Novel recessive PDZD7 biallelic mutations associated with hereditary hearing loss in a Chinese pedigree

Gene 709 (2019) 65–74

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Research paper

Novel recessive PDZD7 biallelic mutations associated with hereditary hearing loss in a Chinese pedigree Hualei Luo, Reem N. Hassan, Jin Yan, Jie Xie, Peng Du, Qiuyue Hu, Yue Zhu, Weiying Jiang

T ⁎

Department of Medical Genetics, Zhongshan School of Medicine, Sun Yat-sen University, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Autosomal recessive non-syndromic hearing loss Next-generation sequencing Novel mutations PDZD7 gene Pathogenicity assessment

Background: Autosomal recessive non-syndromic hearing loss (ARNSHL) is a highly heterogeneous genetic disease. PDZD7 is a new ARNSHL associated gene. Until now, nine PDZD7 biallelic mutation families with ARNSHL have been reported. Here we report a case of Chinese patient with ARNSHL linked to novel mutations in PDZD7 genes. Method: The pathogenic mutations were detected by whole exome sequencing for hereditary deafness-related genes of both the proband and his parents. We used kinship detection, mutational hazard prediction, genotypephenotype correlation analysis and variation screening for potential pathogenic mutations. Re-sequencing was used to confirm the mutations by Sanger sequence. Real time quantitative PCR (RT-qPCR) was used to analyze the PDZD7 gene expression. Population-based screening for variation frequency, evolutionary conservation comparisons, pathogenicity evaluation, and protein structure prediction were conducted to assess the pathogenicity of the novel mutations of PDZD7 gene. Results: We determined three variants of the PDZD7 gene that contributed to the deafness of the patient (PDZD7 c.192G > A, p. Met64Ile; c.1648C > T p. Gln550* and c.2341_2352delCGCAGCCGCAGCp. Arg781_Ser 784del). Pathogenic analysis in accordance with the ACMG/AMP Standards and Guidelines identified two novel mutations as Likely Pathogenic. The expression level of PDZD7 gene in the patient was decreased compared to the normal control (P < 0.001). Conclusion: Three mutations in PDZD7 gene linked to ARNSHL were identified in a Chinese pedigree. The findings expand not only our knowledge of genetic causes of ARNSHL, but also PDZD7 genes mutation spectrum of the disease. They will aid personalized genetic counseling, molecular diagnostics and clinical management of this condition.

1. Introduction Hearing loss is a common sensory neurological disorder, with at least half of all cases caused by genetic factors (Mahboubi et al., 2012). The hereditary type of deafness has high clinical and genetic heterogeneity (Guan et al., 2018). Depending on whether a patient has other symptoms, the condition can be classified into syndromic deafness (30%) and non-syndromic deafness (70%) (Morton and Nance, 2006). The most common genetic pattern is autosomal recessive (AR)

inheritance, which accounts for approximately 75–80% of all cases. Common genes linked to this inheritance pattern include MYO7A, USH1C, CDH23, PCDH15, USH2A, VLGR1, USH3A, PDZD7, CLCNKA and others. Autosomal dominant (AD) inheritance accounts for about 10–20% of clinical cases and can be associated with the genes PAX3, MITF, EDRRB, TCOF1, COL2A1, NF2, DFNA5 and others. In addition, there is a sex-linked inheritance, which accounts for 1–5% of cases and linked to the genes COL4A5, TIMM8a, GLA and POU3F4. Finally, a very rare (0–2% of cases) inheritance mode is mitochondrial one, which is

Abbreviations: ARNSHL, autosomal recessive non-syndromic hearing loss; RT-qPCR, real time quantitative polymerase chain reaction; AR, autosomal recessive; AD, autosomal dominant; USH2C, Usher syndrome 2C; OMIM, Online Mendelian inheritance in man; PND, prenatal diagnosis; ACMG/AMP, American College of Medical Genetics and Genomics and the Association for Molecular Pathology; NGS, next generation sequencing; EDTA, Ethylene diamine tetraacetic acid; PolyPhen, Polymorphism phenotyping; SIFT, Sorting intolerant from tolerant; ORF, Open Reading Frame; SNP, Single Nucleotide Polymorphism; NHLBI, National Heart, Lung, and Blood Institute; ESP, Exome Sequencing Project; HGMD, Human Gene Mutation Database; PVS, Very strong evidence of pathogenicity; PM, Moderate evidence of pathogenicity; PP, Supporting evidence of pathogenicity; BS, Strong evidence of benign impact; BP, Supporting evidence of benign impact ⁎ Corresponding author at: Department of Medical Genetics, Zhongshan School of Medicine, Sun Yat-sen University, No. 74, Zhongshan Second Road, Guangzhou 410080, China. E-mail address: [email protected] (W. Jiang). https://doi.org/10.1016/j.gene.2019.05.045 Received 23 February 2019; Received in revised form 26 April 2019; Accepted 22 May 2019 Available online 23 May 2019 0378-1119/ © 2019 Elsevier B.V. All rights reserved.

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Fig. 1. Pedigree chart of the family with hereditary deafness. IV1 is the proband. His genotype is PDZD7 exon2 c.192G > A p. Met64Ile, PDZD7 exon11 c.1648C > T p. Gln550* and PDZD7 exon15 c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser 784del. III2, III4, III3, IV2, IV3, II3 are carriers. They genotype are PDZD7 exon11 c.1648C > T p. Gln550*. III1 is also a carrier. His genotype is PDZD7 exon2 c.192G > A p. Met64Ile, and PDZD7 exon15 c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser 784del. I1 and II2 have passed away, their genotypes are unknown.

192G > A p. Met64Ile and c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser 784del are inherited from patient's mother. They were never reported before. PDZD7 c.1648C > T p. Gln550* is inherited from the father. The mutation has already been identified and has been linked to the autosomal recessive genetic hearing loss (Vona et al., 2016). Findings reported in this article extend the pathogenic map of PDZD7 and enhance our understanding of the mechanism by which PDZD7 causes ARNSHL. We also established a rapid and precise prenatal diagnosis (PND) procedure with pathogenicity analysis based on the standards and guidelines for the interpretation of sequence variant by the American College of Medical Genetics and Genomics and the Association for Molecular Pathology (ACMG/AMP Standards and Guidelines)(Richards et al., 2015).

associated with the genes, MTTL1, MTTQ MTTH, MTTK MTTS1, MTND1 MTND5, MTND, MTTS2, and MTATP6 (Mahboubi et al., 2012; Shearer et al., 1993). The PDZD7 gene encodes a scaffold protein containing a PDZ domain. The gene was mapped and localized to chromosome 10q24.3 by positional cloning (Schneider et al., 2009). The intact PDZD7 protein consists of three PDZ domains, a harmonious N-like domain and a proline-rich region. The shorter PDZD7 subtype containing the first two PDZ domains can be detected in the inner ear (Du et al., 2018; Ebermann et al., 2010; Schneider et al., 2009; Vona et al., 2016). The gene was discovered as a modifier and candidate gene in Usher syndrome. One of the types of Usher syndrome is Usher syndrome 2C (USH2C, OMIM: 605472), which can be caused by biallelic digenic mutation in the ADGRV1 and PDZD7 genes (Ebermann et al., 2010). The main clinical features of USH2C are moderate-to-severe congenital sensorineural hearing loss and progressive retinitis pigmentosa (Ebermann et al., 2009; Malm et al., 2011). PDZD7 is also associated with autosomal recessive non-syndromic hearing loss (ARNSHL) (Booth et al., 2015; Le Quesne Stabej et al., 2017; Schneider et al., 2009; Vona et al., 2016). Its homozygous or compound heterozygous mutations can cause autosomal recessive deafness-57 (DFNB57, OMIM: 618003) (Guan et al., 2018). The main clinical phenotype associated with these mutations is sensorineural symmetric peripheral hearing loss without vestibular dysfunction and progressive retinitis pigmentosa (Booth et al., 2015). The PDZD7 gene was the first gene found to be associated with ARNSHL in a consanguineous German family in 2009 (Schneider et al., 2009). Till date, only nine biallelic mutations of the monogenic PDZD7 have been confirmed, which were identified in two German families, four Iranian families, one Pakistani family and two Chinese families with ARNSHL (Booth et al., 2015; Guan et al., 2018; Le Quesne Stabej et al., 2017; Vona et al., 2016). Here, we report a case of Chinese family with ARNSHL, with a patient that carries three variations in the gene PDZD7 (NM_001195263.1 exon11 c.1648C > T p. Gln550*; exon2 c. 192G > A p. Met64Ile; exon15 c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser 784del) representing compound heterozygous mutations. Among them, PDZD7 c.

2. Materials and methods 2.1. Clinical evaluation of patient and his family This study was approved by the Ethics Committee of Sun Yat-Sen University and the informed consents were obtained from the involved individuals, and consent was obtained from the parent on behalf of the proband as they are a minor. The patient and his family members attended the First Affiliated Hospital of Sun Yat-Sen University. The family members performed hearing and vision tests. The pedigree chart is shown in Fig. 1. There was only one patient in this Chinese family, a seven years old boy. He was diagnosed with mild to moderate sensorineural hearing loss five months after birth. However, his hearing improved after wearing a hearing aid. During the next few years, the sensorineural deafness persisted, especially at 3000, 4000 and 5000 Hz (Fig. 2a). At the age of 4, an eye test was performed. The color ultrasound examination was normal (Fig. 2b). At the age of 7, the results of the review did not change. The patient did not have night blindness. Initially, there were no manifestations of progressive retinitis pigmentosa in the patient.

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Fig. 2. Clinical data of proband. (a): distortion product otoacoustic emissions (DPOAE) of proband's left and right ear; (b): the results of color ultrasound examination of proband's left and right eyes.

designed using Primer Premier 5.0 software. Primers with the following sequences were used: exon 2 of PDZD7: 5′-CCCTCCCTCATCCCTGC TCT-3′ and 5′- CCCAATCATACTGGCTCTGC-3′; exon 11 of PZDZ7: 5′-GATGAGGGAGCCTGTGC-3′ and 5′-GCTGCGGATGGGATGAA-3′; exon 15 of PZDZ7: 5′-TGGTGCCTGAACAAACTCG-3′ and 5′-CGCCTGG CCCAATACTC-3′. In this family, DNA samples from 11 individuals were subjected to Sanger sequencing. The process of amplification, sequencing and analysis of target fragments has been previously described (Xu et al., 2014).

2.2. Next generation sequencing (NGS) The samples of peripheral blood were collected from patient and his family members. EDTA was used to prevent coagulation. Extraction of whole-genome DNA from peripheral blood was done using HiPure Blood DNA Mini Kit (Magen, China) according to the manufacturer's instructions. The extracted samples of genomic DNA were tested to ensure that they meet the requirements for NGS. Whole exon sequencing of the patient's hereditary deafness-related genes was done using the extracted whole genomic DNA. Whole exon sequencing was performed using HiSeq 2500 Illumina Genome Analyzer II platform (Illumina Inc., Santiago, CA) with a depth of 150-fold. The data obtained by NGS can be used in kinship detection, mutational hazard prediction, genotype-phenotype correlation analysis and variation screening to screen for potential pathogenic mutations.

2.4. RT-qPCR analysis We isolated total RNA from the peripheral blood of patient using Hipure Blood/Liquid RNA Kit (Magen, China). To obtain cDNA, reverse transcription was performed by the PrimeScript RT reagent KIT (Takara Biomedical Technology, Beijing, China). The RT-qPCR primers for PDZD7 gene (5′-TGCCGGGCATCAAGTTCTC-3′ and 5′-ACCGCACTTCT CCACTACCA-3′) were identified using PrimerBank (pga.mgh.harvard. edu/primerbank/)(Spandidos et al., 2010). BLAST tool (blast.ncbi.nlm. nih.gov/) was used to determine the specificity of both primers. HieffTMqPCR SYBR Green Master Mix (YEASEN, China) was used according to the manufacturer's instruction. Relative RNA expression was

2.3. Sanger sequencing Because of the high error rate of NGS, the potential pathogenic sites revealed by NGS should be identified by Sanger sequencing. We screened three variants of one candidate gene by NGS. Primers complimentary to the second, eleventh and fifteenth exons of PDZD7 were 67

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generations of individuals III1 and III2. Second, the exome data of three individuals (IV1, III2, and III1) were analyzed by filtering annotation, such as quality control and screening process. The former included sequencing quality score ≥ 30, typing quality score ≥ 20, base reads ≥4, etc. The latter included non-synonymous variation of exon regions, not being in the mutation database or having a frequency ≤ 3%, genetic pattern filtering, etc. Third, the variation of harmfulness notes was done through tools such as VarCards (http://varcards.biols.ac.cn/). The pathogenicity identification score of VarCards ranges from 0 to 1, and the higher score represent the stronger pathogenicity (Li et al., 2018). We performed a comprehensive comparison of the scores of each candidate variant sites. Consequently, only four variants from DFNA5 and PDZD7 genes matched our selection criterion. (Selected variants: DFNA5 c.2 T > C p. Met1; PDZD7 c.1648C > T p. Gln550-; PDZD7 c. 192G > A p. Met64Ile; PDZD7 c. 2341_2352delCGCAGCCGCAGC p. Arg781_Ser 784del). Finally, the correlation between phenotype and candidate genes was ranked by Phenolyzer (http://phenolyzer.wglab. org/) (Yang et al., 2015). The higher is the score, the higher is the genotype-phenotype correlation. In this study, we used the sensorineural hearing loss as a keyword to analyzed the genotype-phenotype correlation of the candidate genes of PDZD7 and DFNA5. The results showed that the PDZD7 gene passed the known diseases Usher syndrome type 2 and autosomal recessive deafness-57 related to the sensorineural hearing loss. In addition, DFNA5 was also predicted to be associated with sensorineural hearing loss. PDZD7 is a gene with a relatively high predicted score among the two candidate pathogenic genes. Based on these findings, we determined three variants of the PDZD7 gene that contributed to the deafness of the patient. The results of NGS screening are shown in Table 1.

Table 1 NGS analysis results. Individual

IV1

III2

III1

Nucleotide change

DFNA5 c.2 T > C PDZD7 c.192G > A PDZD7 c.1648C > T PDZD7 c.2341_2352del CGCAGCCGCAGC DFNA5 c.2 T > C PDZD7 c.1648C > T PDZD7 c.192G > A PDZD7 c.2341_2352del CGCAGCCGCAGC

Amino acid change

State

Computational prediction VarCards damaging score

Phenolyzer score

p. Met1

Het

0.86

0.008

p. Met64Ile

Het

0.70

0.248

p. Gln550*

Het

1

p. Arg781_Ser 784del p. Met1

Het

0

Het

0.86

0.008

p. Gln550*

Het

1

0.248

p. Met64Ile

Het

0.70

0.248

p. Arg781_Ser 784del

Het

0

Het, heterozygous.

calculated using 2-ΔΔCt method. β-actin was employed as the housekeeping gene. All experiments were performed in triplicates. 2.5. Variants analysis of PDZD7 gene The PDZD7 variant c.192G > A p. Met64Ile is a missense mutation. Polyphen-2 (genetics.bwh.harvard.edu/pph2/), SIFT and PROVEAN (provean.jcvi.org) tools were used to analyze its hazards (Adzhubei et al., 2013; Choi et al., 2012). In order to determine species' evolutionary conservation of amino acids encoded by variant sites, UniProt (www.uniprot.org/uniprot/) and Clustalx software for multi-species protein sequence alignments were utilized (Larkin et al., 2007; UniProt Consortium, 2018). SWISS-MODEL was used to construct the 3D structure of normal and mutated proteins (Waterhouse et al., 2018). The variant of PDZD7 c.1648C > T p. Gln550* is a nonsense mutation which has already been reported in association with the autosomal recessive genetic hearing loss (Vona et al., 2016). To analyze the hazards associated with the PDZD7 variant c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser 785del, UniProt and Clustalx software were used to estimate the level of evolutionary conservation.

3.2. Mutation detection The Sanger sequences for PDZD7 gene's exons 2, 11 and 15 of the patient and other family members were obtained. The novel variations identified in exon 2 and exon 15 of the PDZD7 gene were inherited from the proband's father. The previously reported variation identified in the exon 11 of the PDZD7 gene was inherited from the proband's mother. The Sanger sequencing results of the family members are shown in Table 2. The Sanger sequencing map of the patient and his parents are shown in Fig. 3. 3.3. Pathogenicity Assessment The variant of PDZD7 c.192G > C, p. Met6Ile was neither reported in the SNP database, the NHLBI Exome Sequencing Project (ESP), the 1000 Human Genome Database, nor in the HGMD. As a novel variant, its hazard was predicted by various bioinformatics methods including SIFT and PolyPhen-2. The results are shown in Fig. 4(a, b). We also performed a multi-species alignment of the amino acids encoded by PDZD7. The results of alignment near the p.M64I variant site are shown in Fig. 4(c). The species to be aligned included Homo sapiens (Human), Canis lupus familiaris (Dog), Macaca mulatta (Rhesus macaque), Mus musculus (Mouse), Nomascus leucogenys (Northern white-cheeked gibbon), Otolemur garnettii (Small-eared galago), Pan troglodytes

3. Results 3.1. NGS results analysis The analysis of NGS results of the patient and his parents revealed1139 variations. For these variations, first, we calculated the kinship coefficient and ruled out the close relatives within the three Table 2 The Sanger sequencing results of the family members. Individual

IV1 III2, III4, III3, IV2, IV3, II3 III1 II1, I2, III5

PDZD7 exon2c.192G > A p. Met64Ile and exon15c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser 784del Het Normal Het Normal

Het: Heterozygote. 68

PDZD7 exon11c.1648C > T p. Gln550*

Statement

Het Het Normal Normal

Patient Carrier Carrier Normal

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①

②

③

④

⑤

⑥ (caption on next page)

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Fig. 3. Sanger sequencing results of the three new alternations in PDZD7 gene. Sequencing of E2 in the PDZD7 gene. ①Normal control and single line shows normal G/G. ②Double lines show heterozygote mutation G/A (c.192G > A p. Met64Ile). Sequencing of E11 in the PDZD7 gene. ③Normal control and single line shows normal C/C. ④Double lines show heterozygote mutation C/T (c.1648C > T p. Gln550*). Sequencing of E15 in the PDZD7 gene. ⑤Normal control and single line shows normal CGCAGCCGCAGC/CGCAGCCGCAGC. ⑥Double lines show heterozygote mutation CGCAGCCGCAGC/−−———————— (c.2341_2352delCGCAG CCGCAGC p. Arg781_Ser 784del).

2009; Vona et al., 2016). In this study, we reported a Chinese family with a history of hereditary deafness. We used NGS to identify possible pathogenic mutation sites. From the results of NGS, we obtained multiple variations, which were screened and analyzed step by step. Finally, four variants of two genes were identified as the most likely pathogenic sites. They all were related to deafness. Two of the three variants in the PDZD7 gene were novel variants. According to the ACMG/AMP Standards and Guidelines, they all were identified as likely pathogenic. Another nonsense variant of PDZD7 (c.1648C > T p. Gln550*) was previously reported in association with the hearing loss. The variant of DFNA5 gene (NM_ 04403 c.2 T > C) is an initiation codon mutation. To assess its pathogenicity, we used the NCBI ORF finder tool to locate the potential translation initiation site. We found that the initiation of translation at the next available initiation site truncates the protein by 164 amino acids at the N-terminus. It was previously reported that DFNA5 protein can be cleaved by caspase-3 at the amino acid position 270 to produce DFNA5N (1–270) and DFNA5-C (271–496). The DFNA5-N fragment can penetrate the membrane and lead to the induction of pyrophosphorylation, which in turn triggers pyroptosis (Wang et al., 2017). So, the ORF finder result which truncates the protein by 164 amino acids at the N-terminus maybe beneficial for the cell, as it helps to avoid pyroptosis. Moreover, the skipping of exon eight during transcription was observed in all reported hearing loss families with DFNA5 variants (Bischoff et al., 2004; Chai et al., 2014; Cheng et al., 2007; Li-Yang et al., 2015; Nishio et al., 2014; Park et al., 2010; Van Laer et al., 1998; Yu et al., 2003). This exon encodes amino acids 331 to 394 of the DFNA5 protein, the remainder of the protein contains residues 1–330 and 41 residues at the C-terminus. The alteration will cause the Cterminal (271–496) domain of DFNA5 protein to be affected, but the function of the DFNA5 N-domain will be revealed and lead to pyroptosis. However, this protein is unstable in mammals, so it only affects the sensitive hearing system leading to hearing loss (Wang et al., 2017). With the patient, we performed the RT-PCR of mRNA, and the results showed that the eighth exon was not skipped. This again reduces the hazard of the initiation codon mutation in the DFNA5 gene detected by NGS in this study. Therefore, we can rule out the relationship between this variation and the hereditary deafness of the patient. Although NGS has been widely used in the clinical treatment and diagnosis of genetic disorders, the identification of pathogenic diseases is still a difficult problem. When the novel variants are found by NGS, a variety of methods including the segregation analysis of characterized mutations and pathogenicity evaluation (SIFT, PloyPhen-2, Mutation Taster, PROVEAN) as well as protein structure prediction are used to analyze the pathogenicity of novel mutations. Compared with Sanger sequencing, the NGS has a higher error rate (0.1–15%) and a shorter reading length. However, in clinical practice, this step is essential for discovering variants (Goodwin et al., 2016). The results of NGS can only be used for reference in clinical application. Each variant in each candidate gene needs to be analyzed. PDZD7 gene was discovered as a modifier and candidate gene in Usher syndrome (Ebermann et al., 2010). The biallelic digenic mutation of the ADGRV1 and PDZD7 genes can cause USH2C. PDZD7 is also an ARNSHL-associated gene. Its homozygous or compound heterozygous mutations can lead to DFNB57. The difference between the clinical manifestations of DFNB57 and USH2C is that the former does not lead to progressive retinitis pigmentosa while the latter does. Although the current clinical manifestations of the proband including the recently eye examination showed normal are more in line with DFNB57, Usher

(Chimpanzee), Papio anubis (Olive baboon), Propithecus coquereli (Coquerel's sifaka), and Rattus_norvegicus (Rat). In addition, we constructed 3D protein structures of PDZD7 p. Met wild type and p. Met64Ile variant using the SWISS-MODEL (Fig. 5(a,b)). The modelling shows that once the amino acid 64 is changed from methionine to isoleucine, the 3D protein structure undergoes some changes include the addition of two beta folds and a small alpha helix. In addition, it also includes some random crimp modifications. These changes may cause changes in protein's functions and lead to a damage. To sum up, absent from controls in Exome Sequencing Project or 1000 Genomes and detected in trans with a pathogenic variant are two moderate evidence of pathogenicity (PM2 and PM3). Moreover, the co-segregation with disease in multiple affected family members in a gene definitively known to cause the disease, and multiple lines of computational evidence support a deleterious effect on the gene (conservation, bioinformatics analysis). The patient's phenotype and family history highly specific for a disease with a single genetic etiology are supporting evidence of pathogenicity (PP1, PP3 and PP4). According to ACMG/AMP Standards and Guidelines, this variant meets the criteria for likely pathogenic, as this conclusion is supported by two moderate evidences and three supporting evidences. So, this novel missense variant in PDZD7 was identified as likely pathogenic. The variant of PDZD7 c.2341_2352del, p. Arg781_Ser 784del was also not found in the SNP database, the NHLBI Exome Sequencing Project (ESP), the 1000 Human Genome Database and HGMD. The results of the amino acid alignments near p. Arg781_Ser784del variant sites are shown in Fig. 4(d). Human PDZD7 p. Arg773-Ser784 is a string of ‘RS’ (Arg-Ser) repeats. From the results of the Clustalx analysis, this string of ‘RS’ repeats is highly conserved, indicating that these repeats have important biological functions. In addition, PDZD7 c.2319_2336del, p. Ser 774_ Arg779del and PDZD7 c.2331_2342del, p. Ser 778_ Arg781del variants were detected in patients with deafness (Sommen et al., 2016). Both were identified as pathogenic variants, and both were missing two repeats of ‘RS’. In short, this variation also has two moderate evidence (PM2 and PM3) and three supporting evidence (PP1, PP3 and PP4) of pathogenicity. According to ACMG/AMP Standards and Guidelines, this variant also meets the criteria for likely pathogenic. So, this novel deletion variant in PDZD7 was identified as likely pathogenic. The variation of PDZD7 c.1648C > T p. Gln550* is a nonsense mutation which has already been reported in association with the autosomal recessive genetic hearing loss. We used RT-qPCR to measure the expression of PDZD7 gene in the proband and normal controls. Our analysis showed that the patient's PDZD7 gene expression was 30% lower than the expression in controls (P < 0.001). The results are shown in Fig. 6. This indicates that the variation detected in this study can reduce the expression of the gene.

4. Discussion Most genes that cause hereditary deafness are autosomal recessive, with only homozygotes expressing a specific phenotype. Hereditary deafness is divided into syndromic and non-syndromic. In non- syndromic hereditary deafness, the loss of hearing is the only symptom. In syndromic hereditary deafness, there are also pathological changes to the eyes, bone, kidney, skin and other parts of the body, in addition to deafness (Shearer et al., 1993). ARNSHL is a non-syndromic hereditary deafness, and PDZD7 has been implicated as an ARNSHL-associated gene (Booth et al., 2015; Le Quesne Stabej et al., 2017; Schneider et al., 70

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(b)

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Fig. 4. Bioinformatics analysis. (a): SIFT Provean program prediction analysis of the PDZD7 c.192G > C, p. Met6Ile scored 0.036. (b): the polyphen-2 prediction analysis of the PDZD7 c.192G > C, p. Met6Ile scored 0.994. (c) and (d): the result of the multi-species alignment. (c): alignment near the site of p.64 M; (d): alignment near the site of p.781_784RSRS.

(a)

S0 (b)

S0,

Fig. 5. The variant type of c.192G > A p. Met64Ile and wild type tertiary structures of PDZD7 protein. (a) The position of p. M64 wild type. (b) The position of p. M641 variant type.

family and two Chinese families. In the family analyzed in this work we found a compound heterozygous mutation (c.192G > A, c.1648C > T, c.2341_2352delCGCAGCCGCAGC). The RT-qPCR results demonstrated that the PDZD7 gene expression was decreased in the patient, indicating that the variant observed in this study can affect the expression of the gene and its normal function. PDZD7 is expressed in the stereocilia ankle-link region of the mechanosensitive structure (Guan et al., 2018). The intact PDZD7 protein consists of three PDZ domains, an HNL domain and a PR region. The shorter PDZD7 subtype containing the first two PDZ domains can be detected in the inner ear (Du et al., 2018; Ebermann et al., 2010; Schneider et al., 2009; Vona et al., 2016).

syndrome should not be definitively excluded. We will be tracking him and his family for a long time. The inheritance of PDZD7 is consistent with the genetic disease co-segregation, and the patient is heterozygous for this gene. Two of the three variations of the gene are novel variants, and we have made a pathogenic analysis of the factors influencing its pathogenicity using the ACMG/AMP Standards and Guidelines. These novel mutations were identified as Likely Pathogenic. This prediction was supported by the fact that the mutation co-segregated with the disease status. PDZD7 mutations cause autosomal recessive deafness. Only nine biallelic mutations have been reported with nine family, including two German families, four Iranian families, one Pakistani 72

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Authors' contributions Hualei Luo conceived and designed the experiments, performed the experiments, performed the data analyses and wrote the manuscript; Reem N. Hassan performed the experiments, performed the data analyses and revised the manuscript; Jin Yan revised the data and the figures; Peng Du, Jie Xie, Qiuyue Hu and Yue Zhu performed the experiments and analyzed the results; Weiying Jiang conceived and designed the experiments, and contributed reagents and materials, helped perform the analysis with the manuscript revised and approved the manuscript and made the final decision to submit the manuscript to Gene. Consent for publication Written consent was obtained from all family members for the publication of this study and the clinical images, and consent was obtained from the parent on behalf of the proband as they are a minor. Appendix A. Supplementary data Fig. 6. PDZD7 gene expression in patient and normal control. Data represent the mean ± standard error of the mean (SEM); n = 3. ***P < 0.001 compared with control group.

Supplementary data to this article can be found online at https:// doi.org/10.1016/j.gene.2019.05.045. References

Multi-PDZ domain-containing scaffolds could play a complementary role in the structure of the ankle-links for the mutant type (Grati et al., 2012). In our study, the mutation c.192G > A is not inside the PDZ domain, but this variant can lead to some changes around the position include the addition of two beta folds and a small alpha helix. Which possibly perturbing the amino acid chain (Fig. 4). The previously reported variant c.1648C > T p. Gln550* can cause formation of a truncated protein and could affect the PR region and PDZ3 domain. The variant c.2341_2352delCGCAGCCGCAGC p. Arg781_Ser784del result in the absence of two Arg-Ser repeats which could affect the PR region. Overall, our data support the contribution of PDZD7 biallelic mutations to the etiology of ARNSHL in humans.

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5. Conclusions We found three pathogenic variants, including two novel ones, in PDZD7 gene that cause ARNSHL. The findings will expand not only our knowledge of genetic causes of ARNSHL, but also PDZD7 genes mutation spectrum of the disease. However, we ruled out the possibility that the DFNA5 gene initiation codon variations can cause hereditary deafness. The findings will help to study the relationship between the genotype and phenotype of PDZD7 and DFNA5 genes and enhance our understanding of these genes' functions and roles. A better understanding of the underlying genetic, molecular and cellular mechanisms of hereditary deafness will be advantageous for the development of effective treatments and will help in improving molecular diagnosis and genetic counseling of hereditary deafness. Funding This study was supported by the Science and Technology Planning Project Grant of Guangdong Province (No. 2014A020213020, Recipient Weiying Jiang) and the Science and Technology Program Project of Guangzhou (No. 201604020020, Recipient Weiying Jiang). Acknowledgements We thank patients, their families and healthy volunteers for participating in this study, donating blood samples and providing detailed medical history. 73

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