The current study was approved by the Ethics Committee of Zhoushan Hospital, and each participant provided written informed consent. A four-generation Han Chinese family (Fig. 1), comprising 30 individuals and 6 deaf patients, was enrolled from the Department of Otolaryngology, Zhoushan Hospital, the age, sex of each patient was summarized in Table I. First, a comprehensive physical examination of each patient was performed, including the determination of whether they were taking aminoglycosides. In addition, the levels of hearing loss were tested in each participant using pure-tone audiometry (PTA) as described (3). The average PTA level was measured using the total audiometric thresholds at 125, 250, 500, 1,000, 2,000, 4,000 and 8,000 Hz. The levels of hearing loss were divided into the following degrees: i) Normal hearing (PTA <26 dB); ii) mild hearing loss (PTA 26–40 dB); iii) moderate hearing loss (PTA 41–70 dB); iv) severe hearing loss (PTA 71–90 dB); and v) profound hearing loss (PTA >90 dB). In addition, 200 healthy subjects were enrolled from the Physical Examination Center of Zhoushan Hospital. The inclusion criteria for the healthy subjects were that they were age- and sex-matched, healthy, and had no family history of mitochondrial disorders.

To analyze the mtDNA mutations, PCR and direct sequencing analysis were performed. The genomic DNA was first isolated from blood samples using Puregene DNA Isolation kits (Gentra Systems; Thermo Fisher Scientific, Inc.). The complete mtDNA of the matrilineal relatives from this family were then PCR amplified using 24 oligonucleotides, as previously described (14). The PCR product was purified and analyzed by Sanger sequencing as described (3). The data were then compared with the updated mitochondrial genome sequence to detect mtDNA mutations (GenBank accession no. NC_001807) (15).

Variants in the GJB2 gene were further investigated in matrilineal relatives. For amplification of the GJB2 coding region, PCR was performed using the following primers: Forward, 5′-TTGGTGTTTGCTCAGGAAGA-3′ and reverse, 5′-GGCCTACAGGGGTTTCAAAT-3′, as previously described (16). PCR fragments were sequenced and the data were analyzed with the wild-type GJB2 sequence (accession no. NM_004004.5).

SPSS 19.0 software (IBM Corp., Armonk, NY, USA) and Student's t-test were used to perform the statistical analysis; we compared the significance between deaf patients carrying mitochondrial A1555G and A7551G mutations and control subjects. P<0.05 was considered to indicate a statistically significant difference.

The proband (III-12), aged 35, lived in Zhoushan (Zhejiang, China). He attended to the Otology Clinic of Zhoushan Hospital for deafness treatment. Clinical examination suggested that he had manifested bilateral hearing loss at 30 years old. As shown in Fig. 2, it was noted that the proband exhibited profound hearing loss (right ear, 100 dB; left ear, 90 dB). However, he did not have any associated medical history.

In addition, a physical examination was performed for each deaf patient from this pedigree, including the level of hearing loss, aminoglycosides usage, as well as other disorders. It was revealed that the family members did not have other common disorders, such as cardiovascular events, diabetes mellitus, visual impairment or neurological disorders. It was also noted that several individuals had a history of using aminoglycosides, such as individual II-8. She received kanamycin for fever when she was 58 years old, unfortunately, three weeks later, she developed bilateral deafness. As presented in Table I and Fig. 2, a variable degree of hearing loss was determined in this Chinese pedigree, ranging from mild (IV-6) to profound (III-12). Furthermore, the deafness exhibited a pattern of maternal transmission (Fig. 1).

Owing to the apparent maternal transmission of deafness in this family, the proband's complete mtDNA was analyzed for mutations, together with those of other matrilineal relatives. A total of 24 overlapping DNA fragments spanning the 16,569 bp of mtDNA were PCR amplified and sequenced. It was shown that compared with the standard mitochondrial genome sequences, the proband and other matrilineal relatives exhibited 24 genetic polymorphisms (Table II), which were as follows: Seven variants in the D-loop gene, three in the 12S rRNA gene, two in the 16S rRNA gene and one in a tRNA gene (Fig. 3A and B), as well as a nine bp deletion in the non-coding region between tRNA^Lys and cytochrome C oxidase II (CO2). These polymorphisms have all been previously reported (www.mitomap.org). The other variants were located in oxidative phosphorylation (OXPHOS) encoding genes. In addition, six missense mutations were identified, namely, the NADH:Ubiquinone (ND2) C5178A (Leu→Met) and T5442C (Phe→Leu) mutations, the ATPase 6 A8701G (Thr→Ala) and A8860G (Thr→Ala) mutations, the NADH:Ubiquinone oxidoreductase core subunit 5 (ND5) G13928C (Ser→Thr) mutation, and the cytochrome B (Cyt b) A15326G (Thr→Ala) mutation. Phylogenetic analysis of these mutations in 11 vertebrates was performed, including mouse (17), bovine (18), and Xenopus laevis (19) (Table III). The results revealed that only the A1555G and A7551G mutations exhibited a high level of conservation. Further, the A1555G and A7551G mutations were not detected in the 200 control subjects, suggesting that these mutations may have a functional impact associated with deafness (P<0.05; Table IV). Notably, the homoplasmic A7551G mutation occurred at position 37 in the anticodon stem of tRNA^Asp, which may have caused tRNA metabolism failure.

To examine whether the GJB2 gene had critical function in deafness expression, the coding regions of GJB2 were analyzed. However, no functional variants in this gene were identified (data not shown).