Hearing loss is a genetically heterogeneous sensory defect, and the frequent causes are biallelic pathogenic variants in the GJB2 gene. However, patients carrying only one heterozygous pathogenic (monoallelic) GJB2 variant represent a long-lasting diagnostic problem. Interestingly, previous results showed that individuals with a heterozygous pathogenic GJB2 variant are two times more prevalent among those with hearing loss compared to normal-hearing individuals. This excess among patients led us to hypothesize that there could be another pathogenic variant in the GJB2 region/DFNB1 locus. A hitherto undiscovered variant could, in part, explain the cause of hearing loss in patients and would mean reclassifying them as patients with GJB2 biallelic pathogenic variants. In order to detect an unknown causal variant, we examined 28 patients using NGS with probes that continuously cover the 0.4 Mb in the DFNB1 region. An additional 49 patients were examined by WES to uncover only carriers. We did not reveal a second pathogenic variant in the DFNB1 region. However, in 19% of the WES-examined patients, the cause of hearing loss was found to be in genes other than the GJB2. We present evidence to show that a substantial number of patients are carriers of the GJB2 pathogenic variant, albeit only by chance.

Centre for Medical Genetics and Reproductive Medicine GENNET 17000 Prague Czech Republic

Department of Medical Genetics Masaryk Hospital in Usti nad Labem Regional Health Corporation 40011 Ústí nad Labem Czech Republic

Department of Otorhinolaryngology Head and Neck Surgery Faculty of Medicine and University Hospital Comenius University 85107 Bratislava Slovakia

Diabgene Laboratory Institute of Experimental Endocrinology Biomedical Research Center Slovak Academy of Sciences 84505 Bratislava Slovakia

Neurogenetic laboratory Department of Paediatric Neurology 2nd Faculty of Medicine Charles University and University Hospital Motol 15006 Prague Czech Republic

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Morton C.C., Nance W.E. Newborn hearing screening—A silent revolution. N. Engl. J. Med. 2006;354:2151–2164. doi: 10.1056/NEJMra050700. PubMed DOI

Shearer A.E., Hildebrand M.S., Smith R.J.H. Hereditary Hearing Loss and Deafness Overview. In: Adam M.P., Ardinger H.H., Pagon R.A., Wallace S.E., Bean L.J.H., Stephens K., Amemiya A., editors. GeneReviews®. University of Washington; Seattle, WA, USA: 1993.

Seeman P., Malikova M., Raskova D., Bendova O., Groh D., Kubalkova M., Sakmaryova I., Seemanova E., Kabelka Z. Spectrum and frequencies of mutations in the GJB2 (Cx26) gene among 156 Czech patients with pre-lingual deafness. Clin. Genet. 2004;66:152–157. doi: 10.1111/j.1399-0004.2004.00283.x. PubMed DOI

Chan D.K., Chang K.W. GJB2-associated hearing loss: Systematic review of worldwide prevalence, genotype, and auditory phenotype. Laryngoscope. 2014;124:E34–E53. doi: 10.1002/lary.24332. PubMed DOI

Tsukada K., Nishio S.Y., Hattori M., Usami S. Ethnic-specific spectrum of GJB2 and SLC26A4 mutations: Their origin and a literature review. Ann. Otol. Rhinol. Laryngol. 2015;124(Suppl. 1):61S–76S. doi: 10.1177/0003489415575060. PubMed DOI

Seeman P., Sakmaryova I. High prevalence of the IVS 1 + 1 G to A/GJB2 mutation among Czech hearing impaired patients with monoallelic mutation in the coding region of GJB2. Clin. Genet. 2006;69:410–413. doi: 10.1111/j.1399-0004.2006.00602.x. PubMed DOI

Karczewski K.J., Francioli L.C., Tiao G., Cummings B.B., Alfoldi J., Wang Q., Collins R.L., Laricchia K.M., Ganna A., Birnbaum D.P., et al. The mutational constraint spectrum quantified from variation in 141,456 humans. Nature. 2020;581:434–443. doi: 10.1038/s41586-020-2308-7. PubMed DOI PMC

Del Castillo I., Moreno-Pelayo M.A., Del Castillo F.J., Brownstein Z., Marlin S., Adina Q., Cockburn D.J., Pandya A., Siemering K.R., Chamberlin G.P., et al. Prevalence and evolutionary origins of the del(GJB6-D13S1830) mutation in the DFNB1 locus in hearing-impaired subjects: A multicenter study. Am. J. Hum. Genet. 2003;73:1452–1458. doi: 10.1086/380205. PubMed DOI PMC

Del Castillo F.J., Rodriguez-Ballesteros M., Alvarez A., Hutchin T., Leonardi E., de Oliveira C.A., Azaiez H., Brownstein Z., Avenarius M.R., Marlin S., et al. A novel deletion involving the connexin-30 gene, del(GJB6-d13s1854), found in trans with mutations in the GJB2 gene (connexin-26) in subjects with DFNB1 non-syndromic hearing impairment. J. Med. Genet. 2005;42:588–594. doi: 10.1136/jmg.2004.028324. PubMed DOI PMC

Seeman P., Bendova O., Raskova D., Malikova M., Groh D., Kabelka Z. Double heterozygosity with mutations involving both the GJB2 and GJB6 genes is a possible, but very rare, cause of congenital deafness in the Czech population. Ann. Hum. Genet. 2005;69:9–14. doi: 10.1046/j.1529-8817.2003.00120.x. PubMed DOI

Feldmann D., Le Marechal C., Jonard L., Thierry P., Czajka C., Couderc R., Ferec C., Denoyelle F., Marlin S., Fellmann F. A new large deletion in the DFNB1 locus causes nonsyndromic hearing loss. Eur. J. Med. Genet. 2009;52:195–200. doi: 10.1016/j.ejmg.2008.11.006. PubMed DOI

Del Castillo I., Villamar M., Moreno-Pelayo M.A., del Castillo F.J., Alvarez A., Telleria D., Menendez I., Moreno F. A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N. Engl. J. Med. 2002;346:243–249. doi: 10.1056/NEJMoa012052. PubMed DOI

Wilch E., Azaiez H., Fisher R.A., Elfenbein J., Murgia A., Birkenhager R., Bolz H., Da Silva-Costa S.M., Del Castillo I., Haaf T., et al. A novel DFNB1 deletion allele supports the existence of a distant cis-regulatory region that controls GJB2 and GJB6 expression. Clin. Genet. 2010;78:267–274. doi: 10.1111/j.1399-0004.2010.01387.x. PubMed DOI PMC

Tayoun A.N., Mason-Suares H., Frisella A.L., Bowser M., Duffy E., Mahanta L., Funke B., Rehm H.L., Amr S.S. Targeted Droplet-Digital PCR as a Tool for Novel Deletion Discovery at the DFNB1 Locus. Hum. Mutat. 2016;37:119–126. doi: 10.1002/humu.22912. PubMed DOI

Rodriguez-Paris J., Schrijver I. The digenic hypothesis unraveled: The GJB6 del(GJB6-D13S1830) mutation causes allele-specific loss of GJB2 expression in cis. Biochem. Biophys. Res. Commun. 2009;389:354–359. doi: 10.1016/j.bbrc.2009.08.152. PubMed DOI

Rodriguez-Paris J., Tamayo M.L., Gelvez N., Schrijver I. Allele-specific impairment of GJB2 expression by GJB6 deletion del(GJB6-D13S1854) PLoS ONE. 2011;6:e21665. doi: 10.1371/journal.pone.0021665. PubMed DOI PMC

Wilch E., Zhu M., Burkhart K.B., Regier M., Elfenbein J.L., Fisher R.A., Friderici K.H. Expression of GJB2 and GJB6 is reduced in a novel DFNB1 allele. Am. J. Hum. Genet. 2006;79:174–179. doi: 10.1086/505333. PubMed DOI PMC

Common J.E., Bitner-Glindzicz M., O’Toole E.A., Barnes M.R., Jenkins L., Forge A., Kelsell D.P. Specific loss of connexin 26 expression in ductal sweat gland epithelium associated with the deletion mutation del(GJB6-D13S1830) Clin. Exp. Dermatol. 2005;30:688–693. doi: 10.1111/j.1365-2230.2005.01878.x. PubMed DOI

Oza A.M., DiStefano M.T., Hemphill S.E., Cushman B.J., Grant A.R., Siegert R.K., Shen J., Chapin A., Boczek N.J., Schimmenti L.A., et al. Expert specification of the ACMG/AMP variant interpretation guidelines for genetic hearing loss. Hum. Mutat. 2018;39:1593–1613. doi: 10.1002/humu.23630. PubMed DOI PMC

Mohiyuddin M., Mu J.C., Li J., Bani Asadi N., Gerstein M.B., Abyzov A., Wong W.H., Lam H.Y. MetaSV: An accurate and integrative structural-variant caller for next generation sequencing. Bioinformatics. 2015;31:2741–2744. doi: 10.1093/bioinformatics/btv204. PubMed DOI PMC

Fan X., Abbott T.E., Larson D., Chen K. BreakDancer: Identification of Genomic Structural Variation from Paired-End Read Mapping. Curr. Protoc. Bioinform. 2014;45:15–16. doi: 10.1002/0471250953.bi1506s45. PubMed DOI PMC

Abyzov A., Urban A.E., Snyder M., Gerstein M. CNVnator: An approach to discover, genotype, and characterize typical and atypical CNVs from family and population genome sequencing. Genome Res. 2011;21:974–984. doi: 10.1101/gr.114876.110. PubMed DOI PMC

Abyzov A., Li S., Kim D.R., Mohiyuddin M., Stutz A.M., Parrish N.F., Mu X.J., Clark W., Chen K., Hurles M., et al. Analysis of deletion breakpoints from 1,092 humans reveals details of mutation mechanisms. Nat. Commun. 2015;6:7256. doi: 10.1038/ncomms8256. PubMed DOI PMC

Ye K., Schulz M.H., Long Q., Apweiler R., Ning Z. Pindel: A pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics. 2009;25:2865–2871. doi: 10.1093/bioinformatics/btp394. PubMed DOI PMC

Chen X., Schulz-Trieglaff O., Shaw R., Barnes B., Schlesinger F., Kallberg M., Cox A.J., Kruglyak S., Saunders C.T. Manta: Rapid detection of structural variants and indels for germline and cancer sequencing applications. Bioinformatics. 2016;32:1220–1222. doi: 10.1093/bioinformatics/btv710. PubMed DOI

Layer R.M., Chiang C., Quinlan A.R., Hall I.M. LUMPY: A probabilistic framework for structural variant discovery. Genome Biol. 2014;15:R84. doi: 10.1186/gb-2014-15-6-r84. PubMed DOI PMC

Kronenberg Z.N., Osborne E.J., Cone K.R., Kennedy B.J., Domyan E.T., Shapiro M.D., Elde N.C., Yandell M. Wham: Identifying Structural Variants of Biological Consequence. PLoS Comput. Biol. 2015;11:e1004572. doi: 10.1371/journal.pcbi.1004572. PubMed DOI PMC

Talevich E., Shain A.H., Botton T., Bastian B.C. CNVkit: Genome-Wide Copy Number Detection and Visualization from Targeted DNA Sequencing. PLoS Comput. Biol. 2016;12:e1004873. doi: 10.1371/journal.pcbi.1004873. PubMed DOI PMC

MacDonald J.R., Ziman R., Yuen R.K., Feuk L., Scherer S.W. The Database of Genomic Variants: A curated collection of structural variation in the human genome. Nucleic Acids Res. 2014;42:D986–D992. doi: 10.1093/nar/gkt958. PubMed DOI PMC

Kerkhof J., Schenkel L.C., Reilly J., McRobbie S., Aref-Eshghi E., Stuart A., Rupar C.A., Adams P., Hegele R.A., Lin H., et al. Clinical Validation of Copy Number Variant Detection from Targeted Next-Generation Sequencing Panels. J. Mol. Diagn. 2017;19:905–920. doi: 10.1016/j.jmoldx.2017.07.004. PubMed DOI

Safka Brozkova D., Poisson Markova S., Meszarosova A.U., Jencik J., Cejnova V., Cada Z., Lastuvkova J., Raskova D., Seeman P. Spectrum and frequencies of non GJB2 gene mutations in Czech patients with early non-syndromic hearing loss detected by gene panel NGS and whole-exome sequencing. Clin. Genet. 2020;98:548–554. doi: 10.1111/cge.13839. PubMed DOI

Likar T., Hasanhodzic M., Teran N., Maver A., Peterlin B., Writzl K. Diagnostic outcomes of exome sequencing in patients with syndromic or non-syndromic hearing loss. PLoS ONE. 2018;13:e0188578. doi: 10.1371/journal.pone.0188578. PubMed DOI PMC

Sloan-Heggen C.M., Bierer A.O., Shearer A.E., Kolbe D.L., Nishimura C.J., Frees K.L., Ephraim S.S., Shibata S.B., Booth K.T., Campbell C.A., et al. Comprehensive genetic testing in the clinical evaluation of 1119 patients with hearing loss. Hum. Genet. 2016;135:441–450. doi: 10.1007/s00439-016-1648-8. PubMed DOI PMC

Gandia M., Del Castillo F.J., Rodriguez-Alvarez F.J., Garrido G., Villamar M., Calderon M., Moreno-Pelayo M.A., Moreno F., del Castillo I. A novel splice-site mutation in the GJB2 gene causing mild postlingual hearing impairment. PLoS ONE. 2013;8:e73566. doi: 10.1371/journal.pone.0073566. PubMed DOI PMC

Stanghellini I., Genovese E., Palma S., Ravani A., Falcinelli C., Guarnaccia M.C., Percesepe A. New and rare GJB2 alleles in patients with nonsyndromic sensorineural hearing impairment: A genotype/auditory phenotype correlation. Genet. Test Mol. Biomark. 2014;18:839–844. doi: 10.1089/gtmb.2014.0185. PubMed DOI

Walsh T., Shahin H., Elkan-Miller T., Lee M.K., Thornton A.M., Roeb W., Abu Rayyan A., Loulus S., Avraham K.B., King M.C., et al. Whole exome sequencing and homozygosity mapping identify mutation in the cell polarity protein GPSM2 as the cause of nonsyndromic hearing loss DFNB82. Am. J. Hum. Genet. 2010;87:90–94. doi: 10.1016/j.ajhg.2010.05.010. PubMed DOI PMC

Bademci G., Abad C., Incesulu A., Rad A., Alper O., Kolb S.M., Cengiz F.B., Diaz-Horta O., Silan F., Mihci E., et al. MPZL2 is a novel gene associated with autosomal recessive nonsyndromic moderate hearing loss. Hum. Genet. 2018;137:479–486. doi: 10.1007/s00439-018-1901-4. PubMed DOI PMC

Wesdorp M., Murillo-Cuesta S., Peters T., Celaya A.M., Oonk A., Schraders M., Oostrik J., Gomez-Rosas E., Beynon A.J., Hartel B.P., et al. MPZL2, Encoding the Epithelial Junctional Protein Myelin Protein Zero-like 2, Is Essential for Hearing in Man and Mouse. Am. J. Hum. Genet. 2018;103:74–88. doi: 10.1016/j.ajhg.2018.05.011. PubMed DOI PMC

Tekin M., Chioza B.A., Matsumoto Y., Diaz-Horta O., Cross H.E., Duman D., Kokotas H., Moore-Barton H.L., Sakoori K., Ota M., et al. SLITRK6 mutations cause myopia and deafness in humans and mice. J. Clin. Investig. 2013;123:2094–2102. doi: 10.1172/JCI65853. PubMed DOI PMC

Morlet T., Rabinowitz M.R., Looney L.R., Riegner T., Greenwood L.A., Sherman E.A., Achilly N., Zhu A., Yoo E., O’Reilly R.C., et al. A homozygous SLITRK6 nonsense mutation is associated with progressive auditory neuropathy in humans. Laryngoscope. 2014;124:E95–E103. doi: 10.1002/lary.24361. PubMed DOI PMC

Du W., Han M.K., Wang D.Y., Han B., Zong L., Lan L., Yang J., Shen Q., Xie L.Y., Yu L., et al. A POU3F4 Mutation Causes Nonsyndromic Hearing Loss in a Chinese X-linked Recessive Family. Chin. Med. J. 2017;130:88–92. doi: 10.4103/0366-6999.196565. PubMed DOI PMC

Scheidecker S., Bar S., Stoetzel C., Geoffroy V., Lannes B., Rinaldi B., Fischer F., Becker H.D., Pelletier V., Pagan C., et al. Mutations in KARS cause a severe neurological and neurosensory disease with optic neuropathy. Hum. Mutat. 2019;40:1826–1840. doi: 10.1002/humu.23799. PubMed DOI

Yu X., Lin Y., Xu J., Che T., Li L., Yang T., Wu H. Molecular epidemiology of Chinese Han deaf patients with bi-allelic and mono-allelic GJB2 mutations. Orphanet. J. Rare Dis. 2020;15:29. doi: 10.1186/s13023-020-1311-2. PubMed DOI PMC

Moisan S., Le Nabec A., Quillevere A., Le Marechal C., Ferec C. Characterization of GJB2 cis-regulatory elements in the DFNB1 locus. Hum. Genet. 2019;138:1275–1286. doi: 10.1007/s00439-019-02068-8. PubMed DOI