Ethics statement
This study was approved by the Chinese PLA General Hospital Research Ethics Committee. Fully informed written consent for participation and publication of clinical data was attained from each subject or their guardians when the age of subjects <18 years old.
Clinical data
A three-generation Chinese family with eight affected members and five unaffected members from Sichuan Province was evaluated. The medical history of each family member was obtained using a questionnaire (Additional file 1) that included the degree of hearing loss, age of onset of hearing loss, progression of hearing loss, symmetry of hearing loss, use of hearing aids, presence of tinnitus, pathological changes in the ear, infection, ototoxicity, noise exposure, and other relevant clinical manifestations to understand the otologic manifestation and exclude any history of other diseases and environmental factors. The proband underwent a number of clinical tests including general physical examinations, chest X-rays, brain MRI, and temporal bone CT. No abnormalities were detected in these tests, thus excluding the possibility that the hearing loss in this family was syndromic.
All genomic DNA (gDNA) was extracted from peripheral blood using a blood DNA extraction kit according to the protocol provided by the manufacturer (TianGen, Beijing, China).
Audiometric analysis
Pure-tone audiometry with air and bone conduction was performed according to standard protocols in a sound-controlled room at frequencies ranging from 250 to 8000 Hz. Audiograms were available for six of the eight affected family members and for the one unaffected member.
Deafness gene capture and Illumina library preparation
Among the affected family members, mutations in the common deafness genes GJB2, SLC26A4, and mtDNA 12SrRNA mutations were excluded, with the exception of homozygous c.109G > A in GJB2, which was found in III:2. Targeted NGS was then used to sequence 129 known deafness genes (http://www.otogenetics.com/forms/Deafness_v3_gene_list.pdf).DNA specimens from five patients and two normal hearing members of the family were sequenced by Otogenetics Corporation (Atlanta, GA, USA) using next-generation sequencingwith the Illumina platform. The quality of gDNA was examined by checking the optical density ratio (260/280 ratio) and performing gel electrophoresis imaging. High-molecular weight gDNA (approximately 3 μg) was fragmented ultrasonically using a Covaris E210 DNA shearing instrument (Covaris, Inc., Woburn, MA, USA) to an average size of 300 base pairs (bp). The Covaris protocol was a 3-min total duration, duty cycle of 10 %, intensity of 5, and 200 cycles per burst.
Exons and their flanking 50 bp from 129 known human deafness genes were selected for capture and NGS sequencing using an Illumina HiSeq2000. Hybridisation probes of 0.5 to 1.6 kilobase pairs (kb) were generated for these genes from either cDNA clones of the genes or by polymerase chain reaction (PCR) amplification from targeted gDNA regions. To ensure reliable capture of shorter exons, we specifically generated longer hybridisation probes from gDNA for those exons that were shorter than 50 bp by including approximately 100 bp genomic DNA flanking the exons on both sides. All PCR products (10 ng each) were purified using the QIAquick PCR Purification Kit (Qiagen, Valencia, CA, USA) before use. Further details of the capture probe validation and preparation can be found in a previous report [26].
Fragmented gDNA libraries for Illumina GAII sequencing were prepared using the NEBNextTM DNA Sample Prep Master Mix set (E6040; NEB Biolab, Ipswich, MA, USA). End repair of DNA fragments, addition of a 3′ adenine (A), adaptor ligation, and reaction clean-up were performed according to the manufacturer’s protocol. The libraries were purified and size-selected using the AMPure DNA Purification kit (Beckman Agencourt, Danvers, MA, USA). The ligated product (20 ng) was amplified over 14 PCR cycles using the Illumina PCR primers InPE1.0 and InPE2.0 and indexing primers according to the manufacturer’s instructions.
For targeted enrichment of deafness genes, the Illumina library DNA was purified using a QIAquickMinElute column and eluted into 50 μL hybridisation buffer (Roche NimbleGen, Madison, WI, USA). The barcoded Illumina gDNA libraries (5 μg) were incubated in 16 μL hybridisation buffer on the surface of hybridisation glass slides on a hybridisation station (BioMicro Systems, Inc., Salt Lake City, UT, USA) at 42 °C for 72 h. Nonspecific DNA fragments were removed after a series of six washing steps in washing buffer (Roche NimbleGen, Madison, WI, USA). The DNA bound to the probes was eluted by a 10-min incubation with NaOH (425 mL, 125 mM). The eluted solution was transferred to a 1.5-mL Eppendorf tube containing 500 μL neutralisation buffer (Qiagen’s PBI buffer). The neutralised DNA was desalted and concentrated on a QIAquickMinElute column and eluted into 30 μL EB buffer. To increase the yield, we typically amplified 5 μL eluted solution by 12 cycles of PCR using the Illumina PCR primers InpE1.0 and 2.0. Enrichment of the targeted deafness genes was examined by comparing the growth curves of captured and noncaptured samples during quantitative PCR [26]. Twelve barcoded libraries of captured samples were pooled, and paired-end Illumina sequencing was performed using the Illumina HiSeq system (Illumina, San Diego, CA, USA). Details of the bioinformatic analysis methods used have been published previously [26].
The sequence data were mapped with BWA (0.7.4) against the human reference genome index (hg19), and then analyzed with Picard to remove duplicates from the mapped reads. Variants in the data (SNPs/indels) were called with SAMtools (0.1.19) across the genome and exported in VCF format; 516.7 ± 28.5 variants were obtained per sample. All of the variants in the target regions were selected based on the bed file provided by Otogenetics, and then annotated with ANNOVAR and the internal mutation database to get information on the impact of each variant, predicted functional changes, 1000 Genome Project population allele frequency, and associated diseases, if applicable. Variants with known disease associations, a deleterious functional impact, or aMAF(Minor Allele Frequency) <0.04 were selected as candidate mutations for analysis and validation; 20.4 ± 5.4 variants were obtained per sample. To identify the pathogenic mutation, a cosegregation analysis of the family members and an in-house database of 481 Chinese normal hearing controls from Otogeneticswas applied.
Whole exome sequencing
Exome capture was performed in the proband and his parents by BGI–Shenzhen using NimbleGen SeqCap EZ Human Exome Library v3.0 (Roche NimbleGen, Inc., Madison, WI, USA) according to the manufacturer’s protocols, and sequencing was performed using a HiSeq2000 platform (Illumina, San Diego, CA, USA). Illumina base calling Software 1.7 was used with default parameters to process the raw image files and to sequence the individual products as 90-bp paired-end reads. The sequenced reads were aligned to the human genome reference (UCSC hg19 version, build37.1) using SOAP aligner/SOAP2[27]. SNP or indels were called using Soapsnp [28] software and BWA [29], respectively. The alignment results were identified using GATK [30] to identify the breakpoints.
Sanger sequencing
After filtering against multiple databases, Sanger sequencing was used to determine whether any of the potential mutations in known genes causing ADNSHI co-segregated with the phenotype in this family. Direct PCR products were sequenced using Bigdyeterminator v3.1 cycle sequencing kits (Applied Biosystems, Foster City, CA, USA) and analysed using an ABI 3700XL Genetic Analyzer.
Mutational analysis
Segregation of the mutations was evaluated in all family members. Genotyping for c.638A > G was performed by PCR and detected by bidirectional sequencing of the amplified fragments using an automated DNA sequencer (ABI3100); the primers were 5′-CAGAGCCCTCCCTTAGTGAT-3′ and 5′-CGAGGCTACAGCTTCACCAC-3′. Nucleotide alterations were identified by sequence alignment with the ACTG1 GenBank sequence (NG_011433) using Genetool software.
Multiple sequence alignment
Multiple sequence alignment was performed across 15 species using ClustalW2 online (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
Model building and structure-based analysis
Three-dimensional modelling of the human wild-type and p.K213R mutation was performed using SWISS-MODEL [31], an automated homology modelling program (http://swissmodel.expasy.org/workspace/). We used the automatic modelling approach to model the complete human ACTG1 protein, including its 375 amino acids (NP_001186883.1) with or without the mutations. Data obtained from the homology models were visualised using Swiss-PdbViewer 4.1. Quality of the structure model were assessed by Verify 3D.
Availability of supporting data
Sequence read data of the affected subject (II:3) has been deposited into Sequence Read Archive ([32]; accession number SRP064631).