The mitochondrial COI/tRNA^SER(UCN) G7444A mutation may be associated with hearing impairment in a Han Chinese family

As a part of genetic screening program for hearing loss, a three-generation Han Chinese family (as shown in Figure 1) was found at the Department of Otolaryngology, Hangzhou First People’s Hospital, Zhejiang Province, PRC. Informed consent was obtained from the participants. Blood samples were obtained from all participants prior to their participation in the study, in accordance with the Ethics Committee of Hangzhou First People’s Hospital. In addition, a comprehensive history and physical examination were performed to identify any syndromic findings, the history of the use of aminoglycosides, as well as the genetic factors related to the hearing impairment in members of this pedigree. An age-appropriate audiological examination was performed, and this examination included pure tone audiometry (PTA) and auditory brainstem response (ABR), immittance testing and distortion product otoacoustic emissions. The PTA was calculated from the sum of the audiometric thresholds at 500, 1000, 2000, 4000 and 8000 Hz. The severity of hearing impairment was classified into five grades: normal <26 dB, mild 26-40 dB, moderate 41-70 dB, severe 71-90 dB and profound >90 dB. Moreover, DNA was obtained from 200 control subjects from a panel of unaffected Han Chinese subjects from the same region who were clinically tested.

Figure 1

Genomic DNA was isolated from whole blood of participants using the Puregene DNA Isolation Kits (Gentra Systems, Minneapolis, MN, USA). First, three matrilineal relatives (I-2, II-5, III-2) and control subject’s DNA fragments spanning the mitochondrial 12S rRNA and tRNA^Ser(UCN) genes were amplified by polymerase chain reaction (PCR) using oligodeoxynucleotides as previously described [12]. Subsequently, the entire mitochondrial genomes of the deaf patients (I-2, II-5, III-2) and controls were PCR-amplified in 24-overlapping fragments using the set of light-strand and the heavy-strand primers [12]. After PCR amplification, each fragment was purified and analyzed by direct sequencing in an ABI PRISM™ 3700 automated DNA sequencer using the BigDye Terminator Cycle sequencing reaction kit (Applied Biosystems Inc., Foster City, CA, USA). The sequence data were compared with the reversed Cambridge sequence to detect the mutations (GenBank accession no. NC_012920) [13].

A total of 17 vertebrates’ mitochondrial genome sequences were used in the interspecific analysis. These include Bos Taurus, Cebus albifrons, Gorilla gorilla, Homo sapiens, Hylobates lar, Lemur catta, Macaca mulatta, Macaca sylvanus, Mus musculus, Nycticebus coucang, Pan paniscus, Pan troglodytes, Pongo pygmaeus, Pongo abelii, Papio hamadryas, Tarsius bancanus, and Xenopus laevis (GenBank). The conservation index (CI) was calculated by comparing the human nucleotide variants with 16 other vertebrates. Notably, the CI ≥75.0% was regarded as having functional potential.

The DNA fragments spanning the entire coding region of the GJB2 gene were amplified by PCR using the following primers: forward (5′-TAT GA CAC TCC CCA GCA CAG-3′) and reverse (5′-GGG CAA TGC TTA AAC TGG C-3 ′). Polymerase chain reaction amplification and sequencing analyses were performed as described elsewhere [14]. The results were compared with the wild-type GJB2 sequence to identify the variants (GenBank Accession No. M86849).

A previous study showed that the TRMU exon 1 A10S variant may modulate the phenotypic manifestation of deafnessassociated mitochondrial 12S rRNA mutations [15]. To see whether TRMU played an active role in deafness expression, we conducted a mutational screening for the TRMU exon 1 in matrilineal relatives in this pedigree and the healthy controls. The primers for detecting the A10S variant were as follows: forward (5′-ACA GCG CAG AAG AAG AGC AGT-3′) and reverse (5′-ACAACG CCA CGA CGG ACG-3′). The PCR segments were analyzed and compared with the TRMU genomic sequence (Accession No. AF_448221).

All patients from the Han Chinese family lived in Hangzhou City of Zhejiang Province. The proband (III-2) was an infant born in Hangzhou First People’s Hospital. As shown in Table 1 and Figure 2, the proband exhibited bilateral hearing impairment (90 dB right ear and 95 dB left ear). A comprehensive history and physical examination were performed to identify any syndromic findings, and the history of use of aminoglycosides. Moreover, we noticed that the proband’s mother (II-5), a young woman at the age of 26 years; had been administered gentamicin (5 mg/kg/dose, 10 days) for fever when she was 18-years-old. She developed the profound hearing loss 2 months after the drug administration. It is interesting to note that two matrilineal relatives (I-2, II-5), who had a history of exposure to gentamicin and streptomycin, exhibited a severe hearing impairment in this maternal kindred, suggesting that the aminoglycosides may play an important role in this disorder.

Table 1

Summary of clinical data for several members of this family

Figure 2

The maternal transmission of hearing loss in this family suggested a mitochondrial involvement and led us to analyze the mitochondrial genome of matrilineal relatives (I-2, II-5, III-2) and the healthy subjects. We first examined the known mtDNA pathogenic variants associated with deafness by PCR amplification (A1555G, C1494T, A7445G, T7510C and T7511C). As shown in Figure 3, the PCR-Sanger sequencing identified two known pathogenic variants: the C1494T in the 12S rRNA gene and the G7444A in the COI/tRNA^Ser(UCN) gene. However, we did not detect the presence of the A1555G pathogenic variant in the 12S rRNA gene or the A7445G, T7510C, T7511C pathogenic variants in the tRNA^Ser(UCN) gene in these matrilineal relatives.

Figure 3

To elucidate the molecular basis for maternally transmitted deafness, 24-overlapping DNA fragments spanning the entire mitochondrial genome were PCR-amplified and sequenced. The comparison of the resultant sequence with the Cambridge consensus sequence identified a set of polymorphisms, as shown in Table 2. Among these, there were five variants in the D-loop, two known variants in the 12S rRNA and two variants in the 16S rRNA genes, while other variants were mainly localized at protein-coding genes. Moreover, we noticed that there were four amino acid substitutions caused by corresponding mtDNA variants occurring in different polypeptides. These missense variants included the ND1 C3497T (A64V), A6 A8860G (T112A), ND3 A10398G (T114A) and Cytb A15326G (T194A). These variants in rRNAs and polypeptides were further evaluated by phylogenetic analysis from other organisms including mouse [16], bovine [17] and Xenopus laevis [18]. However, none of the variants in the polypeptides were highly evolutionarily conserved and implicated to have functional consequences.

Table 2

mtDNA sequence variants in this family with hearing impairment.

To examine the role of the GJB2 and TRMU genes in phe-notypic expression of the C1494T pathogenic variant, we performed the mutational screening of GJB2 and TRMU exon 1 in matrilineal relatives who carried the C1494T pathogenic variant. However, none of the variants were found in the GJB2 and TRMU genes, suggesting that the GJB2 and TRMU genes may not play an important role in this Chinese family.