearman’s correlation between the observers and the algorithm, we concluded that the algorithm performs optimally, relative to the two observers, when the criterion value is set to 0.35 (Fig. S3B, C, and E–G) as it best matched visually-determined ABR thresholds. Further details regarding threshold determination are available within the Supplementary Materials. The ABR recordings for C57, +/+ and awg/+ mice all showed characteristic waveforms with peaks and troughs for tone bursts and click stimuli (Fig. S4 top row). The ABR thresholds for C57 mice (mean ± standard error: 46 ± 0.8 dB SPL) were significantly lower than +/+ (50 ± 1.5 dB SPL) and awg/+ mice (55 ± 2.4 dB SPL) in response to click stimulation (Fig. 3B). However, the tone-evoked hearing thresholds were not statistically different among the C57, +/+, and awg/+ groups. Most awg/awg mice did not exhibit a clear ABR waveform with a detectable threshold at the maximum playback amplitude (ca. 85 dB SPL). Collectively, our results suggest that awg/awg mice have profound hearing loss and vestibular dysfunction.

The Hk1D255N variant exhibited genetic linkage to the AWG phenotype

To identify the causative gene mutation underlying the AWG phenotype, we performed WGS on mouse tail genomic DNA, sequence alignment to the GRCm39 mouse reference genome, and downstream variant calling. The first round of WGS and variant analysis was conducted on the first generation of backcrossed mice and was focused on uncovering candidate variants that were homozygous recessive in AWG mice and heterozygous in unaffected F1 carrier parentals. A total pool of 615 267 variants were detected across all samples prior to filtering (Fig. 4A).

Figure 4 Candidate causative variants for the AWG phenotype were obtained by whole-genome sequencing (WGS) and variant analysis of AWG mice compared to F1 heterozygote parentals. (A) Variant analysis pipeline of whole-genome sequences from AWG (n = 2) and pWT F1 parentals (n = 2). From the total variant pool, 48 136 variants were homozygous in the AWGs and heterozygous in the F1 parentals. All variants on chromosome 11 were filtered because the AWG phenotype is unlinked to the miR-132/212 locus. (B) Of 1395 candidate variants, 114 were either in coding regions or in transcripts that undergo nonsense-mediated decay. (C) After filtering known and unrelated variants, only candidate variants in genes with GO terms from Ensembl’s variant effect predictor related to motor function, hyperactivity, nervous system, neurodegenerative disorders, or SIFT scores < 0.05 were included for downstream analysis. Sanger sequencing candidate variants in 10 AWG and 5 pWT littermate controls revealed only the HK1D255N variant to be linked to the AWG phenotype.

Graphical representation of the variant filtering and analysis pipeline for the whole-genome sequencing data, the predicted effect of the variants on genes or transcripts, and the list of candidate variants based on desired Gene Ontology terms or SIFT scores.

Of the 48 136 candidate variants with the appropriate genotypes, 46 741 were on chromosome 11 and linked to the miR-132/212 gene locus. Monitoring the status of the miR-132/212 genotype of each AWG mouse in later backcrossed generations confirmed that the miR-132/212 null allele was not linked to the AWG phenotype; therefore, the variants on chromosome 11 were discarded. Ensembl’s Variant Effect Predictor (VEP) software was applied to the remaining 1395 candidate variants (Fig. 4B). We focused on 11 variants in coding regions and 103 variants in transcripts that underwent nonsense-mediated decay (NMD), as these were most likely to disrupt protein expression or function. Of these, 25 variants had been previously documented and did not correspond to the AWG phenotype. To further narrow our search to a single causative variant, we evaluated gene ontology (GO) terms provided by Ensembl’s VEP database. From the remaining pool of variants, we retained only 11 because they contained GO terms related to motor function, hyperactivity, nervous system, or neurodegenerative disorders. We also included any variants that had SIFT scores < 0.05 as these are predicted to highly impact gene function (Fig. 4C).

Sanger sequencing revealed only one candidate variant that was homozygous in all 10 AWG mice assayed but not homozygous in any of the 5 pWT littermate controls. This novel point mutation resides in the coding region of hexokinase 1 (Hk1), the enzyme responsible for phosphorylating glucose to produce glucose-6-phosphate (G6P) in the first step of glycolysis, and changes the 255th amino acid from aspartic acid to asparagine. This result suggested that HK1D255N either contributes to the manifestation of the AWG phenotype or is closely linked to the causative genetic variant on chromosome 10. Liquid chromatography–tandem mass spectrometry (LC–MS/MS) assessment of the cerebellum from AWG mice found a significant increase in the early metabolites of glycolysis such as glucose 6-phosphate (G6P), fructose 6-phosphate (F6P), fructose 1,6-bisphosphate (FBP), dihydroxyacetone phosphate (DHAP), and glyceraldehyde 3-phosphate (GAP) relative to pWT controls. (Fig. S5). However, introduction of the HK1D255N mutation on the C57BL/6J background using CRISPR-Cas9 genome editing technology did not recapitulate the AWG phenotype when mice were bred to homozygosity. None of the overt behaviours exhibited by AWG mice, such as circling, head-tossing and hyperactivity, were evident in HK1D255N homozygous mice, which were visually indistinguishable from wild-type littermates. Taken together, these results suggest that the causative mutation of the AWG phenotype is linked to, but is not, the HK1D255N mutation.

A homozygous deletion of exons 11 and 12 in Cdh23 gives rise to the AWG phenotype

To identify new candidate variants of the AWG phenotype, we performed a second round of WGS and focused on loci that may exhibit genetic linkage to Hk1, which is situated on chromosome 10. Three AWG mice and three littermate controls that were wild-type for Hk1 from the 3rd generation backcross against C57BL/6J were analyzed. Based on OtoSCOPE v9 [27], 7 known deafness-associated genes mapped to chromosome 10 (Fig. 5A). Only three of these genes had candidate variants that were homozygous in AWG mice and absent in the littermate controls. Of these, 9 variants are in Cdh23, 1 in Pcdh15 and 2 in Msrb3. We focused on Cdh23 due to its proximity to Hk1 and the large number of variants and because previously documented mice with defects in Cdh23 exhibited behaviours and physiology reminiscent of the AWG phenotype [16, 17, 28].

Figure 5 Investigation of deafness-associated genes linked to HK1D255N on chromosome 10 reveals a decrease in Cdh23 transcript abundance and ~300 bp of missing Cdh23 coding sequence from cDNA. (A) Mouse chromosome 10 ideogram labelled with seven known deafness-associated genes and their position relative to Hk1. (B) A representation of the Cdh23 locus. Exons are represented by alternating blue and purple bars while black bars represent introns. The relative position of nine candidate variants (V1-V9) for the AWG phenotype are shown. (C) qRT-PCR experiments on cDNA from awg/awg (blue traces) and +/+ littermate controls (green traces) show a dramatic reduction in abundance of mRNA transcripts containing exons 12–14 (V5-V6 target). (D and E) PCRs of cDNA from Cdh23 mRNA transcripts targeting (D) exons 10–16 result in amplicons that are ~300 bp smaller in awg/awg mice and (E) exons 11–15 yield no amplification in awg/awg mice, suggesting loss of either exon 11 or 15 from the Cdh23 mRNA transcript.

Graphical ideogram of mouse chromosome 10 and locations of deafness-associated genes relative to the hexokinase 1 (Hk1) gene. Magnified view of the Cdh23 locus shows the locations of candidate variants and quantitative RT-PCR data analysis of the effects of each variant cluster on the mRNA of Cdh23. Data from conventional RT-PCR suggest the loss of either exon 11 or 15 from the Cdh23 mRNA transcript.

Cadherin-related 23 (Cdh23) is highly expressed during development and is critical for the formation and maintenance of stereocilia of inner ear hair cells, which are required for the transduction of mechanical vibrations to electrical signals that the brain can interpret as sound or balance information [29, 30]. We investigated the 9 variants (V1-V9) within Cdh23: V1 was within the 3′ UTR and V2-V9 were within intronic regions (Fig. 5B). Although none of these variants were predicted to be deleterious by Ensembl’s VEP, we postulated that unpredictable alternative splicing or mRNA processing may give rise to non-functional CDH23 protein. As Cdh23 is highly expressed during development [28], we performed qRT-PCR on whole head tissue from postnatal day 1 (P1) mice, targeting Cdh23 exons flanking the variant-harbouring introns. AWG mice exhibited a dramatic reduction in Cdh23 transcripts containing exons flanking introns 12 and 13, corresponding to V5 and V6 (Fig. 5C). Using primers complementary to exons 10 and 16 in Cdh23, PCRs on cDNA acquired from whole head tissue from P1 mice revealed WT littermate controls amplified the expected band size of 666 bp whereas AWG mice amplified a band ~300 bp shorter (Fig. 5D). When targeting exons 11 to 15, WT littermate controls amplified the expected 494 bp band whereas no amplification was detected in AWG mice (Fig. 5E), suggesting that either exons 11 (113 bp) and 12 (189 bp) are absent or, alternatively, exons 14 (150 bp) and 15 (159 bp) are absent in the Cdh23 transcript.

In a second round of WGS, we broadened our variant detection beyond conventional pipelines that focus on SNP and indel discovery. We performed a thorough investigation of CNVs to investigate the possibility that a genomic deletion went undetected within the Cdh23 locus. Using CNVpytor to perform read depth CNV detection with established, conservative calling parameters of 10 kb bin sizes and p values of < 0.01 yielded no calls on chromosome 10 in any awg/awg mouse or +/+ control (Fig. 6A). We sought to maximize sensitivity of CNV detection by decreasing bin sizes to the minimum of 100 bp and relaxing all filtering of parameters, which predictably yielded a large quantity of false-positive CNV calls on chromosome 10 for both awg/awg and +/+ littermate controls (Fig. 6B). However, manual investigation of 20 kb read depth plots revealed a complete absence of signal in the region of chromosome 10 containing Cdh23 exons 11 and 12 for awg/awg mice (n = 3), corresponding to a putative 10 400 bp deletion not observed in +/+ controls (Fig. 6C). Moreover, PCRs targeting exons 11 or 12 failed to amplify the expected band from tail genomic DNA samples from awg/awg mice (Fig. 6D and E).

Figure 6 Putative 10.4 kb CNV deletion encompassing exons 11 and 12 of Cdh23 in AWG mice is observed by manual inspection of 100 bp sliding window read depth plots. (A–C) representative CNVpytor read-depth plots for an awg/awg mouse and a +/+ control. (A) CNV sliding window of 10 000 bp and filtering of CNVs with P < 0.05 does not reveal any CNVs on chromosome 10 for awg/awg or +/+ mice. (B) Use of 100 bp sliding windows and no filtering of CNVs results in many false-positives for both awg/awg and +/+ mice. (C) Using 100 bp sliding windows and manual inspection of Cdh23 exons 11 and 12 show a ~10.4 kb deletion in awg/awg mice. (D and E) PCRs targeting exons 11 and 12 in Cdh23 of genomic DNA fail to produce the expected 182 bp band in awg/awg mice, confirming the putative CNV deletion.

Data from whole-genome sequencing read-depth plots reveal the presence of a 10.4 kb genomic deletion encompassing exons 11 and 12 of Cdh23 in AWG mice, but not in wild-type controls. Gel images from PCR analysis of genomic DNA from AWG mice confirms the absence of exons 11 and 12.

Deletion of exons 11 and 12 from Cdh23 introduces a premature stop codon within exon 14 that corresponds to the fourth extracellular cadherin (EC) domain in the CDH23 protein (Fig. 7A). To validate the presence of the homozygous CNV deletion of exons 11 and 12, we designed PCR primers spanning the predicted 10.4 kb deletion, resulting in amplification of a ~1.6 kb band only in awg/awg and not in +/+ mice (Fig. 7B). Excision and purification of these bands followed by Sanger sequencing permitted detection of the CNV breakpoints corresponding to a 10 457 bp deletion flanking exons 11 and 12 of Cdh23. Interestingly, an unanticipated 738 bp of additional sequence was found within the breakpoints. Using BLAST from NCBI on this sequence suggests that it is derived from a murine endogenous retrovirus with leucine tRNA primer (MERVL) element that is common throughout the mouse genome. Finally, Western blot analysis of whole head tissue extracts from P1 mice showed a complete absence of the canonical long isoform of CDH23 protein (~370 kDa) in AWG mice and a significant reduction of this isoform in mice heterozygous for the CNV deletion (Fig. 7C). Notably, expression of a lower molecular weight band (~240 kDa) appears to be inversely correlated to the expression of the long isoform of CDH23. Taken together, these results suggest that a novel homozygous CNV deletion in AWG mice, hereby named Cdh23awg/awg, introduces a premature stop codon that results in the absence of functional CDH23 protein and gives rise to the AWG phenotype.

Figure 7 Deletion of exons 11 and 12 within Cdh23 in awg/awg mice results in a premature stop codon that leads to the complete absence of the canonical long isoform CDH23 protein in the whole head of P1 mice. (A) Schematic representation of CDH23 protein. CDH23 is a single-pass transmembrane (TM) protein with 27 extracellular cadherin (EC) repeat domains that are critical for tip link formation of inner ear hair cells. Deletion of exons 11 and 12 results in a premature stop codon introduced into EC4 and prevents the production of full-length, functional CDH23 protein in awg/awg mice. (B) PCRs targeting the putative CNV breakpoints within Cdh23 in awg/awg mice followed by Sanger sequencing confirms the CNV deletion and reveals the presence of 738 bp of unexpected sequence derived from a MERVL element. (C) Western blot targeting CDH23 using the TF7 antibody shows absence of the functional, long (370 kDa) isoform CDH23 protein in whole head extracts from P1 awg/awg mice as well as a marked reduction in heterozygotes.

Graphical representation of the CDH23 protein and the position of the amino acid sequence encoded by exons 11 and 12. Sequencing data confirming the deletion of exons 11 and 12 within the Cdh23 gene of awg/awg mice. Western blot data showing the absence of full-length CDH23 protein in the heads of awg/awg neonates.

Discussion

Here, we report the first mouse model of profound hearing loss to be caused by a spontaneous homozygous pathogenic CNV deletion in Cdh23. In addition to being deaf, Cdh23awg/awg mice are hyperactive, weigh less than littermate controls, and exhibit vestibular dysfunction as evidenced by their circling and head-tossing behaviours and motor incoordination. These phenotypes closely mirror those of several strains of waltzer mice, which harbour single point mutations or small indels in Cdh23 resulting in the introduction of a premature stop codon [22]. The AWG phenotype demonstrates an inheritance pattern slightly less frequent than expected for an autosomal recessive allele (20% observed vs. 25% expected from heterozygous intercrosses). This may be due to incomplete preweaning (e.g. embryonic, perinatal) lethality, incomplete penetrance, or both, as previously documented in the Cdh23tm2b(EUCOMM)Wtsi targeted mouse line [31]. We initially failed to identify the causative variant from WGS data analyzed using a conventional variant analysis pipeline. Ultimately, Sanger sequencing revealed the presence of a ~10 kb genomic deletion encompassing exons 11 and 12 of Cdh23. In addition to introducing a new mouse model for hearing loss research, the present study draws attention to the need for a more comprehensive approach to identifying disease-causing variants from WGS data.

Applying conventional variant detection and filtering to WGS data was insufficient to identify the causative CNV within Cdh23, but a nearby linked variant, HK1D255N, was found to segregate with the AWG phenotype after five generations of backcrossing against the C57BL/6J background. Since Hk1 is only ~2 Mb from Cdh23, it is unsurprising that the HK1D255N variant remains linked to the AWG phenotype as estimations of the rate of recombination on chromosome 10 in mice are approximately 0.64% per Mb without accounting for recombination hotspots [32]. CRISPR/Cas9 introduction of the HK1D255N mutation into a C57BL/6J mouse line and subsequent breeding to homozygosity did not recapitulate the AWG phenotype, necessitating further molecular investigations of hard-to-detect variants in nearby deafness-associated genes. The most proximal candidate was Cdh23, and qRT-PCR experiments revealed a substantial depletion of mRNA transcripts containing exons 12–14 in AWG mice. Manual investigation of the Cdh23 locus using small 100 bp bin read-depth plots generated by CNVpytor revealed a ~10.4 kb homozygous deletion encompassing exons 11 and 12 which was confirmed by genomic PCRs targeting these exons. Genomic PCRs amplifying across the breakpoints of the putative CNV followed by Sanger sequencing confirmed the causative mutation was a loss of 10 457 bp in addition to an insertion of 738 bp derived from a MERVL element. Although we do not know what role, if any, this MERVL element played in the generation of the CNV in Cdh23, recent evidence suggests that endogenous retroviral elements (ERVs) may promote genome instability and play a role in CNV formation [33]. The CNV deletion in Cdh23 results in a premature stop codon introduced early in the transcript coding for extracellular cadherin repeat domain 4 and results in complete absence of the canonical 370 kDa CDH23 protein in whole head tissue from P1 mice when analyzed by Western blot. Curiously, the CDH23 antibody recognized a protein migrating at ~240 kDa that was upregulated in both Cdh23awg/awg and Cdh23awg/+ mice. Although further experiments are needed to determine its identity, this band may represent a protein isoform of CDH23 that is increased in a compensatory manner when full-length CDH23 is absent or reduced.

To confirm that Cdh23awg/awg mice have deficits in auditory function, we recorded behavioural and electrophysiological measures of hearing from these animals and compared them to controls. Overall, our results suggest that Cdh23awg/awg mice do not show an observable ASR because they have profound hearing loss, as evidenced by a lack of clear acoustically-evoked AEPs, compared to Cdh23+/+, Cdh23awg/+, and unrelated C57BL/6J control mice. The click-evoked AEPs recorded from Cdh23awg/awgmice did not have discernible peaks or troughs at expected latencies and instead presented waveforms and auditory thresholds that were similar to those of congenitally deaf mice [34, 35] and mice with sound-induced hearing loss [36].

Our behavioural and physiological data show that the AWG phenotype is consistent with the waltzer phenotype, which has been attributed to loss of CDH23 protein expression. Previous studies have demonstrated that CDH23 is a crucial component of the tip links connecting the stereocilia of sensory hair cells of the inner ear and is necessary for the mechanotransduction of sound waves to neural impulses [17, 29, 30]. In humans, variants in CDH23 are known to be responsible for Usher syndrome type ID and non-syndromic hearing loss [37, 38]. However, the majority of documented variants within Cdh23 are SNPs or indels, although at least one confirmed example of a pathogenic heterozygous CNV deletion has been reported [39]. As of the time of this writing, there are 4534 documented variants within Cdh23 that affect fewer than 50 bp on the ClinVar database. In contrast, only 9 documented structural variants of Cdh23 that affect more than 50 bp have been submitted to ClinVar [40]. This bias also extends to mouse models of deafness caused by spontaneous mutations in Cdh23 documented on the Mouse Genome Informatics database: 11 mouse lines have known mutations that affect fewer than 10 bp and 2 mice have unknown mutations (Table S2). Limitations in current approaches to CNV detection may partly explain the rarity of documented CNVs within Cdh23, in addition to their relatively low prevalence (compared to SNPs and indels) in the human and mouse genomes.

Screening for novel pathogenic mutations in deafness-associated genes is challenging due to the polygenic nature of hereditary hearing loss. Recent attempts at comprehensive genetic screening for patients with hearing loss have yielded success rates of molecular diagnoses ranging from 29% to 57% [12, 41–43]. Screening individual genes using Sanger sequencing can be effective at uncovering novel pathogenic mutations, but applying this process is impractical for large genes such as Cdh23 at a broad scale [44]. Since many pathogenic mutations continue to evade detection through conventional means, there is an urgent need to develop methods that can identify previously undocumented variants. Pathogenic CNVs are likely underreported for congenital hearing loss, with some estimates suggesting that up to 18.7% of non-syndromic hearing loss is due to pathogenic CNVs [23, 45].

The most common approaches deployed for detection of CNVs in a diagnostic setting have been microarray based comparative genomic hybridization (aCGH) and SNP microarrays and multiplex ligation-dependent probe amplification (MLPA) [46, 47]. Although these methods are sensitive, they are low throughput, expensive, and will only detect CNVs in genomic regions targeted by the assays. With the increasing capability of applying next-generation sequencing technology to the task of mutation detection in a clinical setting, it is critical to ensure that methods going forward employ robust methods for variant calling that extend beyond SNPs and indels. The four methods employed for detecting CNVs from next-generation sequencing data is through paired-end mapping-based detection, split read based detection, de novo assembly-based detection, and read depth (RD) based detection [48, 49]. Although these methods can reliably detect large-scale structural mutations (on the order of megabases), they show poor performance for detecting small CNVs that affect one or a few closely spaced exons [50, 51]. Streamlined bioinformatic pipelines that integrate robust CNV detection using NGS data have lagged behind pipelines focused on uncovering pathogenic SNPs and indels. As depth of sequencing increases, modified RD based approaches offer reliable means to uncover previously undetectable CNV mutations and may reduce the cost, labour, and time required to perform genetic diagnoses in the future. The Cdh23awg/awg mouse is an exemplary case study showcasing the need to diversify the detection of pathogenic variants to improve the rate of molecular diagnoses for complex polygenic disorders such as hereditary hearing loss in the future.

Materials and methods

Animals

Animal handling and experimental procedures were performed at the animal facilities at the University of Toronto Mississauga and McMaster University. All animal experiments were approved by the University of Toronto’s Biological Sciences Local Animal Care Committee (LACC) and McMaster University’s Animal Research Ethics Board (AREB), respectively, complying with guidelines established by the University of Toronto’s University Animal Care Committee (UACC), AREB, and the Canadian Council on Animal Care. miR-132/212 germline knockout (KO) mice were obtained from Dr Richard Goodman (Vollum Institute, Oregon Health & Science University), backcrossed for at least 10 generations to C57BL/6J mice, and subsequently maintained in-house as an in-bred colony for unrelated investigations before the AWG phenotype spontaneously arose [52, 53]. AWG mice were backcrossed to C57BL/6J mice and F1 progeny were sibling mated to recover the AWG phenotype in the F2 generation. This backcrossing breeding scheme was repeated for five generations and the miR-132/212 KO genotype was removed without effect on the AWG phenotype.

Behavioural tests

Open field maze (OFM)

An open field maze apparatus measuring 50 cm × 50 cm (l × w) was placed under a brightly lit (150 lux) environment in a quiet room. Mice were brought into the room to acclimatize for > 10 mins before the experiment began. Equal numbers of AWG mice and phenotypically WT littermate controls (pWT; n = 8 each) were assayed, with sex balanced across both groups (n = 4 males, n = 4 females). Each mouse was placed into the center of the open field apparatus and their behaviour was tracked (duration = 10 mins) by video camera positioned directly above the apparatus. Motion tracking software (ToxTrac) was used to measure the total distance travelled [54]. The reported values correspond to total distance traveled corrected for number of frames ToxTrac was able to identify the mouse. One AWG mouse was removed from downstream analysis due to incomplete motion tracking as only 57% of frames captured detected this mouse. Circling events were quantified by manual observation of the video footage and were reported each time a tight 360-degree turn was performed by the mouse.

Rotarod test

A rotarod (Columbus Instruments, Columbus, OH; axle diameter 3.6 cm) was used to assess differences in motor coordination and muscular endurance as previously described with minor modifications [55]. The paradigm involved 3 consecutive training days paired with habituation, followed by a single day of testing. We selected AWG mice and their respective pWT littermate controls that were 4 weeks of age for the first experimental training day. For training, animals were placed on the spindle after which the instrument was turned on. Revolution speed started at 5 revolutions per minute (rpm) on the first training day and was increased by 5 rpm/day for two successive training days. Animals were returned to their home cage once they had fallen from the rotarod or after a maximum duration (i.e. 3 min) had passed. On the test day, both the constant and accelerated rotarod tests were conducted. For the constant rotarod, animals were placed on the spindle (revolution speed = 15 rpm) and latency to fall was recorded. For animals that did not fall, the maximum duration of 3 min was recorded. For the accelerated rotarod, animals were placed on the spindle with speed initially set at 4 rpm. After 30 s, the rotation was set to accelerate at a rate of +0.1 rpm/sec until the animal fell. The latency to fall (where t = 0 is the start of acceleration) was recorded and used to calculate the rotation speed at which the animal fell. Each animal was subjected to the constant (3 trials) and accelerated rotarod (2 trials), and the mean latency to fall was calculated. The paradigm was repeated every three weeks for the same cohort of mice over a 12-week period.

Acoustic startle response (ASR)

An acoustic startle response (ASR) is a behavioral test used to assess hearing in animals. The ASR is a reflexive behaviour characterized by contractions of major skeletal muscles resulting in freezing, flinching, and limb extension or flexion upon hearing a loud and unexpected sound [56, 57]. Mice were tested in a box arena (40 × 31 × 23 cm; l × w × h). The walls and top of the arena were lined with sound attenuating foam (Sonex® Classic; Pinta Acoustic, USA) and the floor was covered with a surgical towel. A red bulb (240 W) was mounted through the top of the arena, which provided light for the video recordings. Acoustic signals were presented with a loudspeaker (UltraSoundGate Player BL Light, Avisoft Bioacoustics, Berlin, Germany) which was placed on the arena floor and abutted one of the shorter side walls. The total duration of each playback experiment was approximately 4 mins. No acoustic stimuli were broadcast during the first 2 min to allow mice to habituate to the testing arena. To elicit an ASR, mice were presented with a pulse of loud broadband noise (107 decibel sound pressure level [dB SPL] at 10 cm; duration = 200 ms; minimum frequency = 1 Hz, maximum frequency = 11.5 kHz—3 dB bandwidth = 11.2 kHz). In each experiment, a mouse received three presentations (trials) of a single pulse of broadband noise. The timing of each noise pulse was randomly dispersed over the final 2 min of the experiment with 15 s, 30 s or 45 s of silence between trials. Experimental trials were filmed with a Hero8 Action camera (GoPro, San Mateo, CA, USA), which also records audio. At the conclusion of an experiment, the mouse was returned to its home cage. Recorded videos were scored independently by 19 raters using a 4-point Likert scale: 1 = ASR definitely not present, 2 = ASR likely not present, 3 = ASR likely present, and 4 = ASR definitely present. See Supplementary Material for further information.

Auditory brainstem response (ABR)

Auditory Brainstem Response (ABR) recordings are a non-invasive neural recording technique whereby subdermal electrodes are positioned near the auditory bullae, and auditory-evoked neural activity is recorded. The resultant auditory evoked potential (AEP) waveform represents the summated neural activity within nuclei along the ascending auditory pathway (Fig. S4 Top Row). Mice were anesthetized with a Ketamine + Xylazine cocktail (0.1 to 0.25 ml of a 3:1 v/v mixture of 100 mg/ml Ketamine +20 mg/ml Xylazine; final animal dose 3.2 to 8.0 mg/kg) and placed on a foam pad atop a raised plexiglass platform inside an angle-iron Faraday cage that was insulated with sound-attenuating foam. Recordings were made with three needle-electrodes inserted subcutaneously (3-lead disposable, 27-gauge, 13 mm; S83018–19 Rochester Electro-Medical, Lutz, Florida, USA). The reference electrode was placed behind the ipsilateral ear, the recording electrode was placed at the nape of the neck, and the ground electrode was placed at the hip of the mouse. The impedance of the recording and reference electrodes (re-ground electrode) never exceeded 1 kΩ. Recorded signals were amplified 20× by a low impedance head stage (TDT RA4LI) whose output was further amplified 250× and digitized by a preamplifier (TDT RA4PA Medusa, sampling rate 25 kHz) before passing to a multifunction processor (TDT RX6) via a fiber optic cable. Both the head stage and preamplifier were battery operated and located inside the Faraday cage.

The mouse was exposed to repeated presentations of broadband acoustic clicks (duration = 1.0 ms) or pure tones at 8, 16, and 32 kHz (tone duration = 5.0 ms, rise/fall time = 2.0 ms) at 85 dB SPL down to 5 dB SPL to generate AEPs [57]. Stimulus evoked neural responses were collected over a 10.0 ms recording window. Stimuli were repeated 512 times at each SPL at a presentation rate of 21 Hz. The averaged ABR waveform was then bandpass filtered (high-pass cut-off frequency = 300 Hz, low-pass cut-off frequency = 3 kHz; notch frequency = 60 Hz) before and after exporting for visualization and analysis. Upon completion of a recording, the electrodes were removed, cleaned with 95% ethanol, and the mouse was placed on a heating pad until it recovered. Mice were returned to their home cage once they showed full signs of wakeful behaviour.

Hearing thresholds were determined through visual inspection, then standardized using an automatic thresholding algorithm based on normalized cross-covariation of adjacent level ABR waveforms in descending order of a stacked plot display [26]. Further details regarding ABR procedure and threshold determination are available within the Supplementary Materials.

Whole-genome sequencing (WGS) and variant calling

We conducted two rounds of whole-genome sequencing on tail DNA samples from: (1) two F2 AWG mice and two F1 parental controls from the first-generation backcross against C57BL/6J mice (first round); and (2) three F2 AWG mice from the third-generation backcross against C57BL/6J mice and three F2 WT littermate controls (second round). Genomic DNA was purified by phenol/chloroform and precipitated with ethanol. DNA pellets were dissolved in 100 μl TE buffer (10 mM Tris pH 8.0, 1 mM EDTA) and purity and degradation of DNA was assayed via Nanodrop and agarose gel electrophoresis before shipment to Beijing Genomics Institute (BGI) for WGS. Samples were sequenced using the BGISEQ-500 platform to a read depth of approximately 38× for the first round and DNBSEQ-G400 sequencing platform to a read depth of approximately 43× for the second round. Paired read lengths of 150 bp were obtained and raw reads containing adapter sequences or low-quality sequences were filtered using proprietary BGI SoapNUKE software. Clean reads were provided to us in FASTQ format, and all further bioinformatics were conducted in-house.

FastQC quality control was conducted on the received FastQ files using default settings. Adapter trimming via SoapNUKE software was successfully verified using Trimmomatic. The trimmed reads were aligned using Burrows-Wheeler alignment (BWA) against the GRCm39 C57BL/6J mouse reference genome using SAMtools. Mate-pair reads validation and clean up, duplicate marking, and read group identification were conducted using Picard Tools with default settings. Variant calling on the BAM output files was conducted using GATK to produce the final variant calling files (VCFs).

CNVpytor, a Python library for CNV analysis, was utilized for CNV detection via read depth quantification [58]. A custom PYTOR file was created for the GRCm39 C57BL/6J mouse reference genome and aligned BAM files were read into CNVpytor version 1.3.1. Unless otherwise specified, default parameters for CNV detection were used. In the first round of CNV detection, calling parameters were set to 10 kb bin sizes. Calls were filtered from downstream analysis if the CNV had a distance less than 100 000 bp from gaps in the reference genome, contained greater than 50% not uniquely mapped reads, contained greater than 50% Ns in the reference genome, or was a non-confident call with a p value of greater than 0.001. The second round of CNV detection sought to maximize sensitivity of CNV calling at the cost of increasing false positives. The calling parameters for CNV detection in the second round included 100 bp bin sizes and calls were only filtered if the CNV had a distance less than 10 000 bp from gaps in the reference genome, contained greater than 50% not uniquely mapped reads, or contained greater than 50% Ns in the reference genome.

Variant filtering

First round of WGS

Variant filtering was conducted on the first round of WGS data using the final VCF files produced by GATK. Variants were considered candidates if they were found to be homozygous in both AWG mice and heterozygous in F1 parental controls. Variants on chromosome 11 were filtered since backcrossing of AWG mice against the C57BL/6J background resulted in breeding out of the miR-132/212 null allele without affecting the inheritance or severity of the AWG phenotype. The remaining variants were evaluated using Ensembl’s Variant Effect Predictor (VEP) using default recommended settings against the GRCm39 C57BL/6J mouse reference genome to identify candidates with functional consequence. We selectively filtered for variants that were within gene regions or the 3′ or 5′ untranslated regions of genes that undergo nonsense-mediated decay. All previously documented variants were reviewed for phenotypic consequences related to deficiencies in the central nervous system or motor function before being removed from downstream analysis. Finally, we manually investigated gene ontological keywords for the remaining undocumented variants and subjectively identified the most likely candidates based on relevant keywords relating to abnormal gait, hyperactivity, or neurological function. We also included candidates with low SIFT scores, indicative of amino acid substitutions that are predicted to be deleterious to protein function.

Sanger sequencing validation of candidate mutations

PCR primers were designed for the top candidate variants identified through the first round of WGS and targeted the following eight genes for Sanger sequencing validation: Kif12, Ulk4, Gns, Usp40, Hk1, Lgr6, Pnisr, and Ank3 (Table S3). PCR amplification using Phusion High-Fidelity DNA polymerase (New England Biolabs) followed by gel extraction using the QIAquick Gel Extraction Kit (Qiagen) was conducted on five AWG mice from different litters. The concentration and integrity of DNA samples were verified on a 1% agarose gel, purified, and sent to The Center for Applied Genomics (The Hospital for Sick Children, Toronto) for Sanger sequencing. We determined the top candidate variant, Hk1255N, and repeated this process with additional AWG and pWT littermate controls (n = 5 each).

Second round of WGS

We submitted a second batch of DNA extracted from the tails of three pWT littermate controls that were homozygous for the wild-type Hk1D255 allele and three AWG mice homozygous for the Hk1D255N variant to BGI that underwent the same bioinformatics pipeline. These mice came from the third generation of animals backcrossed to the C57BL/6J background. We repeated the same variant filtering process as conducted for the first round of WGS and were unable to identify a strong candidate variant for the AWG phenotype. Given that the Hk1D255N allele occurs on chromosome 10, we filtered for variants in seven genes on chromosome 10 that have been linked to deafness-related phenotypes. In total, nine variants were found in Cdh23, two in Msrb3, and one in Pcdh15.

RNA extraction and qRT-PCR

Total RNA extraction was performed using TRIzol Reagent (Thermo Fisher Scientific) following the manufacturer’s instructions with minor modifications. For staging of neonates, the date of birth was considered P0. Briefly, P1 mouse heads were harvested and flash frozen using liquid nitrogen in 1.5 ml microcentrifuge tubes. The whole head tissue was ground in liquid nitrogen using mortar and pestle before finer homogenization of lysate using a syringe and needle in TRIzol Reagent. cDNA was synthesized using SuperScript IV Reverse Transcriptase (Thermo Fisher Scientific), and qPCR was performed using primers targeting Cdh23 (Table S3) and SsoFast EvaGreen Supermix (Bio-Rad) or SensiFAST Probe No-ROX kit (Bioline) on a Bio-Rad CFX384 Real-Time PCR Detection System. Analysis of Cq values obtained from qRT-PCR was conducted using the comparative ΔΔCt method. Relative gene expression levels were calculated by normalizing the target gene expression to Gapdh and presented as fold changes.

Western blotting

Mice were decapitated at P1 and their heads were immediately transferred into 1.5 ml microcentrifuge tubes and flash frozen in liquid nitrogen. Heads were kept on liquid nitrogen and homogenized into a fine powder using a mortar and pestle. Ground tissue was then lysed in chilled RIPA lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 2 mM EDTA, 1.0% NP-40, 0.5% Na deoxycholate, 0.1% SDS) supplemented with a protease inhibitor cocktail (Sigma-Aldrich, SRE0055) before further homogenization with a needle and syringe. Samples were incubated on ice for 30 min with brief vortexing every 5 min before centrifugation at 16 800 × g for 20 min at 4°C. Supernatant was collected and quantified via Bradford Assay using Coomassie Plus Protein Assay Reagent (Thermo Fisher Scientific). Samples were resolved using a 6% Tris-glycine SDS-PAGE gel and blotted onto PVDF membrane by wet transfer overnight at 4°C. The membrane was washed 5 ×5 min in TBST, blocked for 1 h at room temperature (RT) using 5% skim milk in TBST (10 mM Tris base, 150 mM NaCl, 0.1% Tween-20) and incubated overnight at 4°C with primary antibody (TF7 primary antibody at 1:1000; Lamin [Abcam #133741] primary antibody at 1:15000) diluted in blocking solution. The next day, the membrane was washed 5 × 5 min in TBST, incubated for 2 h at RT in secondary antibody (HRP-conjugated goat anti-rabbit IgG at 1:100000, Invitrogen #31460) diluted in blocking solution, and again washed 5 × 5 min in TBST. Signal was detected by chemiluminescence using SuperSignal West Femto Maximum Sensitivity Substrate (Thermo Fisher Scientific).

Statistical analysis

Statistical analyses were performed in RStudio (version 3.4.2) and included Welch’s t-tests, Wilcoxon rank-sum tests, 2-way ANOVAs and post hoc Tukey’s Honest Significance tests where applicable. The α value was always set to 0.05. For measures of correlation, we used Spearman’s correlation coefficient (r2) to compare visual ABR threshold estimates between two observers (SS and KH). Intraclass correlation coefficient (ICC) scores were calculated to evaluate consistency between raters.

Conflict of interest statement

The authors declare no conflict of interests.

Funding

This work was supported by the Canadian Institutes of Health Research (PJT-166046 and PJT-183946 to H.Y.M.C.) and the Natural Sciences and Engineering Research Council of Canada (RGPIN-2016-05563 to H.Y.M.C. and RGPIN-2020-06906 to P.A.F.) This work was further supported by a John Evans Leadership Fund grant from the Canadian Foundation for Innovation (36131 to M.A.P.)

Data availability

Raw data generated by next-generation sequencing platforms have been deposited at the Sequence Read Archive (SRA) with the accession number PRJNA1072992 and are publicly available.

Supplementary Material

HMG_Submission_Supplementary_Revised_2024_06_03_ddae095

Supplementary_Movie_1_ddae095

Acknowledgements

The authors wish to thank Dr Thomas Friedman for the gift of the TF7 antibody and Dr Ashley Monks for use of the rotarod. Reagents and Cdh23awg/awg mice used in this study are available upon request from the corresponding author.
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