1. Family recruitment and clinical evaluations
The specific procedures in this part refer to my published literature. The proband was a 30-year-old man with progressive hearing loss a decade ago. He visited the Department of Otolaryngology, Head and Neck Surgery, Xijing Hospital in 2020 due to nonsyndromic hereditary sensorineural hearing loss. The patient had a family history of hearing loss. The patient was part of a six-generation Chinese family with 55 members. The family had autosomal dominant, progressive, postlingual, nonsyndromic sensorineural hearing loss, and these features were consistent with ADNSHL. Nine members of this family participated in the present study, which included three affected and six unaffected relatives. Written informed consent was obtained from each participant, and the study was approved by the Medical Ethics Committee of the First Affiliated Hospital of the Air Force Medical University (approval number KY20212002‑C‑1).
The following information was obtained from each study participant: basic information, age of onset, disease progression, mother's pregnancy, study participant's delivery, noise exposure, ototoxic drug use, head trauma, infectious diseases, family history, and other relevant clinical manifestations. Nongenetic causes of hearing loss, such as noise exposure, ototoxic drugs, and head trauma, were excluded. Physical examinations suggested no syndromic deafness in the family. Audiometric evaluations and otological examinations were performed for the proband, including pure-tone audiometry (PTA), acoustic impedance, distortion-product otoacoustic emission (DPOAE), auditory brainstem response (ABR), and temporal bone computed tomography. The other family members were evaluated using PTA. The average values of the thresholds of air conduction were determined at 500, 1000, 2000, and 4000 Hz to determine the degree of hearing loss in the family. The condition of an individual's hearing loss was classified as mild (26–40 dB HL), moderate (41–60 dB HL), severe (60–80 dB HL), or profound hearing loss (≥ 81 dB HL).
2. Targeted genomic capture and variant analysis
Firstly, genomic DNA from members of this family was extracted from the peripheral blood leukocytes using a blood DNA extraction kit (cat. no. CW0544M, Kang Wei Century Blood Genome Non-column Extraction Kit). Secondly, the DNA library was preparated using Standard Library Building kit (MyGenostics GenCap Enrichment Technologies), included DNA fragmentation, end repair, adapter ligation, polymerase chain reaction (PCR) enrichment and pruduct purification. Thirdly, the qualified library was captured using a GenCap Deafness capture kit (MyGenostics GenCap Enrichment Technologies) and sequenced on an Illumina HiSeq X ten sequencer. The DNA probes were designed to tile along the exon regions of the deafness genes. Subsequently, the raw data obtained by sequencing were saved in FASTQ format, followed by the bioinformatics analysis. The Cutadapt (https://cutadapt.readthedocs.io/en/stable/) was used to preprocess data to remove low-quality reads. The clean reads were aligned to the UCSC hg19 human reference genome using BWA (http://bio-bwa.sourceforge.net/). Duplicated reads were removed using Picard (http://broadinstitute.github.io/picard/) tools, and mapping reads were used for variant detection. The variants of SNP and InDel were detected and filtered using GATK (https://www.broadinstitute.org/gatk/). The variants were further annotated using ANNOVAR (http://annovar.openbioinformatics.org/en/latest/) and associated with multiple databases, such as 1000 Genomes databases (http://browser.1000genomes.org/), ESP6500 (http://evs.gs.washington.edu/EVS/), dbSNP (https://www.ncbi.nlm.nih.gov/snp/), EXAC (http://exac.broadinstitute.org), and HGMD (http://www.hgmd.cf.ac.uk/), and predicted using SIFT (http://sift.jcvi.org/), PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/), MutationTaster (http://www.mutationtaster.org/), GERP++ (http://mendel.stanford.edu/SidowLab/downloads/gerp/index.html). And finally, the potential pathogenic variants were filtered based on the monogenic autosomal dominant trait.
3. Co-segregation analysis
The specific procedures in this part refer to my published literature. Following exome sequencing, the segregation analysis of candidate variants was completed by PCR and Sanger sequencing. A GSDME gene fragment was amplified and sequenced with the primers 5'-TTCTTCTTCCCTGCCCTACA-3' and 5’-CTCTGTGTCCCCAGAAGCAT-3’. PCR was performed with 25 µL reaction mixtures containing 100 ng genomic DNA, 1 µl of the forward and reverse primers, and 22 µL of 1.1× Golden Star T6 Super PCR Mix (cat. no. TSE101, TsingKe Biological Technology). Thermocycling was performed using the following program: initial denaturation at 98˚C for 2 min, followed by 30 cycles of 98˚C for 10 s, 62˚C for 10 s, and 72˚C for 10 s, and then final extension at 72˚C for 1 min. PCR products were purified using a Cycle Pure kit (cat. no. D6492 OMEGA Bio‑Tek) and sequenced using an Applied Biosystems 3730 DNA Analyzer (Thermo Fisher Scientific, Inc.). All sequencing chromatograms were compared to the published sequence for GSDME.
4. Evolutionary conservation analysis
The target sequence for alignment contained amino acid residues encoded by exon 7–exon 10. Multiple sequence alignment was performed across 18 species using BLAT on the UCSC Genome Browser (https://genome.ucsc.edu).
5. Minigene splicing assay
A minigene splicing assay was performed to verify whether the variant affected splicing products. A partial sequence of the wild type and mutant type GSDME, including partial intron 6, exon 7, intron 7, exon 8, intron 8, exon 9, and partial intron 9 (5605 base pairs), was PCR amplified with gene-specific primers (F: TTATGGGGTACGGGATCACCAGAATTCgcatcgcagtcatgagactt and R: CGGGATCACCAGATATCTGGGATCCtggatgtctaccccctcatc) that contained EcoRI and BamHI restriction enzyme sites. After restriction enzyme digestion by EcoRI and BamHI, the PCR fragment was ligated into the pSPL3 vector and sequenced confirmed. PCR and Sanger sequencing were used to evaluate whether the wild type and mutant type expression vectors had been successfully constructed. Wild type or mutant type GSDME minigenes were transfected into COS7 cells (the kidney cells of the African green monkey). The cells were harvested 36−48 h after transfection. All cellular RNAs were extracted and used to produce cDNA. PCR amplification was performed using primer SD6-F (TCTGAGTCACCTGGACAACC) and SA2-R (ATCTCAGTGGTATTTGTGAGC). The PCR products were isolated after electrophoresis through 1.5% agarose gels and verified by Sanger sequencing.