Detection Of Hereditary Hearing Loss Genes Based On TaqMan Probe Melting Curve Technology

Deafness is a prevalent disabling disease affecting human health. Roughly two thirds of deafness caused by genetic etiologies and most were sensorineural deafness. Non-syndromic hearing impairment(NSHI) which generally follows simple Mendelian inheritance was the most common type(about 70%). And it passed on to the next generation with different ways, such as autosomal recessive(about 75%-80%), autosomal dominant(about 20%), X-linked(about 2-5%) and mitochondrial mutations(about 1%). Autosomal recessive NSHI has the characteristics of congenital, stable, severity and generally leads to severe-to-profound prelingual deafness. While autosomal dominant NSHI is primarily characterized as post-lingual and progressive. Along with decades of development, the exploration and discovery of deafness genes had laid a solid foundation for clinical diagnosis, prevention and treatment. Nonsyndromic Loci has been positioned to 177(DFNB103, DFNA67, DFNX6, AUNA1, DFNM2, DFNY1), cloned to 97 gene(http://hereditaryhearingloss.org) and had more than one thousand mutation site(http://.deafnessvariationdatabase.org). In recent years, the amount of deafness genes increased perpendicularly due to the rapid development of high throughput sequencing technology, but there are hundreds of genes related with deafness still unknown.GJB2, SLC26A4 and mtDNA 12 S rRNA genes were common deafness genes in China. About 20% of the patients with GJB2 gene mutation in the syndrome type deafness. For now, gene chip technology and MALDI-TOF-MS were generally used to detect four common deafness gene mutations in China, but it is hard to spread in a hospital because of high price. This study chose ordinary TaqMan probe, using the common fluorescent PCR instrument, and established a probe melting curve technology successfully. The NSHI patients, EVA patients and aminoglycoside-induced deafness patients were collected and tested by TaqMan probe melting curve technology. All results were confirmed by Sanger sequencing technology. The results show that about 19.4% of patients carried GJB2 gene among the NSHI patients, and normal person was 1.4%. The detection rate of SLC26A4 gene in EVA patients reached 84.2%. As well as aminoglycoside-induced deafness patients all carried 1555A>G mutation. All results were consistent with that of Sanger sequencing technologies.1555A>G mutation is in mitochondrial DNA 12 S rRNA gene and mostly with homogeneous mutation, but a few families had the characteristics of heterogeneous mutation. In recent years, there are a few study reported 1555A>G family with heterogeneous mutation, which show normal or varying degrees of hearing loss had different mutation loads, and different mutation loads transfer to the next generation also inconsistent. This study quantitate the percentage of mutant mtDNA by TaqMan probe melting curve technology, and the results compared with DHPLC technology and sequencing technology which to explore application of this technology in heterogeneity detection. The proportions of mutant mtDNA 1555A>G copies ranged from 19% to 86% were accurately detected by the TaqMan probe melting curve technology. The results show that the proportions of mutant mtDNA 1555A>G copies ranged from 19% to 86% were accurately detected by the TaqMan probe melting curve technology, and it found one more case of heteroplasmic mutation(85.84%) than Sanger sequencing technology, but three less than DHPLC method(11.90%, 14.80% and 97.90%).TaqMan probe melting curve technology has many advantages in homogeneity mutation detection, such as cheap, simple, sensitive, automation, closed operation and no pollution. That would be popularized in basic hospitals more easily, so as to realize deafness gene clinical screening which could reduce the number of deaf children born and the burden of families and country. TaqMan probe melting curve technology has a certain credibility in the detection of 19 to 86 percent of mutant mtDNA 1555A>G copies. But its sensitivity is lower than DHPLC technology, and still need to be further optimized.