Apart from recessive and dominant mutations in the GJB 2 gene , the simultaneous presence of heterozygous mutations in 2 different genes ( GJB 2 and GJB 6 ) has been described in a small subset of patients

We report on 335 patients (319 families) with mild-to-profound nonsyndromic sensorineural hearing loss. We identified 178 mutated GJB2 alleles representing 29 different sequence changes (including 3 novel mutations: Q7P, N14D, H100Q), and 2 alleles with the deletion del(GJB6-D13S1830) of the GJB6 gene. Eleven GJB2 mutations (119 mutated alleles) were truncating (T), and 18 mutations (59 alleles) were nontruncating (NT). Biallelic GJB2 mutations were found in 71 patients (21.2%; 67 families; 25 different genotypes). Audiograms of 62 patients (56 families) with biallelic GJB2 mutations typically indicated a profound hearing loss with T/T mutations, moderate hearing loss with T/NT mutations, and mild hearing impairment with NT/NT mutations (p ! 0.01, Student’s t test). From 37 patients (34 families) with biallelic GJB2 mutations, audiograms at different ages were available and indicated progressive hearing loss ( 1 15 dB) in 10 patients (27.0%, 10 families). Interestingly, we identified an unexpectedly large subset of patients (n = 29; 8.7%) presenting Received: May 18, 2009 Accepted after revision: January 19, 2010 Published online: March 17, 2010 Neurotology Audiology Prof. Annerose Keilmann Department of Communication Disorders, Johannes Gutenberg University Langenbeckstrasse 1, DE–55131 Mainz (Germany) Tel. +49 6131 172 190, Fax +49 6131 176 623 E-Mail keilmann @ kommunikation.klinik. uni-mainz.de © 2010 S. Karger AG, Basel 1420–3030/10/0156–0375$26.00/0 Accessible online at: www.karger. com/aud Bartsch et al. Audiol Neurotol 2010;15:375–382 376 ticular GJB2 mutation (c.35delG) accounts for up to 80% of GJB2 alleles [Estivill et al., 1998; Janecke et al., 2002; Cryns et al., 2004], being most frequent in Southern European countries close to the Mediterranean [Gasparini et al., 2000; Janecke et al., 2002]. Apart from recessive and dominant mutations in the GJB2 gene, the simultaneous presence of heterozygous mutations in 2 different genes ( GJB2 and GJB6 ) has been described in a small subset of patients [del Castillo et al., 2002; Snoeckx et al., 2005]. The clinical relevance of digenic inheritance of a GJB2 mutation and a 309-kb deletion [del(GJB6-D13S1830)] or the less frequent 232-kb deletion [del(GJB6-D13S854)] of the GJB6 gene has been well documented [del Castillo et al., 2005; Snoeckx et al., 2005]. Here, we report on a 6-year survey of molecular analyses of the GJB2 and GJB6 genes in a cohort of 335 patients studied at Mainz, Germany. Material and Methods This study was approved by the ethics committee of the Medical Council Rhineland-Palatinate, Germany. From April 2002 to March 2008, a total of 335 patients (319 families) with nonsyndromic sensorineural hearing loss, aged 1 month to 46 years, were analyzed at our molecular genetic laboratory. All patients underwent ear microscopy and audiometric testing; most children ( 1 90%) were tested in our department for pediatric audiology. Pure-tone audiometry and speech audiometry were carried out in a sound-proof booth using a MAICO audiometer ST 36 (headphones Holmberg type 9501) with ageand language-adapted tests. Additionally, brain stem response using a Nicolet Pathfinder was measured. Frequency-specific information was assessed using the notched noise option, and the normalized hearing level was used for comparison. Pure tone averages (PTAs) in the conversational frequencies (0.5, 1, 2, and 4 kHz) were calculated for each ear, and the severity of hearing loss was defined using the data from pure tone audiometry in older children and adults, or the results of notched noise brainstem audiometry in children under 2 years of age and in cases of developmental delay and lacking cooperation. Hearing loss was classified by the hearing impairment in the better ear: mild (PTA 21–40 dB), moderate (41–70 dB), severe (71–90 dB), or profound ( 1 90 dB). According to Orzan et al. [1999], progressive hearing loss was diagnosed when audiograms of the initial better ear indicated a PTA loss of 1 15 dB in at least 2 frequencies. All audiograms resulting from audiometric testing with most likely normal middle ear conditions were included in the analysis. All patients underwent standard karyotype analysis (GTG banding at the 400-band level) and were screened for GJB2 mutations and the deletion del(GJB6-D13S1830) of the GJB6 gene as described later. GJB2 Sequence Analysis DNA was isolated from blood by standard salting-out procedures. The GJB2 gene coding sequence was PCR-amplified using primers 5 -TGCTTACCCAGACTCAGAGAA-3 (forward) and 5 -CGACTGAGCCTTGACAGCTGA-3 (reverse). PCR conditions included initial denaturation at 94 ° C for 90 s, followed by 35 cycles of denaturation at 94 ° C for 30 s, annealing at 62 ° C for 30 s and extension at 72 ° C for 1 min, followed by a final extension at 72 ° C for 10 min. PCR products were purified using the ExoSAP method and sequenced on a Beckman CEQ 8000 Genetic Analysis System. The presence of the GJB2 variants c.457G ] A (p.V153I) and c.493C ] T (p.R165W) on the same allele was confirmed by cloning the PCR product with the Topo TA cloning kit (Invitrogen, Karlsruhe, Germany) and sequencing the inserts of individual plasmid clones. PCR Analysis of the Deletion del(GJB6-D13S1830) of the GJB6 Gene To determine the incidence of deaf individuals heterozygous or homozygous for the deletion del (GJB6 -D13S1830) in the German population, all patients without biallelic GJB2 mutations were tested for the presence of the deletion using a duplex PCR. The binding sites of the 2 forward primers Cx30del_for2 (5 -CATTGTTGTGAACTAACCTCC-3 ) and Cx30_for1 (5 GCCATGCATGTGGCCTACTA-3 ) lay 131 bp telomeric from the distal and 170 bp telomeric from the proximal deletion breakpoint, respectively. The reverse primer Cx30_rev1 (5 ACTATCTGAAATCAGCTCATT-3 ) binds 310 bp centromeric from the proximal deletion breakpoint. PCR conditions were: initial denaturation at 94 ° C for 3 min, followed by 35 cycles of 94 ° C for 40 s, 58 ° C for 45 s and 72 ° C for 45 s, and final extension at 72 ° C for 5 min. The wild-type allele produces a 480-bp fragment from primers CX30_for1 and CX30_rev1, and the del(GJB6D13S1830) allele gives a 441-bp product from primers CX30del_ for2 and CX30_rev1.