Front. Genet. Frontiers in Genetics Front. Genet. 1664-8021 Frontiers Media S.A. 780874 10.3389/fgene.2021.780874 Genetics Original Research TMPRSS3 Gene Variants With Implications for Auditory Treatment and Counseling Moon et al. Cochlear Implantation in TMPRSS3 Patients Moon In Seok 1 2 † Grant Andrew R. 3 4 † Sagi Varun 5 6 Rehm Heidi L. 3 7 Stankovic Konstantina M. 1 5 * Department of Otolaryngology—Head and Neck Surgery, Massachusetts Eye and Ear and Harvard Medical School, Boston, MA, United States Department of Otorhinolaryngology, Yonsei University College of Medicine, Seoul, Korea Program in Medical and Population Genetics, Broad Institute of MIT and Harvard, Cambridge, MA, United States New York Medical College, Valhalla, NY, United States Department of Otolaryngology—Head and Neck Surgery, Stanford University School of Medicine, Stanford, CA, United States University of Minnesota Medical School, Minneapolis, MN, United States Center for Genomic Medicine and Departments of Pathology and Medicine, Massachusetts General Hospital and Harvard Medical School, Boston, MA, United States

Edited by: Gavin R. Oliver, Mayo Clinic, United States

Reviewed by: Katarina Trebušak Podkrajšek, University of Ljubljana, Slovenia

Sedigheh Delmaghani, Institut Pasteur, France

Jourdan Holder, Vanderbilt University Medical Center, United States

 *Correspondence: Konstantina M. Stankovic, kstankovic@stanford.edu

These authors have contributed equally to this work

This article was submitted to Human and Medical Genomics, a section of the journal Frontiers in Genetics

 19 11 2021 2021 12 780874 21 09 2021 18 10 2021 Copyright © 2021 Moon, Grant, Sagi, Rehm and Stankovic. 2021 Moon, Grant, Sagi, Rehm and Stankovic

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Objective: To identify and report novel variants in the TMPRSS3 gene and their clinical manifestations related to hearing loss as well as intervention outcomes. This information will be helpful for genetic counseling and treatment planning for these patients.

Methods: Literature review of previously reported TMPRSS3 variants was conducted. Reported variants and associated clinical information was compiled. Additionally, cohort data from 18 patients, and their families, with a positive result for TMPRSS3-associated hearing loss were analyzed. Genetic testing included sequencing and copy number variation (CNV) analysis of TMPRSS3 and the Laboratory for Molecular Medicine’s OtoGenome-v1, -v2, or -v3 panels. Clinical data regarding patient hearing rehabilitation was interpreted along with their genetic testing results and in the context of previously reported cochlear implant outcomes in individuals with TMPRSS3 variants.

Results: There have been 87 previously reported TMPRSS3 variants associated with non-syndromic hearing loss in more than 20 ancestral groups worldwide. Here we report occurrences of known variants as well as one novel variant: deletion of Exons 1–5 and 13 identified from our cohort of 18 patients. The hearing impairment in many of these families was consistent with that of previously reported patients with TMPRSS3 variants (i.e., typical down-sloping audiogram). Four patients from our cohort underwent cochlear implantation.

Conclusion: Bi-allelic variants of TMPRSS3 are associated with down-sloping hearing loss regardless of ancestry. The outcome following cochlear implantation in patients with variants of TMPRSS3 is excellent. Therefore, cochlear implantation is strongly recommended for hearing rehabilitation in these patients.

 TMPRSS3 cochlear implantation sensorineural hearing loss genetic counseling hereditary hearing loss R01 DC015824 National Institutes of Health10.13039/100000002 1 Introduction

Autosomal recessive non-syndromic hearing loss (ARNSHL) is the most common form of hereditary hearing loss. It accounts for about 70–80% of congenital hereditary hearing loss. ARNSHL is an extremely heterogenous condition as more than 98 loci have been mapped and 77 causative genes have been identified to date (http://hereditaryhearingloss.org/).

The TMPRSS3 gene encodes a type III transmembrane serine protease that is structurally defined by four functional domains: a transmembrane domain, low density lipoprotein receptor A domain, scavenger receptor cysteine rich domain, and a carboxyl terminal serine protease domain (Südhof et al., 1985; van Driel et al., 1987; Sarrias et al., 2004; Rawlings et al., 2010). The TMPRSS3 gene is expressed in inner hair cells, spiral ganglion neurons (SGNs), the stria vascularis, and cochlear aqueducts of fetal cochlea (Guipponi et al., 2002). Four alternatively spliced transcripts have been described (DiStefano et al., 2018). The transmembrane serine protease 3 protein is thought to be involved in the development and maintenance of the inner ear, perilymph, endolymph and SGNs (Guipponi et al., 2002). While the function of the TMPRSS3 gene in the auditory system is not fully understood, its alteration has been linked with non-syndromic genetic hearing loss (DiStefano et al., 2018).

The incidence of TMPRSS3-associated ARNSHL is variable amongst different ancestral backgrounds but TMPRSS3 is a significant contributor in some populations. Pathogenic TMPRSS3 variants account for 0.7% of Japanese (Miyagawa et al., 2015), 3% of Pakistani (Ben-Yosef et al., 2001), 4.6% of Chinese (Gao et al., 2017), 5–6% of Tunisian (Masmoudi et al., 2001), 5.9% of Korean (Chung et al., 2014), and 11% of Turkish (Wattenhofer et al., 2005) ARNSHL cases. However, this gene has been reported in less than 1% of non-syndromic genetic deafness in White individuals (Wattenhofer et al., 2002). In contrast, pathogenic variants in the GJB2 gene are found in up to 50% of patients with ARNSHL. Despite the relatively low proportion of ARNSHL cases attributed to TMPRSS3, the gene remains a prime candidate for post lingual progressive ARNHSL in North European populations once GJB2 variants are ruled out (Seligman et al., 2021).

Patients with pathogenic variants in the TMPRSS3 gene have been described as having one of two discrete hearing phenotypes: severe, prelingual or progressive, post-lingual hearing loss. Weegerink et al. (2011) proposed that the phenotypic outcome of hearing loss is dependent on the combination and severity of TMPRSS3 variants (i.e., mild or severe). They assert that having two “severe” pathogenic variants leads to profound deafness with prelingual onset (DFNB10), whereas a single ‘severe’ pathogenic variant in trans with a milder TMPRSS3 pathogenic variant yields an initially less severe, but progressive and post-lingual onset hearing loss (DFNB8) (Weegerink et al., 2011). The TMPRSS3 gene encodes for a transmembrane serine protease which is expressed in SGNs (Guipponi et al., 2002). Therefore, the differential hearing phenotype may reflect the extent of loss of protease activity from a given variant.

In this study, we compile previously reported TMPRSS3 variants and present a novel variant along with their associated hearing phenotypes. We also aggregate reported outcomes and present new findings regarding the therapeutic effects of cochlear implantation (CI) in patients with pathogenic TMPRSS3 variants. Together, this information may assist with genetic counseling and treatment planning for patients with TMPRSS3 variants.

 2 Methods 2.1 Review of the Literature

Literature databases were searched using different combinations of keywords such as “transmembrane serine protease 3,” “TMPRSS3,” “ear,” “hearing loss,” “non-syndromic hearing loss,” and “cochlear implantation.” The databases searched were PubMed, Google Scholar, and two selected gene database websites (https://hereditaryhearingloss.org; https://www.ncbi.nlm.nih.gov/clinvar/). The titles and abstracts were screened using following inclusion criteria: 1) written in English, 2) dealing with non-syndromic hearing loss, and 3) reporting human data.

Based on the search strategy, 39 TMPRSS3-associated papers published from May 2000 to Aug 2021 were reviewed and summarized (Figure 1; Table 1). Among those 39 studies, eleven studies described patients who underwent cochlear implantation (Table 2).

Schematic representation of TMPRSS3 variants identified from literature review as well as our cohort. Benign variants are not displayed. The exons are numbered with coding sequence shaded blue and untranslated regions unshaded. *: variants with reported cochlear implant outcomes. TM, transmembrane domain; LDLRA, LDL receptor-like domain; SRCR, scavenger receptor cysteine-rich domain.

Overview of TMPRSS3 variants resulting in non-syndromic hearing loss, including those identified in the present study.

DNA change Protein change Exon Domain Variant classification Origin Phenotype severity at testing References
Deletion of E1-5 and 13 — E1-5 and E13 — Pathogenic United States Severe This study
c.36delC p.Pro12fs E2 Chinese Severe Gao et al. (2017)
c.36dupC p.Phe13fs E2 Turkish — Diaz-Horta et al. (2012)
c.157G>A p.Val53Ile E3 TM Palestinian — Scott et al. (2001)
Pakistani — Ben-Yosef et al. (2001)
United States
Korean — Lee et al. (2013)
Taiwanese — Wong et al. (2020)
c.205+38C>T — Intron3 — Taiwanese — Wong et al. (2020)
c.207delC p.Thr70fs E4 Dutch — Ahmed et al. (2004)
Newfoundlander —
Dutch — Weegerink et al. (2011)
c.208delC p.Thr70fs*19 E4 Pathogenic Slovenian Severe Battelino et al. (2016)
Polish — Lechowicz et al. (2017)
United States — Shearer et al. (2018)
Slovenian — Likar et al. (2018)
Czech — Safka Brozkova et al. (2020)
United States Severe This study
c.212T>C p.Phe71Ser E4 LDLRA Korean — Lee et al. (2013)
Japanese — Miyagawa et al. (2015)
c.218G>A p.Cys73Tyr E4 LDRLA Polish — Lechowicz et al. (2017)
c.226C>T p.Gln76X E4 Japanese — Miyagawa et al. (2013)
Miyagawa et al. (2015)
c.238C>T p.Arg80Cys E4 LDRLA Likely pathogenic Europe — Capalbo et al., (2019)
United States Mild This study
c.239G>A p.Arg80His E4 LDRLA Taiwanese — Wong et al. (2020)
c.268G>A p.Ala90Thr E4 LDLRA UK Caucasian — Charif et al. (2012)
Moroccan
c.280G>A p.Gly94Arg E4 LDLRA Japanese — Miyagawa et al. (2015)
c.296C>A p.Ser99X E4 Chinese Severe Gu et al. (2015)
c.308A>G p.Asp103Gly E4 LDLRA Greek — Wattenhofer et al. (2005)
c.310G>A p.Glu104Lys E4 LDLRA Pakistani — Lee et al. (2012)
c.310G>T p.Glu104X E4 Pakistani — Lee et al. (2012)
c.316C>T p.Arg106Cys E4 LDLRA Japanese Mild Miyagawa et al. (2013)
Chinese Gao et al. (2017)
c.323-6G>A — Intron4 — Pathogenic Pakistani — Scott et al. (2001)
Korean — Ahmed et al. (2004)
Dutch — Weegerink et al. (2011)
Chinese Mild Gao et al. (2017)
Pakistani — Singh et al. (2020)
United States Severe This study
c.325C>T p.Arg109Trp E5 SRCR Pathogenic Pakistani — Ben-Yosef et al. (2001)
Pakistani — Ahmed et al. (2004)
Korean — Chung et al. (2014)
Czech — Safka Brozkova et al. (2020)
United States Mild This study
c.326G>A p.Arg109Gln E5 SRCR Chinese — Gu et al. (2015)
Polish Mild Lechowicz et al. (2017)
c.331G>A p.Gly111Ser E5 SRCR United States — Ben-Yosef et al. (2001)
c.346G>A p.Val116Met E5 SRCR Indian — Ganapathy et al. (2014)
Korean — Kim et al. (2017)
Czech — Safka Brozkova et al. (2020)
c.371C>T p.Ser124Leu E5 SRCR Polish — Lechowicz et al. (2017)
c.390C>G p.His130Arg E5 SRCR Japanese — Miyagawa et al. (2015)
c.413C>G p.Ala138Glu E5 SRCR Pathogenic British Mild Weegerink et al. (2011)
Korean
United States — Eppsteiner et al. (2012)
Polish — Lechowicz et al. (2017)
United States — Shearer et al. (2018)
Pakistani — Singh et al. (2020)
United States Mild This study
c.432delA p.Gln144fs E5 Chinese — Sang et al. (2019)
c.447-13A>G — Intron 5 — Pakistani — Ben-Yosef et al. (2001)
United States
Taiwanese — Wong et al. (2020)
c.453G>A p.Val151Val E6 SRCR Palestinian — Scott et al. (2001)
Pakistani — Ben-Yosef et al. (2001)
United States
Korean — Lee et al. (2013)
Taiwanese — Wong et al. (2020)
c.551T>C P.Leu184Ser E6 SRCR Chinese — Sang et al. (2019)
Chinese — Li et al. (2019)
Taiwanese — Wong et al. (2020)
c.581G>T p.Cys194Phe E7 SRCR Pakistani — Ben-Yosef et al. (2001)
Severe Ahmed et al. (2004)
c.579dupA p.Cys194Mfs*17 E7 Pathogenic Polish — Lechowicz et al. (2017)
United States Severe This study
c.595G>A p.Val199Met E7 SRCR Dutch Severe Weegerink et al. (2011)
Korean
c.607C>T p.Gln203X E7 Japanese Severe Miyagawa et al. (2013)
c.617-4_-3dupAT — Intron7 — Japanese — Miyagawa et al. (2015)
Taiwanese — Wong et al. (2020)
c.621T>C p.Cys207Cys E8 Serine protease Taiwanese — Wong et al. (2020)
c.636C>T p.Gly212Gly E8 Serine protease Korean — Lee et al. (2013)
c.646C>T p.Arg216Cys E8 Serine protease German Mild Elbracht et al. (2007)
United States (Caucasian) — Eppsteiner et al. (2012)
c.647G>T p.Arg216Leu E8 Serine protease Turkish Severe Wattenhofer et al. (2005)
Japanese — Miyagawa et al. (2015)
c.726C>G p.Cys242Trp E8 Serine protease Pakistani Severe Shafique et al. (2014)
Czech — Safka Brozkova et al. (2020)
c.727G>A p.Gly243Arg E8 Serine protease Indian — Ganapathy et al. (2014)
Pakistani — Khan et al. (2019)
c.734C>T p.Ser245Phe E8 Serine protease Czech — Safka Brozkova et al. (2020)
c.743C>T p.Thr248Met E8 Serine protease Korean Mild Chung et al. (2014)
c.753G>C p.Trp251Cys E8 Serine protease Tunisian Severe Masmoudi et al. (2001)
c.757A>G p.Ile253Val E8 Serine protease Pakistani — Ben-Yosef et al. (2001)
United States
Korean — Lee et al. (2003)
Taiwanese — Wong et al. (2020)
c.767C>T p.Arg256Val E8 Serine protease Pakistani — Lee et al. (2012)
c.778G>A p.Ala260Thr E8 Serine protease Japanese — Miyagawa et al. (2015)
c.782+8insT — Intron8 — Pakistani Severe Ahmed et al. (2004)
c.782+2T>A — Intron8 — Polish — Lechowicz et al. (2017)
c.783-1G>A — Intron8 — Korean — Kim et al. (2017)
c.809T>A p.Ile270Asn E9 Serine protease Chinese Severe Gao et al. (2017)
c.830C>T p.Pro277Leu E9 Serine protease Turkish — Masmoudi et al. (2001)
c.871G>C p.Val291Leu E9 Serine protease Korean — Lee et al. (2013)
Kim et al. (2017)
c.916G>A p.Ala306Thr E9 Serine protease Likely pathogenic German Severe Elbracht et al. (2007)
Dutch — Weegerink et al. (2011)
United States (Caucasian) — Eppsteiner et al. (2012)
Korean — Lee et al. (2013)
— Chung et al. (2014)
Tibetan — Fan et al. (2014)
Chinese — Gao et al. (2017)
Korean — Song et al. (2020)
United States Mild This study
c.933C>T p.Ala311Ala E9 Serine protease Taiwanese — Wong et al. (2020)
c.941T>C p.Leu314Pro E9 Serine protease Pakistani — Zhou et al. (2020)
c.953-5A>G — Intron 9 — Polish — Lechowicz et al. (2017)
c.974T>A p.Leu325Gln E10 Serine protease Polish — Lechowicz et al. (2017)
c.988delA p.Glu330fs E10 Pakistani Severe Walsh et al. (2006)
c.999delC p.Asp334Mfs*24 E10 Polish — Lechowicz et al. (2017)
c.1019C>G p.Thr340Arg E10 Serine protease Italian Severe Vozzi et al. (2014)
c.1025G>A p.Gly342Glu E10 Serine protease Turkish — Duman et al. (2011)
c.1028G>C p.Trp343Ser E10 Serine protease Czech — Safka Brozkova et al. (2020)
c.1039G>T p.Glu347X E10 Korean — Song et al. (2020)
c.1128C>T p.Tyr376Tyr E11 Serine protease United States — Ben-Yosef et al. (2001)
c.1151T>G p.Met384Arg E11 Serine protease Chinese Severe Gao et al. (2017)
c.1156T>C p.Cys386Arg E11 Serine protease Indian — Ganapathy et al. (2014)
c.1159G>A p.Ala387Thr E11 Serine protease Japanese Mild Miyagawa et al. (2013)
c.1180_1187del8ins68 — E11 Serine protease Palestinian Severe Scott et al. (2001)
c.1183G>C p.Asp395His E11 Serine protease Unknown United States Severe This study
c.1192C>T p.Gln398X E11 Pathogenic Turkish Severe Wattenhofer et al. (2005)
United States Severe This study
c.1194+15C>A — Intron 11 — Taiwanese — Wong et al. (2020)
c.1204G>A p.Gly402Arg E12 Serine protease Chinese Severe Gao et al. (2017)
Pakistani — Noman et al. (2019)
United States — Bowles et al. (2021)
c.1211C>T p.Pro404Leu E12 Serine protease Tunisian Severe Masmoudi et al. (2001)
Wattenhofer et al. (2005)
United States — Bowles et al. (2021)
c.1219T>C p.Cys407Arg E12 Serine protease Pakistani Severe Ben-Yosef et al. (2001)
Ahmed et al. (2004)
Lee et al. (2012)
— Khan et al. (2019)
— Zafar et al. (2020)
c.1244T>C p.Leu415Ser E12 Serine protease Chinese Severe Gao et al. (2017)
c.1250G>A p.Gly417Glu E12 Serine protease Chinese Severe Gao et al. (2017)
c.1253C>T p.Ala418Val E12 Serine protease Taiwanese — Wong et al. (2020)
c.1269C>T p.Ile423Ile E12 Serine protease Taiwanese — Wong et al. (2020)
c.1273T>C p.Cys425Arg E12 Serine protease Pakistani — Lee et al. (2012)
c.1276G>A p.Ala426Thr E12 Serine protease Likely pathogenic Dutch Mild Weegerink et al. (2011)
Italian Leone et al. (2017)
Polish — Lechowicz et al. (2017)
United States — Shearer et al. (2018)
Mild This study
c.1291C>T p.Pro431Ser E12 Serine protease Italian Severe Vozzi et al. (2014)
c.1306C>G p.Arg436Gly E12 Serine protease Likely pathogenic Polish — Lechowicz et al. (2017)
Czech — Safka Brozkova et al. (2020)
United States Severe This study
c.1343T>C p.Met448Thr E12 Serine protease Likely pathogenic Polish — Lechowicz et al. (2017)
Czech — Safka Brozkova et al. (2020)
United States Mild This study
c.1345-2A>G — E12 United States — Shearer et al. (2018)

TM, transmembrane domain; LDLRA, LDL receptor-like domain; SRCR, scavenger receptor cysteine-rich domain; serine protease, trypsin-like serine protease domain. Naming of variants and labeling of domains and exons are based on the NM_001256317.3 transcript. Variant classification based on LMM variant classification. Only predicted loss-of-function and coding variants were included in the table. Bolded text refers to variants identified in this study. Of note, the phenotype severity is provided at the time of testing. While some patients may initially have milder phenotypes, the hearing loss can progress and become more severe.

Overview of clinical characteristics and genotypes of patients with TMPRSS3 variants who have received cochlear implantation.

Study (country) DNA change Protein change Exon Domain Hearing loss severity Age at CI (gender) Age at severe-profound HL Pre-operative hearing CI type CI outcomes
Weegerink et al., 2011 (Netherlands) c.207delC p.Thr70fs E4 — 4.5 years — Sloping HL Nucleus Freedom (Cochlear) 91% Phoneme (76% WRS)
c.916G>A p.Ala306Thr E9 Serine protease 40–60–100–110–110 dB (0.25, 0.5, 1, 2, 4 kHz)
c.595G>A p.Val199Met E7 SRCR — 6 years — Sloping HL Nucleus Freedom (Cochlear) 80% Phoneme (65% WRS)
c.916G>A p.Ala306Thr E9 Serine protease 40–50–110–110–110 dB (0.25, 0.5, 1, 2, 4 kHz)
c.413C>G p.Ala138Glu E5 SRCR — 29 years — Decreasing HL Nucleus CI24M (Cochlear) —
c.916G>A p.Ala306Thr E9 Serine protease 80–90–100–110–110 dB (0.25, 0.5, 1, 2, 4 kHz); 5% Phoneme
c.207delC p.Thr70fs E4 — 49 years — Decreasing HL Nucleus Contour CI24R (Cochlear) 89% Phoneme (75% WRS)
c.1276G>A p.Ala426Thr E12 Serine protease 70–95–110–110–110 dB (0.25, 0.5, 1, 2, 4 kHz); 20% Phoneme
c.207delC p.Thr70fs E4 — 45 years — Decreasing HL Clarion AB-5100H (Advanced Bionics) 76% Phoneme (60% WRS)
c.1276G>A p.Ala426Thr E12 Serine protease 80–90–100–110–120 dB (0.25, 0.5, 1, 2, 4 kHz); 5% Phoneme
c.207delC p.Thr70fs E4 — 46 years — Flat Clarion AB-5100H (Advanced Bionics) 82% Phoneme (58% WRS)
c.1276G>A p.Ala426Thr E12 Serine protease 100–100–110–120–120 dB (0.25, 0.5, 1, 2, 4 kHz); 0% Phoneme
c.207delC p.Thr70fs E4 — 43 years — Flat Clarion AB-5100H (Advanced Bionics) 83% Phoneme (62% WRS)
c.1276G>A p.Ala426Thr E12 Serine protease 100–90–110–120–120 dB (0.25, 0.5, 1, 2, 4 kHz); 0% Phoneme
c.413C>G p.Ala138Glu E5 SRCR — 51 years — Decreasing HL Nucleus Contour CI24R (Cochlear) 88% Phoneme (68% WRS)
c.595G>A p.Val199Met E7 SRCR 80–90–100–110–120 dB (0.25, 0.5, 1, 2, 4 kHz); 2.5% Phoneme
c.413C>G p.Ala138Glu E5 SRCR — 30 years — Sloping HL Nucleus Freedom (Cochlear) —
c.323-6G>A — In4 SRCR 50–90–110–110–110 dB (0.25, 0.5, 1, 2, 4 kHz); 10% Phoneme
Eppsteiner et al., 2012 (United States) c.413C>G p.Ala138Glu E5 SRCR Mild 45 years (male) 45 years 93 dB Advanced Bionics CII Poor performance (Combined CNC & HINT Score: 37)
c.646C>T p.Arg216Cys E8 Serine protease Mild (PTA at 0.5, 1, 2, and 4 kHz)
c.413C>G p.Ala138Glu E5 SRCR Mild 32 years (female) 17 years 98 dB Advanced Bionics CII Poor performance (Combined CNC & HINT Score: 23)
c.916G>A p.Ala306Thr E9 Serine protease Severe (PTA at 0.5, 1, 2, and 4 kHz)
Miyagawa et al., 2013 (Japan) c.607C>T p.Gln203X E7 Severe 40 years (female) — Sloping HL MED-EL Pulsar FLEXeas 40–35–30–35–40–40–45 dB (0.125, 0.25, 0.5, 1, 2, 4, 8 kHz)
c.1159G>A p.Ala387Thr E11 Serine protease Mild 25–30–65–100–110–110–100 dB (0.125, 0.25, 0.5, 1, 2, 4, 8 kHz)
Chung et al., 2014 (Korea) c.325C>T p.Arg109Trp E5 SRCR — 12 years (female) — Flat (<sloping) — Mean open set sentence score at 6 months following CI was 88.5%
c.916G>A p.Ala306Thr E9 Serine protease — 100–110–110–110–110–110 dB (0.25, 0.5, 1, 2, 4, 8 kHz)
c.325C>T p.Arg109Trp E5 SRCR — 6 years (male) — Decreasing HL — Mean open set sentence score at 6 months following CI was 88.5%
c.916G>A p.Ala306Thr E9 Serine protease Profound 70–80–90–100–110–100 dB (0.25, 0.5, 1, 2, 4, 8 kHz)
Miyagawa et al., 2015 (Japan) c.390C>G p.His130Arg E5 SRCR — 45 years (male) — Sloping HL MED-EL PULSAR FLEX24 90% discrimination score on Japanese monosyllable test at 24 months
c.647G>T p.Arg216Leu E8 Serine protease — 25–30–65–100–110–110–100 dB (0.125, 0.25, 0.5, 1, 2, 4, 8 kHz); 30% WRS w/HA
c.226C>T p.Gln76X E4 — 39 years (female) — Flat (<-Sloping) MED-EL PULSAR FLEX24 70% discrimination score on Japanese monosyllable test at 12 months
c.778G>A p.Ala260Thr E8 Serine protease — 70–90–100–100–110–110–100 dB (0.125, 0.25, 0.5, 1, 2, 4, 8 kHz); 24% WRS w/HA
c.212T>C p.Phe71Ser E4 LDLRA — 51 years (female) — Sloping HL MED-EL PULSAR FLEX24 80% discrimination score on Japanese monosyllable test at 12 months
c.617-4_-3dupAT p.Thr205fs In7 — — 30–40–40–40–100–110–100 dB (0.125, 0.25, 0.5, 1, 2, 4, 8 kHz); 40% WRS w/HA
Battelino et al., 2016 (Slovenia) c.208delC a p.Thr70fs a 19 E4 — 11 months (male) — 80–110 dB (unclear methodology) — 25 dB (unclear methodology)
c.208delC a p.Thr70fs a 19 E4 — 30 months (male) — 95–110 dB (unclear methodology) — 45 dB (unclear methodology)
c.208delC a p.Thr70fs a 19 E4 — 13 months (male) — 80–100 dB (unclear methodology) — 25 dB (unclear methodology)
c.208delC a p.Thr70fs a 19 E4 — 11 months (male) — 70–85 dB (unclear methodology) — 25 dB (unclear methodology)
Gao et al., 2017 (China) c.916G>A p.Ala306Thr E9 Serine protease Severe 3 years (female) — Decreasing HL — Described as “improved”
c.1250G>A p.Gly417Glu E12 Serine protease Severe 60–80–80–100–100 dB (0.25, 0.5, 1, 2, 4 kHz)
c.916G>A p.Ala306Thr E9 Serine protease Severe 14 years (female) — Sloping HL — Described as “improved”
c.323-6G>A — In4 — Severe 20–20–60–100–100–100 dB (0.25, 0.5, 1, 2, 4 kHz)
Kim et al., 2017 (Korea) c.346G>A p.Val116Met E5 SRCR — 4 years (female) — Decreasing HL — Not described, unofficially good
c.783-1G>A — In8 — Uncertain 90–100–100–1,100–110 dB (0.25, 0.5, 1, 2, 4 kHz)
c.346G>A p.Val116Met E5 SRCR Profound 10 years (female) — Sloping HL — Not described, unofficially good
c.871G>C p.Val291Leu E9 Serine protease Uncertain 45–90–100–100–110 dB (0.25, 0.5, 1, 2, 4 kHz)
Shearer et al., 2018 (United States) c.208delC p.Thr70fs a 19 E4 — 64 years — — Nucelus Hybrid CI L24 Array 80–90–110–110–110 dB (0.125, 0.25, 0.5, 1, 2 kHz)
c.1276G>A p.Ala426Thr E12 Serine protease
c.413C>G p.Ala138Glu E5 SRCR — 53 years — — Nucleus Hybrid CI S8 Array 50–60–90–110–110 dB (0.125, 0.25, 0.5, 1, 2 kHz)
c.1276G>A p.Ala426Thr E12 Serine protease —
c.1345–2A>G a — In12 — — 38 years — — Nucelus Hybrid CI L24 Array 35–30–55–110–110 dB (0.125, 0.25, 0.5, 1, 2 kHz)
Song et al., 2020 (Korea) c.916G>A p.Ala306Thr E9 Serine protease — 17 years (female) 3–5 years Sloping HL — 86% WRS at 12 months following implantation
c.1039G>T p.Glu347Ter E10 Serine protease — 40–90–100–100–110–110 dB (0.25, 0.5, 1, 2, 4, 8 kHz)
Holder et al., 2021 (United States) c.208delC p.Thr70fs a 19 E4 — 54 months (female) — Sloping HL Cochlear Nucleus 522/532 (left/right) CNC 84%; BabyBio Quiet 94%/92% (left/right)
c.916G>A p.Ala306Thr E9 Serine protease 20–25–95–110–100 dB (0.25, 0.5, 1, 2, 4 kHz)
c.208delC p.Thr70fs a 19 E4 — 47 months (female) — Sloping HL Cochlear Nucleus 522/522 (left/right) CNC 72%; BabyBio Quiet 55%
c.916G>A p.Ala306Thr E9 Serine protease 20–20–75–115–115 dB (0.25, 0.5, 1, 2, 4 kHz)
c.208delC p.Thr70fs a 19 E4 — 43 months (female) — Sloping HL Cochlear Nucleus 532/532 (left/right) LNT 92%/82% (left/right); HINT 62%
c.916G>A p.Ala306Thr E9 Serine protease 20–25–15–95–110 (0.25, 0.5, 1, 2, 4 kHz)

HL, hearing loss; CI, cochlear implant; LDLRA, LDL receptor-like domain; dB, decibel; WRS, word-recognition score; SRCR, scavenger receptor cysteine-rich domain; serine protease, trypsin-like serine protease domain; PTA, pure tone average; CNC, consonant-nucleus-consonant; HINT, hearing in noise test; HA, hearing aid; LNT, lexical neighborhood test. Naming of variants and labeling of domains and exons are based on the NM_001256317.3 transcript. Of note, the phenotype severity is provided at the time of testing. While some patients may initially have milder phenotypes, the hearing loss can progress and become more severe.

Patient is homozygous for the specified variant.

Previously reported variants and their associated hearing phenotypes and clinical outcomes following CI, when available, were compiled. Additionally, our own cohort of patients was genetically screened as described below.

 2.2 Cohort Description

Our study included genetic and phenotypic data from 18 patients and their family members (when available), who were largely White, though Family A was a consanguineous White Egyptian family, Family B was “mixed,” and Families M and I were of Hispanic or Latino ethnicity. Of the patients with characterized hearing loss, the severity ranged from moderate to profound with some individuals experiencing congenital onset and others experiencing a childhood onset or an onset in the second decade of life. Patients were referred to the Laboratory for Molecular Medicine (LMM) at Mass General Brigham Personalized Medicine (Cambridge, MA, United States) from 2009 to 2017. Patients were referred from various clinics and hospitals across the United States. The LMM collected information pertinent to the nature of the hearing loss in the patients (if available) including family history of hearing loss and/or disease, audiological testing, temporal bone CT/MRI results, and CI status. Further information was requested through physicians via the Mass General Brigham Human Research Committee’s IRB protocol for the study of the genetics of hearing loss. Patients were selected based on whether they received a positive result for TMPRSS3-associated hearing loss with the intent of follow up of the outcome of CI, if received.

 2.3 <italic>TMPRSS3</italic> Screening and OtoGenome Next-Generation Sequencing Testing

Patient DNA was extracted from whole blood from patients who were referred to the LMM for hearing-loss genetic testing. Our cohort contains patients from 2009 to 2017. The genetic testing varied for each patient based on the judgment of the ordering physician and the nature of the patient’s hearing loss. Testing was performed by single gene sequencing that included TMPRSS3, or LMM’s OtoGenome-v1,-v2, or -v3 panels.

The LMM’s bioinformatics pipeline for targeted next generation sequencing (NGS) panels has been described previously (Pugh et al., 2016). Patients with hearing loss who underwent genetic testing between 2010 and 2014 were tested with the Otogenome-v1 which included the following 71 genes: ACTG1, ATP6V1B1, BSND, CCDC50, CDH23, CLDN14, CLRN1, COCH, COL11A2, CRYM, DFNA5, DFNB31, DFNB59, DIAPH1, ESPN, ESRRB, EYA1, EYA4, GIPC3, GJB2, GJB3, GJB6, GPR98, GPSM2, GRHL2, GRXCR1, HGF, ILDR1, KCNE1, KCNQ1, KCNQ4, LHFPL5, LOXHD1, LRTOMT, MARVELD2, MIR183, MIR96, MSRB3, MTRNR1 (12S rRNA), MTTS1 (tRNAser(UCN)), MYH14, MYH9, MYO15A, MYO1A, MYO3A, MYO6, MYO7A, OTOA, OTOF, PCDH15, PDZD7, POU3F4, POU4F3, PRPS1, RDX, SERPINB6, SLC17A8, SLC26A4 (PDS), SLC26A5, TECTA, TIMM8A, TJP2, TMC1, TMIE, TMPRSS3, TPRN, TRIOBP, USH1C, USH1G, USH2A, and WFS1.

OtoGenome-v2 was used in patients who underwent testing at the LMM from 2014 to 2015. For this iteration, PDZD7 and SLC26A5 genes were removed and the STRC gene was added. In addition, copy number variant (CNV) detection was added using VisCap as previously described (Pugh et al., 2016; Tayoun et al., 2016).

OtoGenome-v3, used from 2015 to 2017, included 87 genes but did not include the following genes included in v2: CRYM, GJB3, MIR182, MYO1A, SLC17A8, and TJP2. The following 23 genes were added CACNA1D, CATSPER2, CEACAM16, CIB2, CLPP, DIABLO, EDN3, EDNRB, HARS2, HSD17B4, KARS, LARS2, MITF, OTOG, OTOGL, P2RX2, PAX3, SIX1, SMPX, SOX10, SYNE4, TBC1D24, and TSPEAR. Parents and other unaffected/affected family members, when available, were tested for detected variants. Variants were confirmed via Sanger sequencing for single-nucleotide variants (SNVs), or droplet digital PCR for CNVs called by VisCap (Pugh et al., 2016; Tayoun et al., 2016).

 2.4 LMM Variant Classification

The LMM’s early variant classification methods are as previously described (Duzkale et al., 2013) and were subsequently updated to conform to more recent professional guidelines (Richards et al., 2015). Data used to classify variants included that from population databases (e.g., Exome Aggregation Consortium (ExAC); gnomAD), internal or external disease databases (e.g., ClinVar, LOVD, HGMD), the literature, functional studies, segregation, allelic observations and in silico missense and splicing prediction tools. Variants were classified as pathogenic (P), likely pathogenic (LP), of uncertain significance (VUS), likely benign, or benign. The VUS category was further subdivided into VUS-5, -4, and -3 where VUS-5 indicated leaning towards pathogenic, and VUS-3 indicated leaning towards benign. Likely benign and benign variants are not reported in this article but were submitted to ClinVar (www.ncbi.nlm.nih.gov/clinvar/) along with all other variants observed at the LMM.

 3 Results

We reviewed the type, position, origin, and variant classification of 87 previously reported TMPRSS3 variants and present one novel variant identified from our cohort (Figure 1; Table 1). Compiled variants are associated with non-syndromic hearing loss in more than 20 ancestral groups worldwide. Fourteen of the identified variants were predicted loss-of-function (pLOF) (frameshift, stop-codon, or splice-site variants) with either prematurely terminated protein products or nonsense-mediated decay of mRNA. Fifty-eight of the identified variants were missense variants. Nearly all variants were predicted to disrupt the proteolytic activity of the protein. Both prelingual and post lingual hearing impairment was reported, with most patients showing a typical ski-slope audiogram configuration. CI outcomes were reported for 32 patients with bi-allelic variants in TMPRSS3 across 11 different studies (Table 2) (Weegerink et al., 2011; Eppsteiner et al., 2012; Miyagawa et al., 2013; Chung et al., 2014; Miyagawa et al., 2015; Battelino et al., 2016; Gao et al., 2017; Kim et al., 2017; Shearer et al., 2018; Song et al., 2020; Holder et al., 2021). While degree of hearing improvement varied between patients, the majority of those who underwent CI had positive outcomes.

Our cohort included 18 patients—7 females and 11 males—with ages ranging from 3 months to 36 years (Figure 2). 15 patients were White with the remaining 3 identifying as Hispanic/Latino or mixed. We identified 12 different TMPRSS3 variants of which 1 has not been previously reported: deletion of Exons 1–5 and 13 (Table 3). This novel variant was classified as pathogenic as it met the criteria outlined by previous professional guidelines (Richards et al., 2015) with specifications provided by ClinGen (https://clinicalgenome.org/working-groups/sequence-variant-interpretation), specifically the combination of PVS1 (predicted loss of function), PM2 (absence in gnomAD), and PM3 (homozygous observation in an individual with phenotype matching the gene). The most commonly identified variants were p.Thr70fs*19 and p.Ala138Glu. Eight patients had congenital hearing loss, four of whom had biallelic pLOF variants.

Pedigree chart for enrolled patient families (A–N). Age at genetic testing, age at onset of hearing loss, and other relevant clinical information is provided, when available, for patients and family members. CI, cochlear implant; HL, hearing loss; HA, hearing aid.

Genotype and phenotype overview of our patient cohort.

Family Age Gender DNA change Protein change Configuration HL onset HL severity
A 16 months F Deletion of Exons 1–5 and13 a — — Congenital —
6 years M Deletion of Exons 1–5 and13 a — — Congenital —
B 3 months M c.208delC a p.Thr70fs a 19 — Congenital Profound
C 9 months F c.208delC; c.1192C>T p.Thr70fs a 19; p.Gln398X — Congenital Profound
D 8 years M c.208delC; c.1276G>A p.Thr70fs a 19; p.Ala426Thr — — Sloping hearing loss
13 years F — — Sloping sensorineural hearing loss
15 years M Trans 10 years old Progressive sloping, moderate left, severe right
E 13 years F c.208delC; c.413C>G p.Thr70fs a 19; p.Ala138Glu — — Progressive, sloping, severe
F 11 years F c.208delC; c.413C>G p.Thr70fs a 19; p.Ala138Glu — 9 years old Sloping, profound
G 22 years F c.208delC; c.413C>G p.Thr70fs a 19; p.Ala138Glu — 19 years old —
H 6 years M c.323-6G>A; c.325C>T -; p.Arg109Trp — Congenital Moderately severe to profound
I 1 year M c.579dupA; c.1183G>C p.Cys194MetfsX17; p.Asp395His Trans Congenital Severe to profound
J 22 years M c.238C>T; c.1343T>C p.Arg80Cys; p.Met448Thr — 12 years old Progressive, moderate-severe left, severe right
24 years M c.238C>T; c.1343T>C p.Arg80Cys; p.Met448Thr — 10–12 years old —
K 3 years F c.310G>A; c.916G>A p.Glu104Lys; p.Ala306Thr — — Moderately severe at low frequencies, profound at high frequencies
L 17 years M c.325C>T; c.413C>G p.Arg109Trp; p.Ala138Glu — 4 years old Moderate-severe
M 12 years M c.413C>G; c.916G>A p.Ala138Glu; p.Ala306Thr — Congenital Progressive, high frequency, moderate
N 36 years M c.208delC; c.1306C>T p.Thr70fs a 19; p.Arg436Gly — Congenital Progressive, profound

HL, hearing loss. Novel variant is bolded. Naming of variants is based on the NM_001256317.3 transcript.

patient is homozygous for the specified variant.

Four patients in our cohort underwent CI, and outcome information was available for two patients. The first patient, from family B, was found to have congenital profound hearing loss and was homozygous for p.Thr70fs*19. It is unclear when the patient underwent CI. However, at a follow up at 4 years of age, the patient had functional speech. Clinical records indicated that the patient had ongoing articulation errors and required speech therapy but was able to maintain adequate hearing. The second patient, from family K, was compound heterozygous for p.Glu104Lys and p.Ala306Thr. Clinical records have suggested positive CI outcome for her moderate-profound hearing loss. The remaining two patients who underwent CI were the siblings from family A who both had profound congenital hearing loss and were homozygous for a deletion of Exons 1–5 and 13. Their current hearing status is unknown.

 4 Discussion

The genotype-phenotype correlations of TMPRSS3 variants have not been well characterized. It has been previously shown that the frequency of TMPRSS3-induced ARSNHL was low in White individuals (Wattenhofer et al., 2002). However, a recent epidemiological study of patients undergoing CI revealed that 10% (13) of patients with positive genetic testing had TMPRSS3 gene variants (Seligman et al., 2021). As adoption of genetic testing in clinical practice continues to grow, it is important to be aware of common TMPRSS3 variants and associated phenotypes to best counsel patients.

In our cohort of 18 patients, 15 of whom were White, the most frequently observed variants were p.Thr70fs*19 and p.Ala138Glu implying that those were either hot spots or founder variants. The combination of the p.Thr70fs*19 frameshift variant with a missense variant appeared to cause sloping hearing loss that varied in severity. Biallelic pLOF variants appeared to cause congenital profound hearing loss. This phenotype information is valuable when trying to understand potential patient prognosis based on genetic testing results.

Previous studies on the role of CI in patients with TMPRSS3 variants have reported variable results. In one study, poor outcomes following CI in patients with TMPRSS3 variants were attributed to the expression of the TMPRSS3 gene in SGNs as opposed to other locations in the cochlea such as the membranous labyrinth (Eppsteiner et al., 2012). These authors also suggested that patients with pathogenic TMPRSS3 variants may have continued loss of SGNs over time which could contribute to ongoing hearing deterioration even after CI. However, recent studies have shown predominantly positive outcomes following CI in patients with TMPRSS3 variants (Weegerink et al., 2011; Miyagawa et al., 2013; Chung et al., 2014; Miyagawa et al., 2015; Battelino et al., 2016; Gao et al., 2017; Shearer et al., 2018; Song et al., 2020; Holder et al., 2021). This discrepancy might be related to the large duration of deafness and older age of the two patients in Eppsteiner et al. (2012) and Holder et al. (2021). In addition, a study of CI outcomes in pediatric patients with TMPRSS3 variants reported positive outcomes with no evidence of SGN degeneration leading to decreased performance over time (Holder et al., 2021). Furthermore, it was suggested that even if SGN degeneration does contribute to a longitudinal decline in performance, early CI may help slow or reverse this process (Holder et al., 2021). Even so, many clinics do not implant patients with precipitously sloping hearing loss as they do not meet labeled indications for CI. However, off-label implantation has been shown to be beneficial and is being employed much more frequently at major academic medical centers (Carlson et al., 2015; Leigh et al., 2016; Carlson et al., 2018).

Taken together with the positive clinical outcomes following CI in two patients from our cohort, it is evident that CI is a promising treatment strategy for patients with TMPRSS3 variants. Active intervention with CI is likely to be beneficial, particularly in patients in whom residual hearing is preserved. It is imperative that the benefits of CI are made clear when counseling patients on their potential treatment options.

 Data Availability Statement

The evidence for all variants classified by the authors is included in submissions to ClinVar by the Laboratory for Molecular Medicine (Organization ID: 21766). All other data supporting the conclusions of this article, if not directly included in the paper, will be made available by the authors, without undue reservation.

 Ethics Statement

The studies involving human participants were reviewed and approved by the Mass General Brigham Human Research Committee’s IRB. Written informed consent to participate in this study was provided by the participants’ legal guardian/next of kin.

 Author Contributions

IM and AG co-wrote the manuscript and prepared the tables and figures, VS edited the manuscript and prepared the tables and figures for submission, HR edited the manuscript and provided technical feedback, KS conceived, designed, and supervised the manuscript writing and editing.

 Funding

We gratefully acknowledge support from the National Institutes of Health grant R01 DC015824 (KMS) and Jennifer and Louis Hernandez (KMS).

 Conflict of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

 Publisher’s Note

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.

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