Patients
A two-generation Chinese family with SNHL was recruited for the present study. This family consisted of four members, including two siblings (II-1 and II-2, Fig. 1a) showing rapidly progressive prelingual SNHL and two normal parents (I-1 and I-2, Fig. 1a). A detailed physical examination and an audiometric assessment were performed on the two affected brothers, including ear electron microscopy, auditory brainstem response (ABR) testing, pure tone audiometry (PTA), computed tomography (CT) of the tympanic membrane and the mastoid process, and magnetic resonance imaging (MRI) of the bilateral inner ear canal and the brain. These tests were used to detect external auditory canal, middle ear, and inner ear malformations as well as auditory nerve abnormalities. The participating parents were assessed through clinical interviews.
Targeted next-generation sequencing and data analysis
Genomic DNA was extracted from the subjects' peripheral blood leukocytes using a Qiagen DNA Blood Kit (Qiagen, Germany) in accordance with the standard extraction protocol, and DNA integrity was detected by agar gel electrophoresis. The target regions of 139 disease-related genes in the patients’ genomic DNA were captured using an Agilent Capture Kit. An Illumina HiSeq sequencing system (Illumina, San Diego, CA, USA) was used for Next-generation sequencing of coding regions, gene regulatory regions, and 10bp flanking introns of target genes (see Additional file 1). The deafness-related sites of the patients’ mitochondrial DNA were detected by Matrix-assisted laser Desorption Ionization-time Of Flight Mass Spectrometry(MALDI-TOF-MS), and the candidate variant sites of the family DNA samples were verified by Sanger sequencing. The sequencing data was analyzed using the Sentieon software suite, while variant annotation and screening were performed using Woxi NextCODE software (Shanghai, China).
Three major databases containing reported or potential pathogenic variants, including ClinVar, OMIM, and HGMD, were used to screen for known pathogenic variants. A population-based database of large-scale sequencing genome Aggregation Database (gnomAD), the Exome Aggregation Consortium (ExAC), 1,000 genomes, and an internal whole exome sequencing(WES) database of 2,114 Han Chinese were used to filter minor allele frequencies (MAFs) <1% of the variation. Further screening for rare variants was conducted according to the interpretive guidelines of the American College of Medical Genetics and Genomics. Phenotypes of selected genes were analyzed to exclude genes not related to the patients’ clinical presentations and genetic patterns.
Validation of variants and inheritance analysis
All candidate pathogenic variants were confirmed by Sanger sequencing. Specific primers were designed to amplify the regions containing the variants by polymerase chain reaction (PCR, see Additional file 1). The PCR products were sequenced on an ABI 3730XL Genetic Analyzer (Applied Biosystems Life Technologies) according to the manufacturer’s protocols.
In silico analysis
A variety of tools were used to predict the pathogenicity of missense variants, including SIFT, Poly-Phen2, the Combined Annotation Dependent Depletion (CADDv1.3) score, and MutationTaster. Multiple amino acid sequence alignments of different species were conducted using ClustalW includeding HARS2 orthologues from Homo sapiens, Pan troglodytes, Macaca mulatta, Felis catus, Mus musculus, Danio rerio, Drosophila melanogaster, Caenorhabditis elegans, Bos taurus, and Rattus norvegicus as well as the human HARS paralogue. The GERP++ score was used to evaluate evolutionary conservatism, and its value ranged from -12.3 to 6.17, with 6.17 as the most conservative score [13]. To investigate the potential effects of each HARS2 variant on protein conformation, the protein structure of the HARS2 variant was predicted using the online protein model prediction server SWISS-MODEL. Analysis of protein structure changes and amino acid interactions were performed using the PyMOL Molecular Graphics System.
Plasmid construction
Human HARS2 cDNA sequence (NM_012208) was cloned into the pcDNA3.1-3xFlag-C vector. Using The Q5® Site-Directed Mutagenesis Kit (New England Biolabs, USA), plasmids carrying missense mutants (p.Asp117Asn or p.Leu303Pro) were constructed on the basis of wild-type plasmids and verified by sequencing.
Western Blot and immunocytochemistry assays
HEK293T cells and COS7 cells were kindly provided by the Key Laboratory for Human Disease Gene Study of Sichuan Province. The plasmid was transfected into HEK293T cells cultured in six-well plates, and the expression of HARS2 was detected by Western Blot. The plasmids were transfected into COS7 cells cultured in 24-well plates, and protein localization was detected by immunocytochemistry 48h after transfection. Anti-FLAG (#14793, Cell Signaling Technology) is used to detect the target protein and Anti-GAPDH (#SA00001-2, Proteintech) was used as a loading control. MitoTracker Red CMXRos (#9082, Cell Signaling Technology) was used to label mitochondria. Each experiment was performed three independent determinations.
Determination of intracellular reactive oxygen species
Intracellular reactive oxygen species (ROS) were determined by dichlorofluorescin diacetate (DCFH-DA, Beyotime, China). DCFH-DA can penetrate cell membranes and be hydrolyzed to DCFH. Intracellular ROS oxidize DCFH to fluorescent-producing DCF. Intracellular ROS levels can be measured by measuring fluorescence intensity. Briefly, the Plasmids were transfected into HEK293T cells cultured in 6-cm culture dishes, and the cells were collected 48h later. The cells were incubated at 37℃ with 10uM DCFH-DA for 20 minutes, washed with PBS twice, and the fluorescence of DCF was detected by flow cytometer (BD Bioscience).
Statistical analysis
Statistical analysis was carried out using Student unpaired double-tailed t test contained in GraphPad Prism software. P <0.05 was considered to be significantly different from the control group.