Patient clinical and audiometry data
Written informed consent was provided from all participating individuals. This study has been approved by the Faculty of Medicine ethics commissions at the University of Würzburg (46/15) and Shahid Chamran University of Ahvaz (#EE/97.24.3 17654). A three generation Iranian family of Lurs ethnicity was ascertained as part of a large ethnically diverse Iranian population rare disease study. Pure-tone audiograms and medical information were collected from participating members. Clinical examination excluded additional syndromic features.
Individuals IV-1, IV-6, and V-1 (Fig. 1) underwent complete ear, nose and throat examination, including binocular ear microscopy and external ear inspection. Routine pure-tone audiometry was performed according to current standards that measured hearing thresholds at frequencies 0.25, 0.5, 1, 2, 4, 6 and 8 kHz. Both air- and bone-conduction thresholds were determined. Severity of hearing loss was defined as previously described (Mazzoli et al. 2003). Individuals IV-1 and IV-6 underwent additional tympanometry and speech recognition threshold testing. Audiometry testing for individuals IV-1, IV-6, and V-1 was performed at ages 29, 44, and 20 years, respectively.
Genotyping, gene mapping, copy number variation and exome sequencing data analyses
Due to parental consanguinity and suspected autosomal recessive mode of inheritance, we assumed that the causal variant would be homozygous and identical by descent in affected individuals in the fourth generation of the family. Blood samples from 14 family members were obtained and genomic DNA was isolated from whole blood using standard procedures. DNA from affected (IV-1, IV-6, and IV-8) and unaffected (IV-2, IV-3, IV-4, and IV-5) individuals were genotyped using the Infinium Global Screening Array-24 v1.0 BeadChip (Illumina, San Diego, CA, USA) according to manufacturer’s protocols. Copy number variation calling was performed using GenomeStudio v.2011.1 and cnvPartition 3.2.0 (Illumina).
From the 618,540 markers on the array, we filtered out InDels, MT- and Y-chromosomal SNPs, multi-allelic SNPs, SNPs with missing genotypes in more than one individual, and SNPs having a minor allele frequency (MAF) lower than 5% in gnomAD European individuals (NFE) resulting in 242,705 bi-allelic SNPs for quality control (QC) and Linkage analysis. Data conversion to Linkage format files and QC was managed with the ALOHOMORA software (Rüschendorf and Nürnberg 2005). The sex of individuals was estimated by counting heterozygous genotypes on the X-chromosome and compared to the given pedigree data. The relationships between family members were verified with the program Graphical Relationship Representation (GRR) (Abecasis et al. 2001). PedCheck (O’Connell and Weeks 1998) was used to detect Mendelian errors (ME) and SNPs with ME were removed from the data set. Unlikely genotypes, e.g., double recombinants, were identified with Merlin (Abecasis et al. 2002) and deleted in the individuals.
Linkage analysis was performed with Merlin (Abecasis et al. 2002) using an autosomal recessive mode of inheritance with complete penetrance and a mutant allele frequency of 0.001. To avoid the problem of Linkage Disequilibrium (LD) between markers, which can lead to inflated LOD scores, we created a less dense, LD-reduced marker set of 91,426 SNPs with a minimal distance of 10,000 bases between markers and a MAF > 0.15. The exact position of the LOD score regions, e.g., the recombination events, were identified with the full marker set of 242,705 SNPs.
Additionally, homozygosity mapping was performed using HomozygosityMapper to identify common homozygous intervals among the affected individuals (Seelow et al. 2009). Runs of homozygosity with a maximum threshold of 0.99 were checked after the exome-wide analysis was completed.
For exome sequencing, DNA samples from two affected individuals (IV-1 and IV-6) were used. The data from individual IV-6 were analyzed exome-wide and data from individual IV-1 were used for determination of allele sharing. Exome capture using genomic DNA was performed using the SureSelect Target Enrichment v6 (Agilent) kit following manufacturer’s recommendations. The libraries were sequenced with a HiSeq4000 (Illumina). Data analysis was performed using the Burrows–Wheeler Alignment (BWA) tool for read mapping to the human reference genome GRCh37 (hg19), Picard for duplicate removal, GATK for local re-alignment, base recalibration, variant calling, and variant annotation, and SnpEff for variant annotation. Variant filtering was based on: coverage > 10X, Phred quality score ≥ 30, and MAF ≤ 0.005 as reported in 1000 Genomes Project and EVS6500. Variants were filtered based on coding effect (non-synonymous, synonymous, indels, and splice site variants), and artifact-prone genes (HLAs, MAGEs, MUCs, NBPFs, ORs, PRAMEs) were excluded. ACMG guidelines were used for variant interpretation (Oza et al. 2018). Visualization was performed using the Integrative Genomics Viewer. Analysis of homozygous and compound heterozygous variants between the two sequenced affected individuals (IV-6 and IV-1) followed. We analyzed missense variants using a combination of criteria that scored conservation using GERP++ and PhyloP, and deleterious or pathogenic scores in Combined Annotation Dependent Depletion (CADD) (Kircher et al. 2014), LRT (Chun and Fay 2009), MutationTaster (Schwarz et al. 2014), PolyPhen-2 (Adzhubei et al. 2010), and SIFT (Ng and Henikoff 2001). Missense variants were excluded when three out of five in silico pathogenicity prediction tools yielded a benign score. Manual MAF analysis used gnomAD (Lek et al. 2016), GME (Scott et al. 2016) and Iranome (Fattahi et al. 2019). Potential effects on splicing were assessed using ESEfinder (Cartegni et al. 2003) and RESCUE-ESE (Fairbrother et al. 2004).
Segregation, sequence and in vitro splicing analyses of the CLRN2 c.494C > A likely pathogenic variant
To confirm segregation of the CLRN2 c.494C > A; p.(Thr165Lys) (NM_001079827.2) homozygous variant, Sanger sequencing was completed in all 14 family members using the following primers (CLRN2 Ex3 F: 5′-AAATGCCACCTCTTACAGAGTTGC-3′ and CLRN2 Ex3 R: 5′-ACCGTGGCCTCTTCGATTTTGGTC-3′) and standard PCR and sequencing parameters.
To document residue conservation, CLRN1 (UniProt: P58418) and CLRN2 (UniProt: A0PK11) were aligned and visualized in Jalview (Waterhouse et al. 2009) with an overview of the pathogenic and likely pathogenic missense and nonsense CLRN1 variants retrieved from the Deafness Variation Database v 8.2 (Azaiez et al. 2018).
In addition, secondary protein structure prediction of human CLRN2 (NP_001073296.1) that included the wild-type (WT) and mutated amino acid residues was performed using I-TASSER (Yang et al. 2015).
To assess the splicing effect of the c.494C > A variant, in vitro splicing assays, also called mini-genes, were carried out as described (Booth et al. 2018a, b). WT CLRN2 exon 3 (266 bp) plus 183 and 51 nucleotides from intron 2 and the 3′UTR were PCR amplified with gene-specific primers containing Sall or SacII restriction enzyme sites, respectively. After PCR amplification, clean up, and restriction enzyme digestion, the PCR fragment was ligated into the pET01 Exontrap vector (MoBiTec) and the sequence was confirmed. Variants were then introduced into the WT sequence using QuikChange Lightning Site-Directed Mutagenesis (Agilent) according to the manufacturer’s protocols via overlapping primers containing the alteration. The WT and mutant mini-genes were sequence confirmed.
WT or mutant mini-genes were transfected in triplicates into COS-7 and ARPE-19 cells using TransIT-LT1 Transfection Reagent (Mirus). Cells were harvested 36 h after transfection and total RNA was extracted using Quick-RNA MiniPrep Plus kit (ZYMO Research). cDNA was transcribed using 750 ng of isolated RNA SuperScript™ III Reverse Transcriptase (ThermoFisher Scientific) using a primer specific to the 3′ native exon of the pET01 vector according to manufacturer’s protocol. PCR amplification followed using primers specific to the 5′ and 3′ native exons of the pET01 vector, and products were visualized on a 1.5% agarose gel. As a negative control, rs117875715 (chr4(GRCh37):g.17,528,480G > A), a benign polymorphism, was used to test and validate the designed mini-gene assay.
Concurrently, the mini-gene splice assay experiment was conducted in a double-blind manner as previously described (Lekszas et al. 2020). Genomic DNAs of an affected homozygous (IV-6) and WT individual (IV-5) were amplified using a forward primer with a XhoI restriction site (CLRN2 Ex3 XhoI F: 5′-aattctcgagTTGCAGTGAGCTGAGATGGT-3′) and a reverse primer with a BamHI restriction site (CLRN2 Ex3 BamHI R: 5′-attggatccGCCTTGCGAAGTTGTTACTG-3′). The 886 bp amplicon included the entire exon 3 sequence plus additional flanking 320 bp (5′) and 306 bp (3′) sequence that was ligated into a multiple cloning site between native exons A and B in the linearized pSPL3b exon-trapping vector. The vector was transformed into DH5α competent cells and plated overnight. All mutant mini-genes were Sanger sequence confirmed.
Homozygous and WT mini-genes were transfected in triplicate into HEK 293 T cells cultured in FCS-free medium in 6 well culture plates with a density of 2 × 105 cells per mL. The mini-genes in the pSPL3b vector were transiently transfected using 6 µl of FuGENE 6 Transfection Reagent (Roche) with 2 µg of vector. An empty vector and HEK 293 T cells were included as controls. The transfected cells were harvested 24–48 h post-transfection. Total RNA was prepared using the miRNAeasy Mini Kit (Qiagen). Approximately, 1 µg of RNA was reverse transcribed using a High Capacity RNA-to-cDNA Kit (Applied Biosystems) following manufacturer’s protocols. The cDNA was used for PCR amplification using a vector specific SD6 forward (5′-TCTGAGTCACCTGGACAACC-3′) and a terminal CLRN2 exon 3 reverse cDNA primer (5′-CAAGATATCCTCAGCTGTGACC-3′). The resulting amplified fragments were visualized on a 1.5% agarose gel. cDNA amplicons were Sanger sequenced. cDNA amplicons from the homozygous individual were cloned following standard protocols for the TA cloning (dual promoter with pCRII) kit (Invitrogen).
CRISPR/Cas9-mediated inactivation of clrn2 in zebrafish
Zebrafish (Danio rerio) were raised and maintained in an AALAC accredited facility at the Oklahoma Medical Research Foundation (OMRF) under standard conditions. Zebrafish embryos/larvae were maintained in embryo medium with 0.00002% methylene blue and raised at 28 °C. All animal experiments were performed as per protocol (17-01) and approved by the Institutional Animal Care Committee of OMRF (IACUC). All zebrafish handling, embryo care, and microinjections were performed as previously described (Westerfield 2000). WT zebrafish strain NHGRI-1 was used for all experiments (LaFave et al. 2014). The zebrafish embryonic staging was determined by morphological features according to (Kimmel et al. 1995).
To produce zebrafish clrn2 crispants, the sgRNA target sequences were selected from the UCSC genome browser tracks generated by the Burgess lab. Five independent targets were chosen and sgRNAs were synthesized by in vitro transcription as described earlier (Varshney et al. 2016). sgRNAs and Cas9 protein complex were used to generate indels. A 6 µL mixture containing 2 µL of 40 µM Spy Cas9 NLS protein (New England Biolabs, MA, USA), 200 ng each of five sgRNAs (in 2 µL) and 2 µL of 1 M potassium chloride was injected into one-cell-stage WT embryos. Injection volumes were calibrated to 1.4 nL per injection. Insertion/deletion (indel) variants were detected by amplifying the target region by PCR and Sanger sequencing as described earlier (Varshney et al. 2016). The sequencing data were analyzed by Inference of CRISPR Edits (ICE) v2 CRISPR analysis tool. The sgRNA target sequences and PCR primer sequences are listed in Supplementary Table S1.
Zebrafish RNA extraction and quantitative reverse transcription PCR (RT-qPCR)
Total RNA at different developmental stages, adult tissues, and CRISPR/Cas9 injected larvae were extracted using the TRIzol Reagent (Thermo Fisher Scientific, CA, USA) and purified by RNA clean and concentrator-5 kit (Zymo Research, CA, USA) according to the manufacturer’s instructions. RNA concentration was measured by DeNovix DS-11 spectrophotometer (DeNovix Inc., USA). The cDNA was synthesized by iScript RT Supermix (Bio-Rad, USA), and was used as a template for performing the RT-qPCR with SYBR Green Supermix (Thermo Fisher Scientific, CA, USA) and the Light Cycler® 96 System (Roche, CA, USA). All RT-qPCR reactions were carried out using three biological and technical replicates. The housekeeping gene 18S was used as a reference gene.
All RT-qPCR primer pairs were designed across exon-exon junctions using NCBI Primer-BLAST program (Supplementary Table S1). PCR cycling conditions were used as per manufacturer instructions. All reactions were carried out using three biological and technical replicates. The housekeeping gene 18S was used as a reference gene. Amplification specificity was assessed by dissociation curve analysis. The cycle threshold values (Ct) data were imported into Microsoft Excel for the relative gene expression analysis. Quantification was based on 2^(−ΔΔCT) method (Livak and Schmittgen 2001), and using 18 h post fertilization (hpf) for clarin 2 temporal expression, muscle for clarin 2 in different tissue expression and the corresponding age-matched control for clarin 2 CRISPR injected F0 larvae as normalization control.
Distribution of clrn2, phalloidin staining and behavioral analysis in zebrafish
To determine clrn2 expression, we used in situ hybridization on larvae and inner ear-containing cryosections. The full-length coding sequence of zebrafish clarin 2 (NM_001114690.1) was PCR amplified from WT zebrafish cDNA using primer pairs with BamHI and XhoI restriction sites cloned into the pCS2+ vector (a kind gift from Dr. Dave Turner, University of Michigan). After restriction digestion, the resulting clones were sequenced and used as templates for riboprobe synthesis. The digoxigenin-UTP-labeled riboprobes were synthesized according to the manufacturer's instructions (Millipore Sigma, MO, USA). Briefly, the clarin 2 and the pvalb9 plasmids (Horizon Discovery) were linearized by BamHI and NotI restriction enzymes, respectively. The linearized plasmid was purified and used as template for in vitro transcription using T7 RNA polymerase to synthesize anti-sense probes. The sense probe was made using XbaI linearized clarin 2 plasmid and SP6 RNA polymerase.
Whole-mount in situ hybridization (WISH) on 3 and 5 dpf zebrafish embryos/larvae was performed following the procedures as described by Thisse et al. with minor modifications (Thisse and Thisse 2008). Age-matched zebrafish embryos were randomly collected by breeding WT zebrafish pairs. The embryos were treated with 0.003% phenylthiourea (PTU) (Millipore Sigma, MO, USA) in embryo medium at 1 day post-fertilization (dpf) until the desired stages reached to reduce the pigment formation that will facilitate color visualization during in situ hybridization. Embryos/larvae were then fixed with 4% (V/V) paraformaldehyde in phosphate-buffered saline (PBS) at 3 and 5 dpf. An additional bleaching step was carried out after fixation by incubating the embryos at room temperature in a 3% hydrogen peroxide and 0.5% potassium hydroxide solution. The permeabilization of 3 dpf embryos and 5 dpf larvae was performed using 2 µg/mL proteinase K for 12 and 18 min, respectively. Color development was conducted using the BM-Purple alkaline phosphatase substrate (Millipore Sigma, MO, USA).
For preparation of cryo-sectioned samples after WISH, the 5 dpf larvae were soaked in 25, 30% (V/V) sucrose/PBS and optimum cutting temperature (OCT) each for at least 2 days, and embedded in OCT, then Cryotome sectioned at a 10-µm thickness.
For phalloidin staining of the zebrafish inner ear, 5 dpf larvae were euthanized with tricaine and fixed in 4% (V/V) paraformaldehyde (PFA) at 5 dpf, fixed embryos were washed by PBSTx (1% PBS, 0.2% triton X-100) and incubated in 2% triton X-100 in PBS at room temperature for overnight with agitation until the otoliths were completely dissolved. The larvae were sequentially washed in PBSTx and incubated with Alexa Fluor 488 Phalloidin (1:50) (Thermo Fisher Scientific, CA, USA) in PBSTw (1% PBS, 0.1% Tween-20) at room temperature for 4 h. The samples were washed in PBSTx after staining and mounted laterally in 75% glycerol on slides. Images were acquired with a Zeiss LSM-710 Confocal microscope.
To perform the acoustic evoked behavioral response (AEBR) test, 6 dpf larvae were placed in a 48-well plate with 200 µL embryo water and placed in a Zebrabox (ViewPoint Life Sciences) and embryos were adapted in the dark for 15–30 min (until spontaneous movements were less frequent). The embryos were subjected to a 100 ms, 1 kHz pure tone at 100% target power every 20 s for 4 min (12 stimuli) in the dark. The Zebrabox recorded the animals using infrared light and measured the activity as pixel changes over time. The burst threshold was set at 50 pixels, the freeze threshold set at 10 pixels, and sensitivity was set at 20 pixels. Movement over the 50 pixel burst threshold within the 2 s after stimulus was considered an evoked response. Responses were excluded if the larvae had spontaneous movement within the 2 s before the stimulus. All responses for a larva were excluded if they had spontaneous movement before 6 or more of the stimuli. The response rate was calculated by how many times an embryo had an evoked response out of the total number of stimuli and converted to a percentage.
Production and phenotyping of clarin 2 deficient mutant in mice
The Clrn2del629 mutant line was generated on a C57BL/6N background by the Molecular and Cellular Biology group at the Mary Lyon Centre (MLC), MRC Harwell Institute, using CRISPR/Cas9 genome editing (Dunbar et al. 2019). The mice were housed and maintained under specific pathogen-free conditions in individually ventilated cages, with environmental conditions as outlined in the Home Office Code of Practice. Animals were housed with littermates until weaned, and then housed with mice of the same sex and of similar ages, which was often their littermates. Both male and female animals were used for all experiments. Animal procedures at the MRC Harwell Institute were licenced by the Home Office under the Animals (Scientific Procedures) Act 1986, UK and additionally approved by the Institutional Animal Welfare and Ethical Review Body (AWERB). The Clrn1−/− mice (Clrn1tm1.2Ugpa, MGI: 6099052) used for comparative scanning electron microscopy analyses were previously described (Dulon et al. 2018).
To screen mice for auditory phenotypes and investigate auditory function, Auditory Brainstem Response (ABR) tests (measured using a click stimulus and frequency-specific tone-burst stimuli (at 8, 16 and 32 kHz) and Distortion Product Oto-Acoustic Emission (DPOAE) tests (measured using frequency-specific tone-burst stimuli from 8 to 32 kHz with the TDT RZ6 System 3 hardware and BioSig RZ software (Tucker Davis Technology, Alachua, FL, USA)) were performed as described in Dunbar et al., 2019. For scanning electron microscopy imaging, fixed inner ear samples were processed by the osmium tetroxide/thiocarbohydrazide (OTOTO) method, as previously described (Dulon et al. 2018; Dunbar et al. 2019). Samples were visualized with a JSM-6010LV Scanning Electron Microscope (JEOL). Micrographs were pseudo-coloured in Adobe Photoshop.
To compare the clrn2 mRNA expression in zebrafish, data are presented as mean values ± standard deviation (SD). Statistical analysis was performed using GraphPad Prism version 8.4 (GraphPad Software, San Diego, CA, USA). The significance level was set to 0.05. The p value was determined using a two-tailed unpaired Student’s t-test for RT-qPCR of clrn2 mRNA expression, and a two-tailed unpaired nonparametric Mann–Whitney U test for AEBR analysis. The statistical significance is represented in the figures as ***p < 0.001.
To assess ABR thresholds and DPOAE responses in the Clrn2del629 mice, one-way ANOVA statistical tests were used. Each frequency was tested for statistical significance separately. A threshold of p > 0.05 was used to determine if differences were statistically significant. Statistical significance is represented in the figure as follows: ***p < 0.001. All data shown are mean ± SD, and all statistical analyses was performed in GraphPad Prism.