title: Allele-specific PCR for detecting the deafness-associated mitochondrial 12S rRNA mutations.
abstract: Mutations in mitochondrial 12S rRNA (MT-RNR1) are the important causes of sensorineural hearing loss. Of these mutations, the homoplasmic m.1555A>G or m.1494C>T mutation in the highly conserved A-site of MT-RNR1 gene has been found to be associated with both aminoglycoside-induced and non-syndromic hearing loss in many families worldwide. Since the m.1555A>G and m.1494C>T mutations are sensitive to ototoxic drugs, therefore, screening for the presence of these mutations is important for early diagnosis and prevention of deafness. For this purpose, we recently developed a novel allele-specific PCR (AS-PCR) which is able to simultaneously detect these mutations. To assess its accuracy, in this study, we employed this method to screen the frequency of m.1555A>G and m.1494C>T mutations in 200 deafness patients and 120 healthy subjects. Consequently, four m.1555A>G and four m.1494C>T mutations were identified; among these, only one patient with the m.1494C>T mutation had an obvious family history of hearing loss. Strikingly, clinical evaluation showed that this family exhibited a high penetrance of hearing loss. In particular, the penetrances of hearing loss were 80% with the aminoglycoside included and 20% when excluded. PCR-Sanger sequencing of the mitochondrial genomes confirmed the presence of the m.1494C>T mutation and identified a set of polymorphisms belonging to mitochondrial haplogroup A. However, the lack of functional variants in mitochondrial and nuclear modified genes (GJB2 and TRMU) in this family indicated that mitochondrial haplogroup and nuclear genes may not play important roles in the phenotypic expression of the m.1494C>T mutation. Thus, other modification factors, such as environmental factor, aminoglycosides or epigenetic modification may have contributed to the high penetrance of hearing loss in this family. Taken together, our data showed that this assay is an effective approach that could be used for detection the deafness-associated MT-RNR1 mutations.
authors.affiliation: Central Laboratory, Hangzhou First People's Hospital, Hangzhou 310006, China; Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China. Electronic address: dingyu.zj@gmail.com.
authors.forename: Yu
authors.initials: Y
authors.lastname: Ding
authors.affiliation: Department of Pharmacy, Hunan University of Traditional Chinese Medicine, Changsha 410208, China.
authors.forename: Bo-Hou
authors.initials: BH
authors.lastname: Xia
authors.affiliation: Department of Laboratory Medicine, Shaoxing People's Hospital, Shaoxing Hospital of Zhejiang University, Shaoxing 312000, China.
authors.forename: Qi
authors.initials: Q
authors.lastname: Liu
authors.affiliation: Analytical Testing Center, Zhejiang University of Traditional Chinese Medicine, Hangzhou 310053, China.
authors.forename: Mei-Ya
authors.initials: MY
authors.lastname: Li
authors.affiliation: Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China; Department of Otolaryngology, Hangzhou First People's Hospital, Hangzhou 310006, China.
authors.forename: Shui-Xian
authors.initials: SX
authors.lastname: Huang
authors.affiliation: Central Laboratory, Hangzhou First People's Hospital, Hangzhou 310006, China; Affiliated Hangzhou Hospital, Nanjing Medical University, Hangzhou 310006, China.
authors.forename: Guang-Chao
authors.initials: GC
authors.lastname: Zhuo
mesh_terms: D000328:Adult; D000368:Aged; D000483:Alleles; D044466:Asian People / Q000235:genetics; D001299:Audiometry; D001483:Base Sequence; D000072259:Connexin 26; D017630:Connexins / Q000235:genetics; D004252:DNA Mutational Analysis; D003638:Deafness / Q000235:genetics*; D005006:Ethnicity / Q000235:genetics; D005260:Female; D054629:Genome, Mitochondrial; D006801:Humans; D008297:Male; D008875:Middle Aged; D008928:Mitochondria / Q000235:genetics*; D024101:Mitochondrial Proteins / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D010375:Pedigree; D016133:Polymerase Chain Reaction / Q000379:methods*; D012335:RNA, Ribosomal / Q000235:genetics*; D012359:tRNA Methyltransferases / Q000235:genetics
chemical_list: D017630:Connexins; C000607042:GJB2 protein, human; D024101:Mitochondrial Proteins; D012335:RNA, Ribosomal; C075526:RNA, ribosomal, 12S; D000072259:Connexin 26; D012359:tRNA Methyltransferases; C496706:TRMU protein, human
keywords: 1494C>T mutation; Allele-specific PCR; Hearing loss; Mitochondrial