G C A T T A C G G C A T genes Article Haplotype Analysis of GJB2 Mutations: Founder Effect or Mutational Hot Spot? Jun Shinagawa 1, Hideaki Moteki 1,2, Shin-ya Nishio 2 , Yoshihiro Noguchi 2,3 and Shin-ichi Usami 1,2,* 1 Department of Otorhinolaryngology, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan; [email protected] (J.S.); [email protected] (H.M.) 2 Department of Hearing Implant Sciences, Shinshu University School of Medicine, 3-1-1 Asahi, Matsumoto, Nagano 390-8621, Japan; [email protected] (S.-y.N.); [email protected] (Y.N.) 3 Department of Otorhinolaryngology, International University of Health and Welfare, Mita Hospital, 1-4-3 Mita, Minato-ku, Tokyo 108-8329, Japan * Correspondence: [email protected]; Tel.: +81-263-37-2666 Received: 2 February 2020; Accepted: 25 February 2020; Published: 27 February 2020 Abstract: The GJB2 gene is the most frequent cause of congenital or early onset hearing loss worldwide. In this study, we investigated the haplotypes of six GJB2 mutations frequently observed in Japanese hearing loss patients (i.e., c.235delC, p.V37I, p.[G45E; Y136X], p.R143W, c.176_191del, and c.299_300delAT) and analyzed whether the recurring mechanisms for each mutation are due to founder effects or mutational hot spots. Furthermore, regarding the mutations considered to be caused by founder effects, we also calculated the age at which each mutation occurred using the principle of genetic clock analysis. As a result, all six mutations were observed in a specific haplotype and were estimated to derive from founder effects. Our haplotype data together with their distribution patterns indicated that p.R143W and p.V37I may have occurred as multiple events, and suggested that both a founder effect and hot spot may be involved in some mutations. With regard to the founders’ age of frequent GJB2 mutations, each mutation may have occurred at a different time, with the oldest, p.V37I, considered to have occurred around 14,500 years ago, and the most recent, c.176_191del, considered to have occurred around 4000 years ago. Keywords: GJB2; congenital hearing loss; haplotype analysis; founder effect; mutational hot spot; genetic clock 1. Introduction Congenital hearing loss affects approximately one in 500–1000 infants in developed countries, and genetic causes account for at least 50% of all childhood onset non-syndromic sensorineural hearing loss [1]. Currently, it is estimated that there are more than 100 causative genes related to non-syndromic hereditary hearing loss [2], with the most frequent deafness-associated gene worldwide being the GJB2 gene. Hearing loss caused by GJB2 gene mutations is divided into autosomal recessive inheritance (DFNB1A) and autosomal dominant inheritance (DFNA3A), but most cases of GJB2-associated hearing loss are autosomal recessive inheritance. The allele frequency of GJB2 gene mutations in the normal Japanese population is approximately 2% [3]. Regarding GJB2 mutations, recurrent mutations are known to differ among ethnic groups. For example, the c.35delG mutation is commonly observed in European, American, North African, and Middle Eastern populations, but this mutation is rarely observed in the Japanese population, whereas the c.235delC mutation is commonly observed in the Japanese population, but this mutation is relatively rare in European and American populations [4]. Therefore, it is important to clarify the mutation spectrum in each population. In particular, the Genes 2020, 11, 250; doi:10.3390/genes11030250 www.mdpi.com/journal/genes Genes 2020, 11, 250 2 of 9 identification of recurrent mutations is crucial for molecular diagnosis to allow decision-making Genes 2019, 10, x FOR PEER REVIEW 2 of 9 with regard to the appropriate intervention. Generally, recurrent genetic mutations occur via two mechanisms:to allow decision one is-making a founder with e ffregardect and to thethe otherappropriate is a mutational intervention. hot Generally, spot. 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Recently, to a founder not effect only GJB2by , buthaplotype mutations analysis in various using other single genes nucleotide have beenpolymorphisms extensively (SNPs) studied [5]. and Recently, the establishment not only GJB2 of, but these recurrentmutations mutations in various due other to a foundergenes have effect been or aextensively mutational studied hot spot and clarified the establishment [6–9]. of these recurrentIn this study,mutations six mutationsdue to a founder in the GJB2effectgene or a mutational commonly hot observed spot clarified in the Japanese [6–9]. population were analyzedIn this bySNP-based study, six mutations haplotype in analysis the GJB2 to gene estimate commonly whether observed the recurring in the Japanese mechanisms population for each mutationwere analyzed were due by toSNP a founder-based haplotype effect or aanalysis mutational to estimate hot spot. whether Also, asthe founder recurring eff ectsmechanism have receiveds for specialeach mutation interest in were terms due of to human a founder migration, effect or toa mutational address questions hot spot. about Also, theas founder origin ofeffect thes founder have received special interest in terms of human migration, to address questions about the origin of the effect, we also calculated the age at which each mutation considered to be established by a founder founder effect, we also calculated the age at which each mutation considered to be established by a effect in this study using the principle of genetic clock analysis [10]. founder effect in this study using the principle of genetic clock analysis [10]. 2. Materials and Methods 2. Materials and Methods 2.1. Subjects 2.1. Subjects We enrolled 7408 sensorineural hearing loss patients, and extracted about 20 patients with We enrolled 7,408 sensorineural hearing loss patients, and extracted about 20 patients with each each homozygous GJB2 mutation frequently identified in the Japanese population (i.e., c.235delC, homozygous GJB2 mutation frequently identified in the Japanese population (i.e., c.235delC, p.V37I p.V37I (c.109G>A), p.[G45E; Y136X] (c.[134G>A; 408C>A]), p.R143W (c.427C>T), c.176_191del, and (c.109G>A), p.[G45E; Y136X] (c.[134G>A; 408C>A]), p.R143W (c.427C>T), c.176_191del, and c.299_300delAT) (Figure1)[ 4]. For the mutations with fewer patients, we also included patients with c.299_300delAT) (Figure 1) [4]. For the mutations with fewer patients, we also included patients with compound heterozygous mutations, including c.235delC. By using these patients, it was possible to compound heterozygous mutations, including c.235delC. By using these patients, it was possible to estimateestimate the the haplotype haplotype for for each each mutationmutation byby eliminatingeliminating the the c.235delC c.235delC haplotype. haplotype. This This study study was was conductedconducted in in accordance accordance with with the the Declaration Declaration of Helsinki,of Helsinki, and and the the protocol protocol was was approved approved by theby Ethicsthe CommitteeEthics Committee of Shinshu of Shinshu University University School ofSchool Medicine of Medicine No. 387—4 No. 387 September—4 September 2012, 2012, and No. and 576—2No. May576 2017.—2 May 2017. Figure 1. A schematic diagram showing the location of each mutation within the GJB2 gene. White Figure 1. A schematic diagram showing the location of each mutation within the GJB2 gene. White boxesboxes indicate indicate exons, exons, and and the the black black box box in exon in exon 2 indicates 2 indicates the coding the coding region. region. All six All mutations six mutations targeted intargeted this study in existthis study in the exist coding in the region. coding region. 2.2. Mutation Analysis 2.2. Mutation Analysis Amplicon libraries were prepared using an Ion AmpliSeq™ Custom Panel (ThermoFisher Amplicon libraries were prepared using an Ion AmpliSeq™ Custom Panel (ThermoFisher Scientific,Scientific, MA, MA, USA), USA), in in accordance accordance withwith thethe manufacturer’s instructions, instructions, for for 68 68 genes genes reported reported to to causecause non-syndromic non-syndromic hereditary hereditary hearing hearing loss.loss. After preparation, preparation, emulsion emulsion PCR PCR and and sequencing sequencing were were performedperformed according according to to the the manufacturer’s manufacturer’s instructions. The The detailed detailed protocol protocol has has been been described described elsewhere [11]. MPS was performed with an Ion Torrent Personal Genome Machine (PGM) system Genes 2020, 11, 250 3 of 9 Genes 2019, 10, x FOR PEER REVIEW 3 of 9 elsewhere [11]. MPS was performed with an Ion Torrent Personal Genome Machine (PGM) system using an Ion PGM™ 200 Sequencing Kit (ThermoFisher Scientific) and an Ion 318™ Chip (Life Technologies). using an Ion PGM™ 200 Sequencing Kit (ThermoFisher Scientific) and an Ion 318™ Chip (Life The sequence data were mapped against the human genome sequence (build GRCh37/hg19) with a Technologies). The sequence data were mapped against the human genome sequence (build Torrent Mapping Alignment Program. After sequence mapping, the DNA variant regions were piled GRCh37/hg19) with a Torrent Mapping Alignment Program. After sequence mapping, the DNA up with Torrent Variant Caller plug-in software (ThermoFisher Scientific).