THE MITOCHONDRIAL COI/tRNASER(UCN) G7444A MUTATION MAY BE ASSOCIATED WITH HEARING IMPAIRMENT IN A HAN CHINESE FAMILY
Ding Y, Xia B-H, Teng Y-S, Zhuo G-C, Leng J-H,
*Corresponding Author: Dr. Jian-Hang Leng, Central Laboratory, Hangzhou First People’s Hospital, Nanjing Medical University, Huansha Road 261, Hangzhou, People’s Republic of China. Tel./Fax: +86-0571-87065701. E-mail: lengjh5@163.com
page: 6

MATERIALS AND METHODS

Subjects. 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. Analysis of the Mutations in the Mitochondrial Genome. 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 tRNASer(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]. Phylogenetic Analysis. 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. Mutational Screening for the GJB2 Gene. 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). Mutational Analysis of the TRMU Gene. 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’-ACA ACG CCA CGA CGG ACG- 3’). The PCR segments were analyzed and compared with the TRMU genomic sequence (Accession No. AF_448221).



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