Novel Homozygous Mutation in PDZD7 Gene in a Family with Nonsyndromic Sensorineural Hearing Loss

Hearing loss is the most common sensory neural disorder in human, and according to WHO estimation, 5.5% (466 million) people worldwide have disabling hearing loss. In this study, a Chinese family with prelingual sensorineural hearing loss was investigated. The affected individuals showed moderately-severe hearing loss at all frequencies. Using target genome enrichment and high-throughput sequencing, a homozygous mutation c.2372delC, p.S791Ffs*17 was identied in PDZD7. The deletion mutation lies in exon 15 of PDZD7 and resultes in a frame shift followed by an early stop codon. Our study expand the mutation spectrum of PDZD7 and strengthens the clinical importance of this gene in patients with moderately-severe hearing loss.


Introduction
Hearing loss is the most common sensory neural disorder in human, and according to WHO estimation, 5.5% (466 million) of the world population have disabling hearing loss (https://www.who.int/healthtopics/hearing-loss). It is estimated that more that half of prelingual hearing loss were caused by genetic factors [1]. Non-syndromic hearing loss (NSHL), which no other symptoms occur, accounts for about 70% of hereditary hearing loss. While syndromic hearing loss (SHL), which associates with other symptoms, accounts for about 30% of hereditary hearing loss [2]. To date, 124 genes has been implicated for NSHL, and more than 400 SHL have been identi ed (https://hereditaryhearingloss.org/) [3]. In the hearing system, most of these pathogenic genes were located at the inner ear hair cells, a polarized epithelial cell with stereocilia bundles at the top, which translate motion to neuronal signals [4].
PDZD7 encodes a scaffold protein that express in the cortex and inner ear. Mutations in PDZD7 have been reported to cause autosomal recessive NSHL [5,6]. Moreover, PDZD7 is suggested to be a contributor to digenic Usher syndrome type IIC and a modi er in patients with Usher Syndrome Type IIA [7]. PDZD7 protein contains three PDZ domains, a proline-rich (PR) region as well as a harmonin-N like (HNL) domain. The PDZ domains enable PDZD7 to interact with SAN, harmonin, USH2A and ADGRV1, all of which were essential components of the ankle-link complex joint the stereocilium to hair bundles [8]. To date, 28 pathogenic mutations in PDZD7 have been identi ed in different regions (https://deafnessvariationdatabase.org/gene/PDZD7).
In this study, we present the genetic characteristics of a Chinese family with congenital sensorineural HL.
The affected individuals had moderately-severe hearing loss at all frequencies. We used targeted genome enrichment (TGE) and high-throughput sequencing (HTS) to identify a novel homozygous deletion mutation in exon 15 of PDZD7. The deletion mutation results in a frame shift followed by an early stop codon, which leads to the loss of several functional domains and results in the pathogenic effect of hearing loss. Our study has enriched the mutation spectrum of PDZD7, and proved that TGE and HTS are a reliable tools for genetic testing of hereditary hearing loss for large genes such as PDZD7.

Subjects
Patients of this study were recruited from outpatient department of A liated Eye and ENT Hospital, Fudan University. All family members were evaluated by audiological tests as previously described [9].
Pure tone audiometry at frequencies of 125, 250, 500, 1000, 2000, 4000, and 8000 Hz was performed on family members above age 6. Romberg and tandem gait tests were performed to evaluate vestibular functions. Auditory brainstem response (ABR) test was performed on family members under age 6. Highresolution computed tomography (HRCT) scans of the temporal bone were obtained to exam inner ear malformations. Written informed consent was obtained from all individuals, and this study was approved by the ethics committee of the Institutional Review Board of the Eye, Ear, Nose and Throat Hospital a liated with Fudan University (Shanghai, China). To screen common deafness mutations in GJB2, SLC26A4, and MT-RNR1 genes, the patients were prescreened by PCR ampli cation and Sanger sequencing according to previous studies [9].

Targeted exome sequencing
Genomic DNA was extracted from whole blood of the participants using a genomic DNA isolation kit (Qiagen, Hilden, Germany). Paired end sequencing library was prepared using library preparation kit (New England Biolabs, Ipswich, MA, catalogue# E6040) as previously reported [9]. A human deafness gene exon enrichment kit including 168 genes was used to capture target genome intervals. High-throughput sequencing was performed on illumine Hiseq 2000 according to the manufacturer's instructions..

Bioinformatics and validation of the variants
Sequencing reads generation, bioinformatic analysis were performed as previously reported [10]. Sequencing reads were generated by the Illumina CASAVA v1.8 pipeline and aligned to the human reference genome (hg19) using the Burrows-Wheeler Aligner (BWA) program. Variants were called using GATK package v4.1.8.1. All variants were annotated and characterized using ANNOVAR software. To validate the variants, Sanger sequencing of the PDZD7 exon 15 was performed on genomic DNA from all family members and 96 normal hearing controls. PCR and sequencing primers were designed by Primer3 online software. Sanger sequencing was performed on a 3730XL sequencer (Applied Biosystems) according to the manufacturer's instructions.

Family and clinical presentations
Family D27 is a non-consanguineous Chinese family that includes three affected siblings and two normal hearing parents (Fig. 1A). The patients were 9, 7 and 2 years old during examine. Audiograms of the two older patients showed bilateral moderate to severe HL with a slightly downward slope (Fig. 1B). ABR test of II:3 revealed bilateral HL with a threshold around 65dB (Fig. 1C). Although new-born hearing screening was not performed, the parents recalled no responses to subtle sounds at 1 to 2 years of age, suggesting a congenital phenotype. A vestibular functional test of the two older patients revealed no abnormalities.
HRCT scan of the II:1 and II:2 revealed no inner ear malformations. We also performed pure tone audiometry on the parents and detected no HL features. Otoscopy and full physical examination with special attention to renal, and ophthalmological evaluations revealed no additional abnormalities.
Targeted massively parallel sequencing Targeted high-throughput sequencing of all exons and exon-intron boundaries for 168 deafness genes was performed on the proband II:1. Sequencing yielded 2.8 million 100 bp paired-end reads. After adaptor trimming and low-quality reads ltering, paired-end fastq les were aligned to the human genome (hg19).
A mean depth of 118.1 for the targeted exons was achieved, and 97.8% of the targeted genome intervals were covered by at least 10 sequencing reads. After ltering against MAFs from various databases (1000 Genome Project, gnomad v2.1.1 and in-house database), we focused on variants in the coding region and intronic variants that might affect splicing. Based on the assumption of an autosomal recessive mode of inheritance, we focused on genes with homozygous or compound heterozygous variants. A homozygous variant, c.2372delC and p.S791Ffs*17 (NM_001195263), was identi ed in exon 15 of PDZD7. This variant was not present in any of the reference databases.
Genetic analysis of the PDZD7 mutation Sanger sequencing of exon 15 of PDZD7 was performed on all family members ( Fig. 2A). The three affected siblings were homozygous for the PDZD7 mutation, while their normal hearing parents were both heterozygous, which suggests that the PDZD7 mutation co-segregated with HL in this family. The deletion mutation resulted in a frame shift followed by an early stop codon and truncated protein p.S791Ffs*17 ( Fig. 2A). Moreover, the mutation was absent in 96 healthy controls screened by sanger sequencing. Discussion PDZD7 locates on chromosome 10q24.31 and was originally identi ed as an autosomal recessive NSHL gene [5,6]. Subsequently, heterozygous mutations in PDZD7 was identi ed as a modi er of retinal disease and a contributor to digenic Usher syndrome [7]. In Usher syndrome patients with biallelic USH2A mutations, another heterozygous PDZD7 mutation causes earlier-onset and more severe retinal disease. Heterozygous mutations in PDZD7 and ADGRV1 together induces Usher Syndrome Type IIC. In this study, using HTS, we identi ed a novel PDZD7 mutation (c.2372delC, p.S791Ffs*17) in a Chinese family with congenital NSHL. The frameshift mutation locates on exon 15 and produces an early stop codon and a truncated protein. Furthermore, no other mutations in the Usher syndrome genes were identi ed in the proband. The affected sibling in the present study were 9, 7 and 2 years of age during examinations. Ophthalmological tests revealed no retinal abnormalities. Newborn hearing screening was not performed, but the parents recalled reduced response to small sounds and mispronunciations of the two older sisters, which indicated congenital or prelingual onset. PDZD7 protein is a paralog of harmonin (USH1C) and whirlin (WHRN), sharing 35% and 55% similarity with harmonin and whirlin, respectively [11]. It contains three PDZ domains, a HNL domain as well as a proline-rich (PR) region. PDZD7 expresses in the inner ear hair cell, and forms the Usher quaternary protein complex with USH2A, ADGRV1 and WHRN, which is essential for the development and organization of ankle-link complex localize at the ankle region of hair cell stereocilia [12,13]. The rst two PDZ domains of PDZD7 mediate interaction with the other Usher quaternary protein complex components USH2A, ADGRV1 and WHRN, whereas the third PDZ domain is only involved in the interaction with WHRN [12].
To date, 16 PDZD7 mutations have been reported in literature, 6 of them are truncating variants including of the present study (Fig. 2B). Four alternative splicing isoforms of PDZD7 have been detected, which encodes either a full-length protein or short isoforms mainly containing the rst two PDZ domains [6]. The mutation identi ed in this study localizes in exon 15 of PDZD7, which is unique to the long isoform (Fig. 2B). Including our research, 3 mutations have been discovered in domains unique to the long isoform (Fig. 2B). Patients with these 3 mutations (p.S703fs, p.R781_S784del and p.S791Ffs*17) showed the same characteristic auditory phenotype, bilateral moderately severe hearing loss at all frequencies with gentle downward sloping, as patients with mutations in other parts of the gene [14,15]. Mouse model lacking exons 2-5 of Pdzd7, which disrupt all isoforms, and mouse model lacking exon 14, which only disrupt the long isoform, both manifest stereocilia disorganization and MET de cits, leading to similar hearing loss phenotype. Moreover, in mouse lacking exon 14 of Pdzd7 gene, PDZD7 short isoforms were not detected in inner ear at the protein level. These ndings suggested that PDZD7 long isoform is indispensable for hair cell function, whereas PDZD7 short isoform may not localize in the stereocilia and not be required for stereocilia function.
Hereditary hearing loss is a genetically and phenotypically heterozygous disorder. To date, 124 genes have been identi ed for NSHL, and 46 genes have been identi ed for the nine most common syndromic HL (https://hereditaryhearingloss.org/). Phenotypes of the patients vary at audiogram, age of onset, progression, vestibular complication, inner ear malformation, retinal complication, etc [16]. These heterogeneities hindered genetic diagnosis of hearing loss, and called for more complicated mutation screening strategy that take phenotype-genotype correlation into consideration. Since most of ARNSHL characterize with prelingual severe to profound HL, the relatively rare moderately-severe audiogram at all frequencies may serve as a reminder for potential causative PDZD7 mutations.
We report a novel frameshift mutation on PDZD7 in a Chinese family with moderately-severe HL. The mutation lies in exon 15, and is unique to the long isoform of PDZD7 protein. Our study extends the mutations spectrum of the PDZD7 gene in Chinese population. The relatively uncommon moderately-severe audiogram with a slightly downward slope is characteristic for PDZD7 patients. The identi cation of a novel PDZD7 mutation may be valuable for genetic consultation and functional research.