TMPRSS3 hearing loss causing variants
We collected all reported TMPRSS3-deafness causing variants via a literature review from PubMed and by using the Deafness Variation Database (https://deafnessvariationdatabase.org/).17 The protein structural domains were determined using NCBI for human TMPRSS3 (assession number AAQ88894). The protease catalytic triad was identified using homology to other serine proteases.
TMPRSS3 Model Building
Three-dimensional (3D) modeling of human wild type TMPRSS3 was performed using SWISS-MODEL protein structure homology-modelling software18-22 and I-TASSER protein structure and function predictions software.23-25 The human TMPRSS3 protein (AAQ88894) was uploaded in FASTA format to each online server and 3D renderings of TMPRSS3 were produced. Missense variants linked to HL were also modeled using both modeling software platforms.
TMPRSS3-associated cochlear implant outcomes
Two independent searches of PubMed for articles published on TMPRSS3-associated hearing loss treated with CI were conducted. Search terms included the combination of “TMPRSS3” with the major subheading topics “hearing loss” and “cochlear implant”. All articles reporting CI outcomes for TMPRSS3-associated hearing loss were included for final review. A total of nine unique articles were identified. Demographics, age of SNHL onset, phenotypic presentation, genetic mutations, patient age at time of CI, and audiologic outcomes were recorded. Individuals with hearing loss prior to two years of age were classified as prelingual hearing loss whereas those with onset two years of age or older were designated as post-lingual hearing loss. Objective outcomes included pre-operative and postoperative pure tone averages (PTA), CNC word recognition scores (WRS), and sentence scores (SS). Subjective outcomes, when reported in published articles, were summarized as either favorable or poor. Postoperative implant-aided CNC WRS scores less than 30% were considered poor outcomes, and scores 50% or greater were considered favorable.
Post-lingual cochlear implant outcomes
Two independent searches of PubMed for articles published on adult cochlear implant outcomes were conducted. Search terms included the combination of “cochlear implant outcomes” with the major subheading topic “hearing loss”. Articles that contained more than 10 patients and had postoperative CNC word scores were searched. Demographics, age of SNHL onset, phenotypic presentation, patient age at time of CI, and audiologic outcomes were recorded.
Statistical Analysis
Objective data were evaluated by measures of central tendency using Microsoft Excel, 2016. Mean age difference between good and poor post-lingual CI performers was evaluated by T-test using SPSS Statistics, 2020. TMPRSS3-associated hearing loss CI outcomes were compared to postoperative outcomes of eight CI studies in the general hearing loss population. Mean postoperative CNC WRS analysis of variance (ANOVA) of the two populations was performed using SPSS Statistics, 2020.
Human Organoids and Human Cochlea
Human stem-cell derived inner ear organoids were a kind give of Eri Hashino, PhD. Human cochlea tissue was harvested according to the IRB approved protocol (2006345852) from an adult male during resection of a skull base cholesteatoma that eroded the cochlea.
Plasmid Construct
The coding sequence of human TMPRSS3 isoform 1 from transcript variant A (NM_024022.4) with added 5’ EcoRI and 3’ KpnI restriction sites was generated as gBlocks™ Gene Fragments (Integrated DNA Technologies, Coralville, IA). The construct p3XFLAG-CMV 7.1_Tmprss3 were generated from the backbone of p3XFLAG-CMV 7.1_syn6 (Addgene, 50012). The syntaxin 6 gene was replaced by TMPRSS3 gBlocks with EcoRI and KpnI restriction enzymes. The NEB® 5-alpha Competent E. coli (New England Biolabs, Ipswich, MA; C2987H) were used for transformation. TMPRSS3 missense mutations were generated using the Q5 site-directed mutagenesis kit (New England Biolabs).
Immunoblotting
HEK293 cells (ATCC, CRL-1573), plated in 6 well plates, were transfected with p3XFLAG-CMV 7.1_Tmprss3 plasmid using Lipofectamine 3000 Reagent (Thermo Fisher, L3000015) for 24 hrs as described by the manufacturer. Cells were washed once with ice-cold PBS and lysed with RIPA Lysis and Extraction Buffer (Thermo Fisher, Waltham, MA; 89901) supplemented with protease (Thermo Fisher, 78430). The protein concentration of the samples was measured using BCA Protein Assay Kit (Thermo Fisher, 23225). 40 μg of total protein per sample were resolved by SDS-PAGE (4%–20% gels) and transferred to Immun-Blot® PVDF Membranes (Bio-Rad, Hercules, CA; 1620174). Membranes were blocked for 1 hour at room temperature in 5% milk with TBST (.05 % Tween-20 in 1x TBS). Primary antibodies were incubated overnight at 4°C. The antibodies used were mouse anti-FLAG (Sigma Aldrich, F1084, 1:16,000) and rabbit anti-Tmprss3 (Thermo Fisher, PA5-35325, 1:200). 1 wash with TBST and followed by HRP-linked goat anti-rabbit (Bio-Rad, 1705046, 1:25,000) and rat anti-mouse (Abcam, Ab131368, 1:1,000) secondary antibodies were incubated for 1 hour at room temperature. Three washes with TBST after incubation followed by development of blots using Clarity Western ECL Substrate (Bio-Rad, 1705061) and imaged with ChemiDoc Touch Imaging System (Bio-Rad, 1708370)
Immunofluorescence
Human cochlea samples were harvested and fixed in 10% formalin for 24 hours. Fixed cochleae were then decalcified for 2 hrs at room temperature with 120 mM EDTA in PBS and cryoprotected in 30% sucrose in PBS. After embedding in TFM™ Tissue Freezing Medium (General Data Company, TFM-5), cochleae were frozen and stored at -30°C. Sections of 6 μm were cut on a cryostat and air-dried for 2 hr. The tissue was blocked with 10% goat serum with 0.1% Tween-20 in PBS for 30 mins at RT. Following this, sections were treated with primary antibodies in 3% goat serum with 0.1% Tween-20 in PBS at room temperature for 1 hr. The antibodies we used were rabbit anti-TMPRSS3 (Thermo Fisher, PA5-35325, 1:50), mouse anti-TUJ1 (BioLegend, 801202, 1:100), rabbit anti-myosin VIIA (Proteus, 25-6790, 1:100), and mouse anti-SOX2 (BD Pharmingen, 561469, 1:50). After washing with PBS, sections were incubated with secondary antibodies or Alexa Fluor™ 568 Phalloidin (Thermo Fisher, A12380, 1:200) in 3% goat serum with 0.1% Tween-20 in PBS at RT for 1 hr. Sections were washed and mounted with ProLong™ Gold Antifade Mountant with DAPI (ThermoFisher, P36931). Staining was visualized on Leica DMi8 microscopy.