In the present study 117 deaf patients, aged between 3 months and 35 years were recruited. These patients were diagnosed with nonsyndromic neurosensory deafness at Nanjing Maternity and Child Health Care Hospital (Nanjing, China) between 2009 and 2013, with hearing loss of >70 dB. Any patients with systemic disease, dysgnosia, syndromic deafness or a history of meningitis, otitis media or trauma of the ear were excluded from the investigations. The present study was performed in accordance with the declaration of Helsinki, and was performed following approval from the ethics committee of Nanjing Maternity and Child Health Care Hospital (Nanjing Medical University, Nanjing, China). Written informed consent was obtained from the patient.

To obtain DNA, 2 ml peripheral blood was collected and then placed into tubes with ethylene diamine tetraacetic acid (EDTA; Greiner Bio-One International GmbH). For the pregnant women offered prenatal diagnosis, 10 ml amniotic fluid was collected at 18–21 weeks gestation, and DNA was extracted using a DNA extraction kit (Xiamen Zhishan Biological Company, Xiamen, China), according to manufacturer's protocol, followed by storage at −20°C.

Primers were designed, according to those previously described (13–15), which were used to perform polymerase chain reaction (PCR) amplification and sequencing. The primer sequences are listed in Table I, and were synthesized by Invitrogen (Thermo Fisher Scientific, Inc., Waltham, MA, USA). The coding sequences of GJB2 were amplified, following which GJB2 genetic mutations were detected by Sanger sequencing (13,16,17). Furthermore, mitochondrial DNA from 1,428 to 1,890 were also amplified and 1,555A>G mutation of the mitochondrial 12S rRNA gene were detected (15,18,19). The hot-spot regions of the SLC26A4 gene, including exons 5, 7, 8, 10, 17 and 19, were also amplified, of which exons 7 and 8 were amplified in one amplicon. Therefore, five fragments were amplified to cover the six exons and flanking sequence, following which the SLC26A4 gene mutation was detected. For patients with one allelic variant in the hot-spot regions, the other exons were sequenced one by one, until two mutant alleles were identified, as previously described (14,20,21).

The GoTaq^® PCR system was purchased from Promega Corporation (Madison, WI, USA). The total volume of the PCR reaction mixture was 50 µl, including 10 µl of 5X Green GoTaq^® Reaction Buffer, 2 µl of 10 mmol/l dNTP, 1 µl of each (10 µmol/l) primer, 0.25 µl of 5 U/µl GoTaq^® DNA polymerase, 5 µl of the 20 ng/µl DNA template and 30.75 µl distilled water. The amplification reaction conditions for exons 7 and 8 of the SLC26A4 gene were as follows: 95°C for 5 min; followed by 35 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 30 sec, and a final step of 72°C for 7 min. For the remaining six exons, the reaction conditions were as follows: 95°C for 5 min; followed by 35 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 1 min, and a final step of 72°C for 7 min. The amplified products were stored at 4°C.

Following PCR, agarose gel electrophoresis was performed, and the products were purified according to TIANgel Midi DNA Purification kits (Tiangen Biotech Co., Ltd., Beijing, China), following which the purified products were sequenced. Sequencing kits were obtained from Life Technologies (Grand Island, NY, USA). The reaction system contained 1 µl Bigdye, 3 µl buffer, 2 µl primers and 5 µl purified products in a total volume of 20 µl. The reaction conditions were as follows: 95°C for 1 min; followed by 35 cycles of 95°C for 10 sec, 50°C for 5 sec and 60°C for 4 min. The amplified products were stored at 4°C. The sequences of the primers are listed in Table I. In addition, the sequencing products were purified with 100 mM EDTA solution and alcohol solution. Following purification, the products were dried and were dissolved in 100% HiDi Formamide (Gibco; Thermo Fisher Scientific, Inc.). Subsequently, all products were sequenced (forward and reverse) using an ABI3130 analyzer (Applied Biosystems; Thermo Fisher Scientific, Inc.). The sequence results were aligned with standard sequences using SeqBuilder procedures on Lasergene software, version 7.1 (DNASTAR, Inc., Madison, WI, USA). The GJB2, SLC26A4 and mitochondrial DNA 12S rRNA genetic standards were NM_004004 [GenBank, National Center for Biotechnology Information (NCBI), www.ncbi.nlm.nih.gov/nuccore/NM_004004], ENSG00000091137 (Ensembl, European Bioinformatics Institute, www.ensembl.org/Homo_sapiens/Gene/Sequence?g=ENSG00000091137) and the Cambridge reference sequence, NC_001807 (GenBank, NCBI, www.ncbi.nlm.nih.gov/nuccore/NC_001807.4), respectively.

In the present study, genetic detection was performed in 117 deaf patients, of which 36 cases were confirmed to carry two pathogenic mutations. The positive rate was 30.77% (Table II), including 19 cases with GJB2 gene mutations (16.24%), 12 cases with SLC26A4 gene mutations (10.26%) and five cases with the 1,555A>G allelic mutation of mitochondrial DNA 12S rRNA (4.27%). In total, four GJB2 gene mutations (Fig. 1), and eight SLC26A4 gene mutations (Fig. 2) were identified.

The 117 deaf patients recruited in the present study included 39 pairs of deaf couples, of which four couples were confirmed to have two deafness-causing mutations (10.26%). Among these, deafness in one couple was caused by SLC26A4 gene mutations, whose children will be deaf. In one deaf couple, deafness was caused by GJB2 and SLC26A4 gene mutations, respectively. The present study hypothesized that their children will have a low risk of deafness with no need for prenatal diagnosis as the parents pathogenic mutations are in different genes, the child will be heterozygous for these two genes which are associated with autosomal recessive hearing loss. Furthermore, in one couple, the husband had two GJB2 gene mutations, whereas the wife had 1,555A>G mutation of mitochondrial DNA 12S rRNA. Their children would carry a GJB2 gene heterozygous mutation and mitochondrial DNA mutation, and may be advised to avoid taking aminoglycoside antibiotics as the mutation 1,555A>G is important in aminoglycoside-induced nonsyndromic deafness (18). In addition, the husband of one couple had 1,555A>G mutation of mitochondrial DNA 12S rRNA, whereas the wife carried two GJB2 gene mutation. The present study hypothesized that their children would be at low risk of deafness due to their child carrying a heterozygous GJB2 gene mutation. The results of genetic sequencing indicated that, in 11 couples, only one of each couple (28.21%) was confirmed to have deafness-causing mutations, which indicated that their offspring were at low risk of deafness. Table III shows the results of genetic analyses.

In the 39 deaf patients, 17 cases were identified with GJB2 gene or SLC26A4 gene mutations. Subsequently, their parents received genetic analysis to confirm whether they were carriers (Table IV). In 7/17 families, prenatal diagnosis was performed during their next pregnancy. The results showed that two fetuses had the same genotype as the proband. These two families decided to terminate the pregnancy. No mutations were identified in one fetus, and the genotypes of the other four fetuses were the same as one parent. The result of clinical follow-up demonstrated that they had normal hearing following birth.