Screening for deafness-associated mitochondrial 12S rRNA mutations by using a multiplex allele-specific PCR method

Abstract

Mitochondrial 12S rRNA A1555G and C1494T mutations are the major contributors to hearing loss. As patients with these mutations are sensitive to aminoglycosides, mutational screening for 12S rRNA is therefore recommended before the use of aminoglycosides. Most recently, we developed a novel multiplex allele-specific PCR (MAS-PCR) that can be used for detecting A1555G and C1494T mutations. In the present study, we employed this MAS-PCR to screen the 12S rRNA mutations in 500 deaf patients and 300 controls from 5 community hospitals. After PCR and electrophoresis, two patients with A1555G and one patient with C1494T were identified, this was consistent with Sanger sequence results. We further traced the origin of three Chinese pedigrees. Clinical evaluation revealed variable phenotypes of hearing loss including severity, age at onset and audiometric configuration in these patients. Sequence analysis of the mitochondrial genomes from matrilineal relatives suggested the presence of three evolutionarily conserved mutations: tRNACys T5802C, tRNALys A8343G and tRNAThr G15930A, which may result the failure in tRNAs metabolism and lead to mitochondrial dysfunction that was responsible for deafness. However, the lack of any functional variants in GJB2, GJB3, GJB6 and TRMU suggested that nuclear genes may not play active roles in deafness expression. Hence, aminoglycosides and mitochondrial genetic background may contribute to the clinical expression of A1555G/C1494T-induced deafness. Our data indicated that the MAS-PCR was a fast, convenience method for screening the 12S rRNA mutations, which was useful for early detection and prevention of mitochondrial deafness.

Maternally transmitted nonsyndromic hearing impairment may be associated with mitochondrial tRNAAla 5601C>T and tRNALeu(CUN) 12311T>C mutations

“…To understand the molecular mechanism underlying mitochondrial deafness, recently, we carried out a mutational analysis for deafness‐related m.1555A>G and m.1494C>T mutations by using a novel multiplex allele‐specific PCR (MAS‐PCR) in 500 patients with NSHL and 300 controls from five hospitals from Zhejiang Province. 23 , 24 We first designed four primers that specifically binding to human 12S rRNA gene, after PCR amplification and electrophoresis, patients carrying the m.1555A>G mutation resulted in two specific bands: 736‐bp and 226‐bp, while subjects with the m.1494C>T mutation created two bands: 736‐bp and 488‐bp, whereas patients without these primary mutations can amplify only one band: 736‐bp, which was consistent with PCR‐Sanger sequencing. 25 During that process, we ascertained a Chinese pedigree with NSHL.…”

“…To understand the molecular mechanism underlying mitochondrial deafness, recently, we carried out a mutational analysis for deafness‐related m.1555A>G and m.1494C>T mutations by using a novel multiplex allele‐specific PCR (MAS‐PCR) in 500 patients with NSHL and 300 controls from five hospitals from Zhejiang Province 23,24 25 .…”

Mitochondrial tRNAGln 4394C>T Mutation May Contribute to the Clinical Expression of 1555A>G-Induced Deafness

“…We noticed that the penetrance of hearing loss in HZD055 was 50% including AmAn and 10% excluding AmAn, while in HZD510 it was 30% including AmAn and 0% excluding AmAn. Compared with previous studies, the penetrances of m.1555A>G-induced hearing loss ranged from 22.2% to 73.9% (average: 50.9%, including AmAn), whereas excluding the effects of AmAn, the penetrances varied from 11.1% to 60.8% (average: 30.8%, Table 5 ) [ 15 , 16 , 20 , 54 ].…”

“…This MAS-PCR is a simple, inexpensive, fast and reliable method, which can be used to detect the deafness-associated 12S rRNA mutations in the general population [ 19 ]. To further assess its accuracy, we screened for the presence of the m.1555A>G and m.1494C>T mutations in a cohort of 500 deaf patients and 300 controls [ 20 ]. During that process, we identified a novel tRNA Gln 4394C>T mutation, together with the m.1555A>G mutation, in a Chinese family with maternal transmission of AINSHL (ID: HZD510), which manifested a much higher penetrance of hearing impairment (50% including AmAn and 10% excluding AmAn) than another pedigree (ID: HZD055) with only the m.1555A>G mutation (30% including AmAn and 0% excluding AmAn) which harbored the same mitochondrial haplogroup D4g2a as HZD510 [ 21 ].…”