G C A T T A C G G C A T genes Case Report Non-Syndromic Autosomal Dominant Hearing Loss: The First Italian Family Carrying a Mutation in the NCOA3 Gene Paola Tesolin 1,* , Anna Morgan 2 , Michela Notarangelo 3, Rocco Pio Ortore 4, Maria Pina Concas 2 , Angelantonio Notarangelo 5 and Giorgia Girotto 1,2 1 Department of Medicine, Surgery and Health Sciences, University of Trieste, 34127 Trieste, Italy; [email protected] 2 Institute for Maternal and Child Health—I.R.C.C.S. “Burlo Garofolo”, 34137 Trieste, Italy; [email protected] (A.M.); [email protected] (M.P.C.) 3 Department of Cellular, Computational and Integrative Biology (CIBIO), University of Trento, 38123 Trento, Italy; [email protected] 4 UOC Otolaryngology, Institute I.R.C.C.S. “Casa Sollievo della Sofferenza”, 71013 San Giovanni Rotondo, Italy; [email protected] 5 UOC Medical Genetics, Institute I.R.C.C.S. “Casa Sollievo della Sofferenza”, 71013 San Giovanni Rotondo, Italy; [email protected] * Correspondence: [email protected]; Tel.: +39-04-0378-5539 Abstract: Hearing loss (HL) is the most frequent sensory disorder, affecting about 1–3 per 1000 live births, with more than half of the cases attributable to genetic causes. Despite the fact that many HL causative genes have already been identified, current genetic tests fail to provide a diagnosis for about 40% of the patients, suggesting that other causes still need to be discovered. Here, we describe a four- Citation: Tesolin, P.; Morgan, A.; generation Italian family affected by autosomal dominant non-syndromic hearing loss (ADNSHL), in Notarangelo, M.; Ortore, R.P.; Concas, M.P.; Notarangelo, A.; Girotto, G. which exome sequencing revealed a likely pathogenic variant in NCOA3 (NM_181659.3, c.2909G>C, Non-Syndromic Autosomal p.(Gly970Ala)), a gene recently described as a novel candidate for ADNSHL in a Brazilian family. A Dominant Hearing Loss: The First comparison between the two families highlighted a series of similarities: both the identified variants Italian Family Carrying a Mutation in are missense, localized in exon 15 of the NCOA3 gene and lead to a similar clinical phenotype, the NCOA3 Gene. Genes 2021, 12, with non-syndromic, sensorineural, bilateral, moderate to profound hearing loss, with a variable 1043. https://doi.org/10.3390/ age of onset. Our findings (i.e., the identification of the second family reported globally with HL genes12071043 caused by a variant in NCOA3) further support the involvement of NCOA3 in the etiopathogenesis of ADNSHL, which should, thus, be considered as a new gene for autosomal dominant non-syndromic Academic Editor: Regie hearing loss. Santos-Cortez Keywords: non-syndromic hearing loss; NCOA3 gene; exome sequencing; Italian family; autosomal Received: 8 June 2021 dominant inheritance Accepted: 2 July 2021 Published: 6 July 2021 Publisher’s Note: MDPI stays neutral 1. Introduction with regard to jurisdictional claims in published maps and institutional affil- Hearing loss (HL) is the most frequent sensory disorder, affecting about 1–3 per iations. 1000 live births [1]. In developed countries, up to 80% of congenital cases are attributable to genetic defects, which can lead to syndromic hearing loss with other organ abnormalities, or non-syndromic HL, which represents the majority of the cases (i.e., 70%, although recent findings suggest that this percentage might be lower) [2]. Non-syndromic hearing loss (NSHL) can be inherited as an autosomal recessive Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. disease (i.e., 80%), autosomal dominant (i.e., 15–20%) or X-linked, mitochondrial or Y- This article is an open access article linked, which, however, account for less than 1% of the total cases [1]. Definition of the distributed under the terms and molecular cause of NSHL is hampered by the high clinical and genetic heterogeneity of conditions of the Creative Commons the disease, with more than 123 genes and 170 loci described thus far (Hereditary Hearing Attribution (CC BY) license (https:// Loss Homepage, https://hereditaryhearingloss.org/, accessed on 25 May 2021). Despite creativecommons.org/licenses/by/ technological advances, the current genetic tests fail to provide a diagnosis for about 40% 4.0/). of patients, suggesting that novel genes and mutations still need to be identified [3]. In Genes 2021, 12, 1043. https://doi.org/10.3390/genes12071043 https://www.mdpi.com/journal/genes Genes 2021, 12, 1043 2 of 9 particular, several studies demonstrated a lower detection rate for autosomal dominant NSHL families (ADNSHL) [4,5], which might be due to the phenotype variability among patients that leads to difficulties in defining a correct genotype–phenotype correlation and, therefore, a proper molecular diagnosis [6]. In light of this, the application of next-generation sequencing techniques, such as exome sequencing (ES), offers a powerful approach to identify and characterize new causative genes. Nevertheless, only the identification of additional families carrying mutations in the same genes provides definitive proof of their pathogenic role. Here, we describe an Italian family affected by ADNSHL carrying a likely pathogenic variant in NCOA3, a gene recently described as a novel candidate for autosomal dominant progressive hearing loss in a Brazilian family [7]. 2. Materials and Methods The Italian family was recruited at the Institute for Maternal and Child Health— I.R.C.C.S. “Burlo Garofolo” (Trieste, Italy). All the patients provided written informed consent, and all research was conducted according to the ethical standard defined by the Helsinki Declaration. The individuals of the family underwent a detailed clinical evaluation, including pure tone audiometric testing. Subsequently, genomic DNA was extracted from peripheral whole blood samples using the QIAsymphony® SP instrument with QIAsymphony® Certal Kits (Qiagen, Venlo, The Netherlands), and DNA concentration was measured using a Nanodrop ND 1000 spec- trophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA). A first round of genetic testing was carried out, including Sanger sequencing to analyze the entire coding region of the GJB2 gene, multiplex PCR to search for GJB6 deletions and Multiplex Ligation-dependent Probe Amplification (MLPA) analysis to identify deletion/duplication in the STRC, CATSPER2 and OTOA genes with SALSA® MLPA® probe mixes P461-A1 DIS (MRC-Holland, Amsterdam, The Netherlands) according to the manufacturer’s protocol. Finally, ES was carried out on an Illumina NextSeq 550 instrument (Illumina Inc., San Diego, CA, USA). According to the manufacturer’s instructions, genomic libraries were prepared using the Twist Human Core Exome + Human RefSeq Panel kit (Twist Bioscience, South San Francisco, CA, USA). The process leads to the creation of FASTQ files, the standard file format for DNA sequencing data. The FASTQ files were processed through a custom pipeline (Germline-Pipeline), developed by enGenome srl. This workflow allows the generation of a final VCF file containing information regarding variants such as single-nucleotide variants (SNVs), short insertion/deletions (INDELs) and exon-level copy number variations (CNVs). Thus, the VCF files were analyzed on enGenome Expert Variant Interpreter (eVai) software (evai.engenome.com). All the variants of interest were confirmed by Sanger sequencing. Finally, a linkage analysis was performed using Merlin software (version 1.1.2) [8], applying a dominant model with a disease allele frequency of 0.01% and penetrance 0,1.0,1.0. 3. Results A four-generation Italian family affected by ADNSHL was recruited at our institute (Figure1). Genes 2021, 12, 1043 3 of 9 Genes 2021, 12, x FOR PEER REVIEW 3 of 9 Figure 1.1. Pedigree of thethe ItalianItalian family.family. PedigreePedigree ofof thethe four-generationfour-generation familyfamily investigatedinvestigated in thethe presentpresent study.study. FilledFilled symbolssymbols representrepresent affectedaffected individuals.individuals. The age of each individual and the co-segregation of the NCOA3 variant with HL (NM_181659.3,(NM_181659.3, c.2909G>C,c.2909G>C, p.(Gly970Ala))p.(Gly970Ala)) areare reported.reported. TheirTheir ages are indicated followed by thethe terms y.o., meaning years old. The labels used to report the subjects’ genotypes are G/G for homozygous wild type and G/C for the individuals old. The labels used to report the subjects’ genotypes are G/G for homozygous wild type and G/C for the individuals carrying the variant herein discussed in heterozygosis. Individuals with Roman numeric labels were analyzed in this carrying the variant herein discussed in heterozygosis. Individuals with Roman numeric labels were analyzed in this study. study. Individuals underwent a careful clinical examination and dysmorphology assessment, Individuals underwent a careful clinical examination and dysmorphology assess- which did not highlight any significant anomaly. Afterwards, a hearing evaluation by pure ment, which did not highlight any significant anomaly. Afterwards, a hearing evaluation tone audiometry was performed on selected members of the family (II:4, III:1, III:3, III:6, by pure tone audiometry was performed on selected members of the family (II:4, III:1, III:8, III:10, III:11, III:13 and III:15), showing a bilateral, symmetric and moderate to severe III:3, III:6, III:8, III:10, III:11, III:13 and III:15), showing a bilateral, symmetric and moderate hearing loss with a medium and high frequencies drop threshold profile (Figure2A). The toage severe of onset hearing of the loss hearing with a impairment medium and ranged high frequencies from the fourth drop tothreshold the fifth profile decade (Figure of life. 2A).Moreover, The age patients of onset complained of the hearing of a impairment worsening ofranged their hearingfrom the performance fourth to the overfifth decade time, a offeature life. Moreover, objectively patients verified complained only in patient of a III:10 worsening (Figure 2ofB). their They hearing also did performance not refer to over any time,other a comorbidities. feature objectively verified only in patient III:10 (Figure 2B).