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A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family
Accepted Manuscript A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family Yuan Liang, Kangwei Wang, Qi Peng, Pengyuan Zhu, Chunqiu Wu, Chunbao Rao, Jiang Chang, Siping Li, Xiaomei Lu PII:
S0165-5876(17)30552-9
DOI:
10.1016/j.ijporl.2017.11.009
Reference:
PEDOT 8773
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 15 August 2017 Revised Date:
8 November 2017
Accepted Date: 10 November 2017
Please cite this article as: Y. Liang, K. Wang, Q. Peng, P. Zhu, C. Wu, C. Rao, J. Chang, S. Li, X. Lu, A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.11.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family Yuan Liang1, 2¶, Kangwei Wang2¶, Qi Peng 2, Pengyuan Zhu4, Chunqiu Wu4, Chunbao
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Rao2, Jiang Chang5, Siping Li3**, Xiaomei Lu2*
1. Department of neurology, Dongguan Children's Hospital, Dongguan, Guangdong, China
2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
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Dongguan, Guangdong, China
3. Medical laboratory, Dongguan Children's Hospital, Dongguan, Guangdong, China
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4. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China
5. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan, Guangdong, China
¶ These authors contributed equally to this work.
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* Corresponding author. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, Guangdong, China. E-mail address: lxm020 @126.com (X. Lu).
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** Corresponding author. Medical laboratory, Dongguan Children's Hospital, Dong -guan, Guangdong, China. E-mail address: lsp020 @163.com (S. Li).
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We declare that we have no conflicts of interest.
ACCEPTED MANUSCRIPT A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family Yuan Liang1, 2¶, Kangwei Wang2¶, Qi Peng 2, Pengyuan Zhu4, Chunqiu Wu4, Chunbao
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Rao2, Jiang Chang5, Siping Li3**, Xiaomei Lu2*
1. Department of neurology, Dongguan Children's Hospital, Dongguan, Guangdong, China
2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
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Dongguan, Guangdong, China
3. Medical laboratory, Dongguan Children's Hospital, Dongguan, Guangdong, China
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4. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China
5. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan, Guangdong, China
¶ These authors contributed equally to this work.
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* Corresponding author. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, Guangdong, China. E-mail address: lxm020 @126.com (X. Lu).
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** Corresponding author. Medical laboratory, Dongguan Children's Hospital, Dong
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-guan, Guangdong, China. E-mail address: lsp020 @163.com (S. Li).
ACCEPTED MANUSCRIPT Abstract: Objectives： ：To explore the pathogenic causes of a proband who was diagnosed with non-syndromic hearing loss. Methods： ：We performed targeted capture of 159 known deafness-related genes and
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next-generation sequencing in the proband who was tested negative for the twenty hotspot variants in four common deafness-related genes（GJB2, GJB3, SLC26A4 and MTRNR1 ） ; Clinical reassessments, including detailed audiological and ocular examinations were performed in the proband and his normal parents.
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Results: We identified a novel heterozygous variant of CDH23:c.4567A>G (p.Asn1523Asp) in exon 37 (NM_022124), in conjunction with a reported mutation of
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CDH23:c.5101G>A (p.Glu1701Lys) in exon 40, to be a potentially pathogenic compound heterozygosity in the proband. The unaffected father has a heterozygous variant of CDH23:c.4567A>G, and the normal mother has another heterozygous variant, CDH23:c.5101G>A. The novel variant was absent in the 1000 Genomes Project. The clinical reassessments revealed binaural profound sensorineural hearing
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loss (DFNB12) without retinitis pigmentosa in the proband.
Conclusions This study demonstrates that the novel variant c.4567A>G (p.Asn1523Asp) in compound heterozygosity with c.5101G>A (p. Glu1701Lys) in the
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CDH23 gene is the main cause of DFNB12 in the proband. Simultaneously, this study provides a foundation to further elucidate the CDH23-related mechanisms of
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DFNB12.
Keywords: CDH23; DFNB12; Congenital hearing loss; Novel variant
ACCEPTED MANUSCRIPT Introduction Hearing loss is the most common type of sensory impairment and seriously impacts patients' normal daily life [1,2]. Although hearing loss is etiologically heterogeneous, it is estimated that congenital deafness is caused by genetic factors in
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more than 50% of affected patients [3,4]. To date, more than 150 genes have been verified to be associated with deafness (http://deafnessvariationdatabase.org/). Some of those genes can form different phenotypes with homozygous or heterozygous genotypes, which may result in non-syndromic hearing loss [5].
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CDH23 (NM_022124) has been reported to be a susceptibility-relevant gene linked to noise-induced hearing loss (NIHL); hearing loss can result when a
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detrimental variant occurs in this gene [6,7]. Usually, recessive, hereditary variants of CDH23 are associated with Usher Syndrome type 1D (USH1D) and non-syndromic hearing loss (DFNB12) [8,9]. USH1D is associated with severe manifestations, including visual problems due to retinitis pigmentosa, congenital profound deafness and vestibular areflexia. Conversely, DFNB12 is characterized by prelingual-onset
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non-syndromic sensorineural hearing loss (SNHL) but no impairment of vestibular or visual functions [7]. I In a Japanese study of children with hearing impairment, variants in GJB2 and SLC26A4 were most frequent, followed by variants in CDH23
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[10]. Recently, the genetic load of CDH23 variants and its implications were reported in a Korean pediatric deaf population [11,12]. In addition, a study mimicked the variants of CDH23 by destroying hair cell stereocilia in animal experiments, which
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resulted in noise-induced hearing loss (NIHL) and senile deafness [13]. In the present study, we performed gene capture and targeted next-generation
sequencing (NGS) to identify 159 deafness-associated genes in the family according to
the
Deafness
Variation
Database
(http://deafnessvariationdatabase.org/).
Interestingly, we found the compound heterozygous variants of CDH23:c.4567A>G and CDH23:c.5101G>A in the proband, who was diagnosed with non-syndromic hearing loss but who had negative genetic testing results for a panel of 20 common deafness-associated variants. However, his father had the same heterozygous variant of CDH23 c.4567A>G but none of the same clinical symptoms, which indicates that
ACCEPTED MANUSCRIPT the father was a heterozygous carrier of the c.4567A>G (p.Asn1523Asp) variant. We also found that the proband and his mother had an identical heterozygous variant of CDH23:c.5101G>A (p.Glu1701Lys), which had been reported as a potentially pathogenic allele, leading to DFNB12 in India, but the pathogenesis keeping unknown
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[14]. To our knowledge, this paper is the first report to identify the variant of CDH23:c.4567A> G (p.Asn1523Asp) in a patient with hearing loss. In addition, the compound heterozygous variants of CDH23 may jointly contribute to congenital
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hearing impairment.
Materials and methods
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Patients
This study was approved by Dongguan Children’s Hospital and conformed to the tenets of the Declaration of Helsinki. Written informed consent was obtained from the parents or legal guardians of the subjects.
All participants were from Guangdong, China. The proband, a 5-year-old boy with
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congenital non-progressive deafness, and his normal-hearing parents were invited to participate in this study. The family history and detailed medical history were obtained. Audiometric tests, including otoscope examination, tympanometry
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audiometry, auditory brainstem response (ABR), pure-tone audiometry (PTA), acoustic stapedial reflex and distortion product otoacoustic emission (DPOAEs), were also performed. Hearing impairment criteria established by the United States Joint
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Committee on Infant Hearing (JCIH) were used to evaluate the audiological data. The different degrees of hearing loss were classified as mild (30～40 dB HL), moderate (41～70 dB HL), severe (71～90 dB HL) and profound (>91 dB HL).
Targeted deafness gene capturing and NGS Genomic DNA was isolated from peripheral blood samples of the proband and his parents using a Blood DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. DNA amplification and quality inspection were performed using a NanoDrop 8000 ultraviolet visible spectrophotometer (Thermo
ACCEPTED MANUSCRIPT Fisher Scientific, Wilmington, DE, USA) and 1% agarose gel electrophoresis according to the protocol. Firstly, twenty hotspot variants of four common deafness-associated genes (GJB2, GJB3, SLC26A4, and MTRNR1) were screened by matrix-assisted laser desorption ionization time-of-flight mass spectrometry
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(MALDI-TOF-MS), and the negative genomic DNA results were used to prepare a library with a SureSelectXT Reagent Kit (Agilent Technologies, Santa Clara, CA, USA; Cat. no. G9611A). In total, 159 known deafness gene sequences, including all exons and some adjacent flanking intronic sequences, were captured by a custom
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array (Agilent Technologies Santa Clara, CA, USA) and were sequenced using single-end 160 bp reads by a BES 4000 sequencer. Then, variant call analyses were
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conducted by using TMAP and Torrent Mutation Caller (TVC) to map to HG19.
Sanger sequencing
Potential mutations, which were detected by next generation sequencing, were verified by PCR amplification and Sanger sequencing in the proband and
were
synthesized:
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corresponding variant region of his parents. The following sequence-specific primers 5’
CTTGCAGGTTGTGGCTTCTGA-3’
and
5’
-GCTGCCCTCACCTCATTCAC-3’ for human CDH23:c.4567A>G (p.Asn1523Asp);
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5’ -ATCGTCGCAGGCAACATCG-3’ and 5’ -GAGGTGAGGCGGTGGGATA-3’ for human CDH23:c.5101G>A (p. Glu1701Lys). The PCR was performed in a 50 µl reaction mixture, and the PCR products were purified by a PCR purification kit
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(TaKaRa, Japan) and sequenced with an ABI 3100 DNA sequencing machine (ABI, Foster City, CA, USA) and ABI 3100 Analysis Software (ver. 3.7 NT).
Bioinformatics and validation of the variants Sequence data were analyzed by DNA Star 5.0 software and aligned with the National Center for Biotechnology Information (NCBI) reference sequence of CDH23. Additionally, the NCBI dbSNP database and the 1000 Genomes Project database (http://www.1000genomes.org/) were used as references to assess the novelty of the variants found in this study. The possible pathogenicity was predicted according to the
ACCEPTED MANUSCRIPT online tool MutationTaster. Results Molecular genetic diagnosis and clinical features of the proband Hearing loss was diagnosed when the proband was 20 months old and characterized
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by significant delays in speech development by his parents. A clinical evaluation of the family was performed, including a detailed family medical history and a physical examination (Fig. 1A and 1B). Hearing loss was identified as congenital and resulted from the compilation of pathogenic variants carried by normal-hearing parents. In
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addition, his parents and medical staff haven't found the visual problems so far , as determined by detailed ophthalmologic examinations. And the detections of acoustic
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stapedial reflex, DPOAE, vestibular and visual functions were normal. Conclusively, the results of clinical detection indicated severe prelingual-onset non-syndromic hearing loss (DNFB12).
The audiograms of his parents were normal. ABR did not show any evoked response at or below 100 dB on bilateral of the proband. Moreover, PTA of the
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proband was determined. As shown in Fig. 1B, the clinical examination of the PTA showed that the left detectable evoked response was focused on 106 dB, with 0.5k Hz 89 dB, 1k Hz 99 dB, 2k Hz 107 dB, 4k Hz 115 dB and 8k Hz 120 dB. Similarly, the
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right detectable evoked response was focused on 117 dB, with 0.5k Hz 108 dB, 1k Hz 120 dB, 2k Hz 120 dB, 4k Hz 120 dB and 8k Hz 120 dB. These results led to the diagnosis of binaural profound sensorineural hearing loss. However, his language
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function well-developed because of a cochlear implant (CI) received at the age of two and a half years old.
Genetic analysis of the heterozygous variant We screened 20 hotspot variants in four common deafness-related genes, GJB2,
GJB3, SLC26A4 and MTRNR1; the results were negative (data not shown). However, we performed targeted capture of 159 known deafness-related genes and next-generation sequencing, and we identified a novel heterozygous variant of CDH23:c.4567A>G (p.Asn1523Asp) in exon 37 (NM_022124) of and CDH23: c.5101G>A (p.Glu1701Lys) in exon 40, suggesting a potentially pathogenic
ACCEPTED MANUSCRIPT compound heterozygosity in the proband. The two variants came from the proband’s father, who has the variant CDH23:c.4567A>G (p.Asn1523Asp) in exon 37, and mother, who has the variant CDH23:c.5101G>A (p.Glu1701Lys) in exon 40 (Fig. 2A). In addition, we found that the parents both have a heterozygous variant of CDH23:c.
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5100C>T, but it was not detected in the proband. Fortunately, this variant does not lead to a change in the amino acid sequence and is considered a benign variant.
The variant CDH23:c.4567A>G (p.Asn1523Asp) is predicted to be probably damaging, with a score of 0.995 (sensitivity: 0.68; specificity: 0.97), by prediction of
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functional effects of human non-synonymous SNPs (http: //genetics.bwh.harvard. edu/pph2/) and is predicted to be disease causing, with a score of 1 by MutationTaster,
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which strongly suggests that the novel variant is closely associated with DFNB12. The novel missense variant of CDH23:c.4567A>G (p.Asn1523Asp) led to the substitution of an AAC codon with a GAC codon at position 1523. At the amino acid level, Asn was replaced by Asp in site 1523 when the variant CDH23:c.4567A>G is present. However, due to the identified coding variants (p.Asn1523Asp and
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Glu1701Lys) located in structurally or functionally important protein domains (Fig. 3A), this residue was highly conserved in different species as shown by multiple sequence alignment analyses in PolyPhen-2 (Fig. 2B). Therefore, the changes in
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amino acid sequences may seriously impact the normal development of hearing (Fig. 2B). We show a scheme of the mutant protein structure as shown in Fig3B, in the CDH23 isoform X1 protein sequence, the DXNDN motifs in all the adherin repeats
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are highly conserved. Sequence alignment with the cadherin repeat in CDH23 with the crystal structure available protein neural (N-) cadherin (PDB ID: 1NCJ) shows that the DXNDN motif chelates a calcium ion in which the main chain of the last Asn residue, which is corresponding Asn1523 in CDH23, involves in chelating calcium ion, together with the side chains of two negatively-charged Asp residues and another Asn in the DXNDN motif, yet it is not very unclear why Asn1523Asp mutation in CDH23 affects the function of the protein. Discussion In the present study, we captured the targeted sequence, used next-generation
ACCEPTED MANUSCRIPT sequencing and identified a novel heterozygous variant of CDH23 in a Chinese family that included a proband who was diagnosed with non-syndromic autosomal recessive hearing loss and his normal parents. To the best of our knowledge, this is the first report to find that the compound heterozygous variants of CDH23 in sites of
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c.4567A>G (p.Asn1523Asp) and c.5101G>A (p.Glu1701Lys) may result in hearing impairment. Based on American College of Medical Genetics and Genomics (ACMG) criteria [14], there are several evidences for pathogenicity of the novel variation c.4567A>G including two moderate evidences (located in a well-established
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functional domain without benign variation and absent from controls in population databases) and three supporting evidences (missense variant in a gene that has a low
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rate of benign missense variation and where missense variants are a common mechanism of disease, multiple lines of computational evidence support a deleterious effect on the gene product and the family history is highly specific for hearing loss with a single genetic etiology). Therefore, this novel variation can be classified as a likely
pathogenic
variant.
Regarding
that,
clinical
diagnosis
(PND)
and
of the pedigree.
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pre-implantation genetic testing (PGD) can be recommended in the future pregnancies
The CDH23:c.5101G>A (p.Glu1701Lys) variant was first reported by Ganapathy A
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et al. [15] based on studying the spectrum of CDH23 variants in Indian patients with hearing loss. However, another variant, CDH23:c.4567A>G (p.Asn1523Asp), was reported for the first time in this study. This variant identified in our study is in the
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highly conserved domain of the CDH23 gene and predicted to be pathogenic. This variant leads to the p.Asn1523Asp transition at amino acid position 1523, which may result in serious dysfunction of CDH23 (Figs. 2B and 3) and affect normal hearing development.
Cadherins are calcium-dependent cell adhesion proteins that preferentially interact with themselves in a homophilic manner in connecting cells. CDH23 is required for establishing and maintaining the proper organization of the stereocilia bundle of hair cells during late embryonic or early postnatal development in the cochlea and the vestibule. Hence, this functional domain maintains normal hearing by mediating
ACCEPTED MANUSCRIPT mechanotransduction in cochlear hair cells. In addition, the variants of CDH23 are associated with sensory impairment via destruction of tip links, which are formed through interactions among CDH23, PCDH15 and lower parts of the tip link complex [7, 16]. Moreover, variants in the CDH23 gene have been linked to autosomal
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recessive deafness [8]. CDH23 is an essential component of assembling the hair cell tip link complex required for hair-cell mechanotransduction, and it has a decisive role in ensuring the structural integrity of the tip link but not of the hair bundle, depending on whether
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there are weak alleles of CDH23 [13, 17, 18]. Accordingly, in the present study, potentially pathogenic compound heterozygous alleles of CDH23 may affect hair-cell
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mechanotransduction, causing deformation and resulting in hearing loss. Related report indicated that CDH23 pathogenic variants were identified in patients with a phenotype more consistent with type II than type I Usher syndrome [19]. Simultaneously, recent data showing that one DFNB12 allele in trans configuration to an USH1D allele results in non-syndromic deafness [20]. However, the proband, who
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is 5 years old, although his parents and medical staff haven't found the visual problems so far, but retinitis pigmentosa in the patient may manifest later in life, we do not rule out the possibility of the proband with Usher syndrome yet, so we will
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follow-up examination to adulthood.
In future studies, we will focus on the construction of a transgenic mouse model
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carrying these variants to reveal the mechanism of hearing impairment.
Figures and legends
Fig. 1. Molecular genetic diagnosis and clinical features of the proband. (A) Pedigree of the family. Arrow indicates the proband. (B) PTA results of the proband.
Fig. 2. Genetic analysis of the heterozygous variant. (A) The DNA sequencing profile shows the c.4567A>G (p.Asn1523Asp) and c.5101G>A (p. Glu1701Lys) variants in CDH23. (B) Protein alignment of CDH23 in
ACCEPTED MANUSCRIPT different species, which shows the conservation of residue p. Asn1523Asp.
Fig. 3. Schematic representation of the structure of CDH23. (A) Schematic structure of CDH23. (B) the variant CDH23 protein structure. Black
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arrow (above) points to the previously identified variant, and the red arrow (below)
transmembrane domain.
Conflicts of interest We declare that we have no conflicts of interest.
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Acknowledgments
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points to the variant identified in this study. N: N-terminal; C: C-terminal; TM:
This work was supported by the Dongguan Bureau of Science and Technology for the City Key Program of Science and Technology (Project Number: 2013108101018) and the Science and Technology Planning Project of Guangdong Province (Project Number: 2014A020213001). We also thank all the neonates and their parents for their
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References
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cooperation during this work.
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S0165-5876(17)30552-9
DOI:
10.1016/j.ijporl.2017.11.009
Reference:
PEDOT 8773
To appear in:
International Journal of Pediatric Otorhinolaryngology
Received Date: 15 August 2017 Revised Date:
8 November 2017
Accepted Date: 10 November 2017
Please cite this article as: Y. Liang, K. Wang, Q. Peng, P. Zhu, C. Wu, C. Rao, J. Chang, S. Li, X. Lu, A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family, International Journal of Pediatric Otorhinolaryngology (2017), doi: 10.1016/j.ijporl.2017.11.009. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family Yuan Liang1, 2¶, Kangwei Wang2¶, Qi Peng 2, Pengyuan Zhu4, Chunqiu Wu4, Chunbao
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Rao2, Jiang Chang5, Siping Li3**, Xiaomei Lu2*
1. Department of neurology, Dongguan Children's Hospital, Dongguan, Guangdong, China
2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
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Dongguan, Guangdong, China
3. Medical laboratory, Dongguan Children's Hospital, Dongguan, Guangdong, China
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4. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China
5. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan, Guangdong, China
¶ These authors contributed equally to this work.
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* Corresponding author. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, Guangdong, China. E-mail address: lxm020 @126.com (X. Lu).
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** Corresponding author. Medical laboratory, Dongguan Children's Hospital, Dong -guan, Guangdong, China. E-mail address: lsp020 @163.com (S. Li).
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We declare that we have no conflicts of interest.
ACCEPTED MANUSCRIPT A novel variant in the CDH23 gene is associated with non-syndromic hearing loss in a Chinese family Yuan Liang1, 2¶, Kangwei Wang2¶, Qi Peng 2, Pengyuan Zhu4, Chunqiu Wu4, Chunbao
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Rao2, Jiang Chang5, Siping Li3**, Xiaomei Lu2*
1. Department of neurology, Dongguan Children's Hospital, Dongguan, Guangdong, China
2. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics,
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Dongguan, Guangdong, China
3. Medical laboratory, Dongguan Children's Hospital, Dongguan, Guangdong, China
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4. CapitalBio Genomics Co.,Ltd, Dongguan, Guangdong, China
5. Department of otorhinolaryngological, Dongguan Children's Hospital, Dongguan, Guangdong, China
¶ These authors contributed equally to this work.
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* Corresponding author. Department of Medical and Molecular Genetics, Dongguan Institute of Pediatrics, Dongguan, Guangdong, China. E-mail address: lxm020 @126.com (X. Lu).
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** Corresponding author. Medical laboratory, Dongguan Children's Hospital, Dong
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-guan, Guangdong, China. E-mail address: lsp020 @163.com (S. Li).
ACCEPTED MANUSCRIPT Abstract: Objectives： ：To explore the pathogenic causes of a proband who was diagnosed with non-syndromic hearing loss. Methods： ：We performed targeted capture of 159 known deafness-related genes and
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next-generation sequencing in the proband who was tested negative for the twenty hotspot variants in four common deafness-related genes（GJB2, GJB3, SLC26A4 and MTRNR1 ） ; Clinical reassessments, including detailed audiological and ocular examinations were performed in the proband and his normal parents.
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Results: We identified a novel heterozygous variant of CDH23:c.4567A>G (p.Asn1523Asp) in exon 37 (NM_022124), in conjunction with a reported mutation of
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CDH23:c.5101G>A (p.Glu1701Lys) in exon 40, to be a potentially pathogenic compound heterozygosity in the proband. The unaffected father has a heterozygous variant of CDH23:c.4567A>G, and the normal mother has another heterozygous variant, CDH23:c.5101G>A. The novel variant was absent in the 1000 Genomes Project. The clinical reassessments revealed binaural profound sensorineural hearing
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loss (DFNB12) without retinitis pigmentosa in the proband.
Conclusions This study demonstrates that the novel variant c.4567A>G (p.Asn1523Asp) in compound heterozygosity with c.5101G>A (p. Glu1701Lys) in the
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CDH23 gene is the main cause of DFNB12 in the proband. Simultaneously, this study provides a foundation to further elucidate the CDH23-related mechanisms of
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DFNB12.
Keywords: CDH23; DFNB12; Congenital hearing loss; Novel variant
ACCEPTED MANUSCRIPT Introduction Hearing loss is the most common type of sensory impairment and seriously impacts patients' normal daily life [1,2]. Although hearing loss is etiologically heterogeneous, it is estimated that congenital deafness is caused by genetic factors in
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more than 50% of affected patients [3,4]. To date, more than 150 genes have been verified to be associated with deafness (http://deafnessvariationdatabase.org/). Some of those genes can form different phenotypes with homozygous or heterozygous genotypes, which may result in non-syndromic hearing loss [5].
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CDH23 (NM_022124) has been reported to be a susceptibility-relevant gene linked to noise-induced hearing loss (NIHL); hearing loss can result when a
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detrimental variant occurs in this gene [6,7]. Usually, recessive, hereditary variants of CDH23 are associated with Usher Syndrome type 1D (USH1D) and non-syndromic hearing loss (DFNB12) [8,9]. USH1D is associated with severe manifestations, including visual problems due to retinitis pigmentosa, congenital profound deafness and vestibular areflexia. Conversely, DFNB12 is characterized by prelingual-onset
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non-syndromic sensorineural hearing loss (SNHL) but no impairment of vestibular or visual functions [7]. I In a Japanese study of children with hearing impairment, variants in GJB2 and SLC26A4 were most frequent, followed by variants in CDH23
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[10]. Recently, the genetic load of CDH23 variants and its implications were reported in a Korean pediatric deaf population [11,12]. In addition, a study mimicked the variants of CDH23 by destroying hair cell stereocilia in animal experiments, which
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resulted in noise-induced hearing loss (NIHL) and senile deafness [13]. In the present study, we performed gene capture and targeted next-generation
sequencing (NGS) to identify 159 deafness-associated genes in the family according to
the
Deafness
Variation
Database
(http://deafnessvariationdatabase.org/).
Interestingly, we found the compound heterozygous variants of CDH23:c.4567A>G and CDH23:c.5101G>A in the proband, who was diagnosed with non-syndromic hearing loss but who had negative genetic testing results for a panel of 20 common deafness-associated variants. However, his father had the same heterozygous variant of CDH23 c.4567A>G but none of the same clinical symptoms, which indicates that
ACCEPTED MANUSCRIPT the father was a heterozygous carrier of the c.4567A>G (p.Asn1523Asp) variant. We also found that the proband and his mother had an identical heterozygous variant of CDH23:c.5101G>A (p.Glu1701Lys), which had been reported as a potentially pathogenic allele, leading to DFNB12 in India, but the pathogenesis keeping unknown
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[14]. To our knowledge, this paper is the first report to identify the variant of CDH23:c.4567A> G (p.Asn1523Asp) in a patient with hearing loss. In addition, the compound heterozygous variants of CDH23 may jointly contribute to congenital
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hearing impairment.
Materials and methods
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Patients
This study was approved by Dongguan Children’s Hospital and conformed to the tenets of the Declaration of Helsinki. Written informed consent was obtained from the parents or legal guardians of the subjects.
All participants were from Guangdong, China. The proband, a 5-year-old boy with
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congenital non-progressive deafness, and his normal-hearing parents were invited to participate in this study. The family history and detailed medical history were obtained. Audiometric tests, including otoscope examination, tympanometry
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audiometry, auditory brainstem response (ABR), pure-tone audiometry (PTA), acoustic stapedial reflex and distortion product otoacoustic emission (DPOAEs), were also performed. Hearing impairment criteria established by the United States Joint
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Committee on Infant Hearing (JCIH) were used to evaluate the audiological data. The different degrees of hearing loss were classified as mild (30～40 dB HL), moderate (41～70 dB HL), severe (71～90 dB HL) and profound (>91 dB HL).
Targeted deafness gene capturing and NGS Genomic DNA was isolated from peripheral blood samples of the proband and his parents using a Blood DNA Kit (Tiangen Biotech, Beijing, China) following the manufacturer’s instructions. DNA amplification and quality inspection were performed using a NanoDrop 8000 ultraviolet visible spectrophotometer (Thermo
ACCEPTED MANUSCRIPT Fisher Scientific, Wilmington, DE, USA) and 1% agarose gel electrophoresis according to the protocol. Firstly, twenty hotspot variants of four common deafness-associated genes (GJB2, GJB3, SLC26A4, and MTRNR1) were screened by matrix-assisted laser desorption ionization time-of-flight mass spectrometry
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(MALDI-TOF-MS), and the negative genomic DNA results were used to prepare a library with a SureSelectXT Reagent Kit (Agilent Technologies, Santa Clara, CA, USA; Cat. no. G9611A). In total, 159 known deafness gene sequences, including all exons and some adjacent flanking intronic sequences, were captured by a custom
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array (Agilent Technologies Santa Clara, CA, USA) and were sequenced using single-end 160 bp reads by a BES 4000 sequencer. Then, variant call analyses were
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conducted by using TMAP and Torrent Mutation Caller (TVC) to map to HG19.
Sanger sequencing
Potential mutations, which were detected by next generation sequencing, were verified by PCR amplification and Sanger sequencing in the proband and
were
synthesized:
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corresponding variant region of his parents. The following sequence-specific primers 5’
CTTGCAGGTTGTGGCTTCTGA-3’
and
5’
-GCTGCCCTCACCTCATTCAC-3’ for human CDH23:c.4567A>G (p.Asn1523Asp);
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5’ -ATCGTCGCAGGCAACATCG-3’ and 5’ -GAGGTGAGGCGGTGGGATA-3’ for human CDH23:c.5101G>A (p. Glu1701Lys). The PCR was performed in a 50 µl reaction mixture, and the PCR products were purified by a PCR purification kit
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(TaKaRa, Japan) and sequenced with an ABI 3100 DNA sequencing machine (ABI, Foster City, CA, USA) and ABI 3100 Analysis Software (ver. 3.7 NT).
Bioinformatics and validation of the variants Sequence data were analyzed by DNA Star 5.0 software and aligned with the National Center for Biotechnology Information (NCBI) reference sequence of CDH23. Additionally, the NCBI dbSNP database and the 1000 Genomes Project database (http://www.1000genomes.org/) were used as references to assess the novelty of the variants found in this study. The possible pathogenicity was predicted according to the
ACCEPTED MANUSCRIPT online tool MutationTaster. Results Molecular genetic diagnosis and clinical features of the proband Hearing loss was diagnosed when the proband was 20 months old and characterized
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by significant delays in speech development by his parents. A clinical evaluation of the family was performed, including a detailed family medical history and a physical examination (Fig. 1A and 1B). Hearing loss was identified as congenital and resulted from the compilation of pathogenic variants carried by normal-hearing parents. In
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addition, his parents and medical staff haven't found the visual problems so far , as determined by detailed ophthalmologic examinations. And the detections of acoustic
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stapedial reflex, DPOAE, vestibular and visual functions were normal. Conclusively, the results of clinical detection indicated severe prelingual-onset non-syndromic hearing loss (DNFB12).
The audiograms of his parents were normal. ABR did not show any evoked response at or below 100 dB on bilateral of the proband. Moreover, PTA of the
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proband was determined. As shown in Fig. 1B, the clinical examination of the PTA showed that the left detectable evoked response was focused on 106 dB, with 0.5k Hz 89 dB, 1k Hz 99 dB, 2k Hz 107 dB, 4k Hz 115 dB and 8k Hz 120 dB. Similarly, the
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right detectable evoked response was focused on 117 dB, with 0.5k Hz 108 dB, 1k Hz 120 dB, 2k Hz 120 dB, 4k Hz 120 dB and 8k Hz 120 dB. These results led to the diagnosis of binaural profound sensorineural hearing loss. However, his language
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function well-developed because of a cochlear implant (CI) received at the age of two and a half years old.
Genetic analysis of the heterozygous variant We screened 20 hotspot variants in four common deafness-related genes, GJB2,
GJB3, SLC26A4 and MTRNR1; the results were negative (data not shown). However, we performed targeted capture of 159 known deafness-related genes and next-generation sequencing, and we identified a novel heterozygous variant of CDH23:c.4567A>G (p.Asn1523Asp) in exon 37 (NM_022124) of and CDH23: c.5101G>A (p.Glu1701Lys) in exon 40, suggesting a potentially pathogenic
ACCEPTED MANUSCRIPT compound heterozygosity in the proband. The two variants came from the proband’s father, who has the variant CDH23:c.4567A>G (p.Asn1523Asp) in exon 37, and mother, who has the variant CDH23:c.5101G>A (p.Glu1701Lys) in exon 40 (Fig. 2A). In addition, we found that the parents both have a heterozygous variant of CDH23:c.
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5100C>T, but it was not detected in the proband. Fortunately, this variant does not lead to a change in the amino acid sequence and is considered a benign variant.
The variant CDH23:c.4567A>G (p.Asn1523Asp) is predicted to be probably damaging, with a score of 0.995 (sensitivity: 0.68; specificity: 0.97), by prediction of
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functional effects of human non-synonymous SNPs (http: //genetics.bwh.harvard. edu/pph2/) and is predicted to be disease causing, with a score of 1 by MutationTaster,
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which strongly suggests that the novel variant is closely associated with DFNB12. The novel missense variant of CDH23:c.4567A>G (p.Asn1523Asp) led to the substitution of an AAC codon with a GAC codon at position 1523. At the amino acid level, Asn was replaced by Asp in site 1523 when the variant CDH23:c.4567A>G is present. However, due to the identified coding variants (p.Asn1523Asp and
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Glu1701Lys) located in structurally or functionally important protein domains (Fig. 3A), this residue was highly conserved in different species as shown by multiple sequence alignment analyses in PolyPhen-2 (Fig. 2B). Therefore, the changes in
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amino acid sequences may seriously impact the normal development of hearing (Fig. 2B). We show a scheme of the mutant protein structure as shown in Fig3B, in the CDH23 isoform X1 protein sequence, the DXNDN motifs in all the adherin repeats
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are highly conserved. Sequence alignment with the cadherin repeat in CDH23 with the crystal structure available protein neural (N-) cadherin (PDB ID: 1NCJ) shows that the DXNDN motif chelates a calcium ion in which the main chain of the last Asn residue, which is corresponding Asn1523 in CDH23, involves in chelating calcium ion, together with the side chains of two negatively-charged Asp residues and another Asn in the DXNDN motif, yet it is not very unclear why Asn1523Asp mutation in CDH23 affects the function of the protein. Discussion In the present study, we captured the targeted sequence, used next-generation
ACCEPTED MANUSCRIPT sequencing and identified a novel heterozygous variant of CDH23 in a Chinese family that included a proband who was diagnosed with non-syndromic autosomal recessive hearing loss and his normal parents. To the best of our knowledge, this is the first report to find that the compound heterozygous variants of CDH23 in sites of
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c.4567A>G (p.Asn1523Asp) and c.5101G>A (p.Glu1701Lys) may result in hearing impairment. Based on American College of Medical Genetics and Genomics (ACMG) criteria [14], there are several evidences for pathogenicity of the novel variation c.4567A>G including two moderate evidences (located in a well-established
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functional domain without benign variation and absent from controls in population databases) and three supporting evidences (missense variant in a gene that has a low
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rate of benign missense variation and where missense variants are a common mechanism of disease, multiple lines of computational evidence support a deleterious effect on the gene product and the family history is highly specific for hearing loss with a single genetic etiology). Therefore, this novel variation can be classified as a likely
pathogenic
variant.
Regarding
that,
clinical
diagnosis
(PND)
and
of the pedigree.
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pre-implantation genetic testing (PGD) can be recommended in the future pregnancies
The CDH23:c.5101G>A (p.Glu1701Lys) variant was first reported by Ganapathy A
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et al. [15] based on studying the spectrum of CDH23 variants in Indian patients with hearing loss. However, another variant, CDH23:c.4567A>G (p.Asn1523Asp), was reported for the first time in this study. This variant identified in our study is in the
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highly conserved domain of the CDH23 gene and predicted to be pathogenic. This variant leads to the p.Asn1523Asp transition at amino acid position 1523, which may result in serious dysfunction of CDH23 (Figs. 2B and 3) and affect normal hearing development.
Cadherins are calcium-dependent cell adhesion proteins that preferentially interact with themselves in a homophilic manner in connecting cells. CDH23 is required for establishing and maintaining the proper organization of the stereocilia bundle of hair cells during late embryonic or early postnatal development in the cochlea and the vestibule. Hence, this functional domain maintains normal hearing by mediating
ACCEPTED MANUSCRIPT mechanotransduction in cochlear hair cells. In addition, the variants of CDH23 are associated with sensory impairment via destruction of tip links, which are formed through interactions among CDH23, PCDH15 and lower parts of the tip link complex [7, 16]. Moreover, variants in the CDH23 gene have been linked to autosomal
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recessive deafness [8]. CDH23 is an essential component of assembling the hair cell tip link complex required for hair-cell mechanotransduction, and it has a decisive role in ensuring the structural integrity of the tip link but not of the hair bundle, depending on whether
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there are weak alleles of CDH23 [13, 17, 18]. Accordingly, in the present study, potentially pathogenic compound heterozygous alleles of CDH23 may affect hair-cell
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mechanotransduction, causing deformation and resulting in hearing loss. Related report indicated that CDH23 pathogenic variants were identified in patients with a phenotype more consistent with type II than type I Usher syndrome [19]. Simultaneously, recent data showing that one DFNB12 allele in trans configuration to an USH1D allele results in non-syndromic deafness [20]. However, the proband, who
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is 5 years old, although his parents and medical staff haven't found the visual problems so far, but retinitis pigmentosa in the patient may manifest later in life, we do not rule out the possibility of the proband with Usher syndrome yet, so we will
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follow-up examination to adulthood.
In future studies, we will focus on the construction of a transgenic mouse model
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carrying these variants to reveal the mechanism of hearing impairment.
Figures and legends
Fig. 1. Molecular genetic diagnosis and clinical features of the proband. (A) Pedigree of the family. Arrow indicates the proband. (B) PTA results of the proband.
Fig. 2. Genetic analysis of the heterozygous variant. (A) The DNA sequencing profile shows the c.4567A>G (p.Asn1523Asp) and c.5101G>A (p. Glu1701Lys) variants in CDH23. (B) Protein alignment of CDH23 in
ACCEPTED MANUSCRIPT different species, which shows the conservation of residue p. Asn1523Asp.
Fig. 3. Schematic representation of the structure of CDH23. (A) Schematic structure of CDH23. (B) the variant CDH23 protein structure. Black
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arrow (above) points to the previously identified variant, and the red arrow (below)
transmembrane domain.
Conflicts of interest We declare that we have no conflicts of interest.
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Acknowledgments
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points to the variant identified in this study. N: N-terminal; C: C-terminal; TM:
This work was supported by the Dongguan Bureau of Science and Technology for the City Key Program of Science and Technology (Project Number: 2013108101018) and the Science and Technology Planning Project of Guangdong Province (Project Number: 2014A020213001). We also thank all the neonates and their parents for their
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