A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment

et al. (2013) A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment. PLoS ONE 8(9): e73566. doi:10.1371/journal.pone.0073566 Editor: Andreas R. Janecke A Novel Splice-Site Mutation in the GJB2 Gene Causing Mild Postlingual Hearing Impairment Marta Ganda 0 Francisco J. del Castillo 0 Francisco J. Rodrguez-lvarez 0 Gema Garrido 0 Manuela Villamar 0 Manuela Caldern 0 Miguel A. Moreno-Pelayo 0 Felipe Moreno 0 Ignacio del Castillo 0 0 1 Unidad de Genetica Molecular, Hospital Universitario Ramon y Cajal, Instituto Ramon y Cajal de Investigacion Sanitaria (IRYCIS) , Madrid , Spain , 2 Centro de Investigacion Biomedica en Red de Enfermedades Raras (CIBERER) , Madrid , Spain , 3 Servicio de Otorrinolaringologia, Hospital Universitario Ramon y Cajal , Madrid , Spain The DFNB1 subtype of autosomal recessive, nonsyndromic hearing impairment, caused by mutations affecting the GJB2 (connection-26) gene, is highly prevalent in most populations worldwide. DFNB1 hearing impairment is mostly severe or profound and usually appears before the acquisition of speech (prelingual onset), though a small number of hypomorphic missense mutations result in mild or moderate deafness of postlingual onset. We identified a novel GJB2 splice-site mutation, c. -22-2A>C, in three siblings with mild postlingual hearing impairment that were compound heterozygous for c. -22-2A>C and c.35delG. Reverse transcriptase-PCR experiments performed on total RNA extracted from saliva samples from one of these siblings confirmed that c. -22-2A>C abolished the acceptor splice site of the single GJB2 intron, resulting in the absence of normally processed transcripts from this allele. However, we did isolate transcripts from the c. -22-2A>C allele that keep an intact GJB2 coding region and that were generated by use of an alternative acceptor splice site previously unknown. The residual expression of wild-type connection-26 encoded by these transcripts probably underlies the mild severity and late onset of the hearing impairment of these subjects. - Hereditary non-syndromic hearing impairment (NSHI) is a heterogeneous genetic condition. To date, mutations in 73 different genes have been shown to cause NSHI. At least as many genes remain to be identified, as indicated by the number of known NSHI loci for which the underlying causative mutations have not been found yet (http:// hereditaryhearingloss.org/) [1]. This high genetic heterogeneity mirrors the structural and functional complexity of the hearing process and it is a major hurdle for the successful genetic diagnosis of subjects with NSHI. The DFNB1 subtype of autosomal recessive NSHI is remarkable for being highly prevalent in most of the populations tested so far (reviewed in 2). The underlying locus, DFNB1, lies on 13q12 and encompasses the GJB2 gene (MIM # 121011), which encodes the gap-junction protein connection-26 (Cx26). GJB2 consists of a 160-bp non-coding exon, a single 3,179-bp intron and a 2,134-bp exon that contains 22 bp of the 5-untranslated region (UTR), the complete 678-bp coding sequence and the 3-UTR (UniGene Hs.524894) [3]. Mutations at the DFNB1 locus can be classified in two groups: (i) those that affect the coding sequence of GJB2; and (ii) those that lie outside the coding sequence of GJB2 and affect the expression and/or regulation of this gene [2]. Since screening of GJB2 is considered the gold standard of genetic diagnosis of hereditary HI, it is not surprising that more than 100 pathogenic mutations in the GJB2 coding sequence have been identified so far (http://davinci.crg.es/deafness/) [4]. A few GJB2 mutations predominate in particular populations due to demonstrated founder effects [511]. By contrast, only six pathogenic mutations are known outside the GJB2 coding sequence. Two of them are point mutations: c. -23+1G > A (originally named IVS1+1G>A), the only mutation known to affect the donor splice site of the single intron [12], and g. -77C>T (originally named -3438C>T), which abolishes the activity of the basal promoter of the gene [13]. The remaining four mutations are large deletions. One of them is a deletion of about 920 kb that encompasses the complete GJB2 gene [14], whereas the three other deletions (del(GJB6D13S1830) [15], del(GJB6-D13S1854) [16] and del(131-kb) [17], respectively) are thought to eliminate a hypothesized cisacting regulatory element located far upstream of GJB2. While the 920-kb and del(131-kb) deletions and the g. -77C>T promoter mutation seem to be private mutations, the del(GJB6D13S1830) and del(GJB6-D13S1854) deletions and the c. -23+1G > A splice-site mutation are frequent in specific populations [16,1822]. All of these mutations have been isolated in compound heterozygosity with GJB2-coding sequence mutations. DFNB1 hearing impairment is clinically heterogeneous because of intrafamiliar and interfamiliar phenotypic variability, even in association with a same genotype [23]. The most common form is prelingual (onset before the acquisition of speech), non-progressive and severe or profound, affecting all frequencies. However, postlingual, progressive and moderate or mild hearing losses have also been reported, often associated with a few specific mutations [23]. Identification of these genotype-phenotype correlations is important to improve the accuracy of genetic counselling. In this work, we studied a Spanish pedigree with three siblings affected by a mild, postlingual NSHI. We identified a novel GJB2 mutation, which is the first one shown to alter the acceptor splice site of the single intron of the GJB2 gene. Investigation of its effects on GJB2 expression provided a likely explanation of the molecular mechanism underlying this mild DFNB1 phenotype. Materials and Methods Ethics statement This study was approved by the Ethical Committee for Clinical Research of Hospital Universitario Ramn y Cajal. The study complied with the Spanish laws for biomedical research currently in force and adhered to the tenets of the Declaration of Helsinki. Subjects and clinical tests Written informed consent was obtained from all the subjects included in the study. Syndromic features or putative environmental causes of HI were excluded in all affected subjects. HI was evaluated by pure-tone audiometry, testing for air conduction (frequencies 1258,000 Hz) and for bone conduction (frequencies 2504,000 Hz). The degree of HI was determined by calculating the binaural mean of the hearing thresholds for air conduction at frequencies 0.5, 1, and 2 kHz, and it was classified as mild (average thresholds in the range of 2140 dB), moderate (4170 dB), severe (7190 dB), or profound (>90 dB). The HI of subject III:2 was also evaluated by auditory brainstem response (ABR) recording. DNA purification and assay procedures DNA was extracted from peripheral blood samples by standard procedures. Genetic tests for mutations in the GJB2 gene and for the large deletions affecting the GJB6 g (...truncated)