インベーダー法によるミトコンドリア遺伝子1555変異ヘテロプラスミー定量測定法の開発 Development of quantitative assay for detecting heteroplasmy of mitochondrial 1555 mutation using Invader Assay
Perception deafness caused by the mitochondrial 1555 A>G mutation has been observed frequently in Japan. Recent studies indicate that patient having this 1555 mutation is more likely to be suseptaible to aminoglycoside antibiotics. Therefore, if the presence of 1555 mutation can be detected, administration of aminoglycoside antibiotics can be prevented. This genetic examination to detect 1555 mutation could be one of the most significant items in personalized medicine. We have developed a qualitative assay to detect 1555 mutation using Invader system.<BR>While 1555 mutations were assumed to be all homoplasmic, recent studies have indicated that heteroplasmy exist among 1555 mutation. Using PCR-RFLP, Abe et al reported that two out of 341 congenital deafness patients examined were heteroplasmic. In order to examine the heteroplasmy in detail, we have modified the current qualitative Invader Assay system for detection of 1555 mutation to be a more quantitative system to detect heteroplasmy.<BR>Standard curve was generated from serial dilutions of wild and mutant type plasmids by measuring the fluorescence signal for conversion to quantitative coefficient.The copy number of mutant and wild type plasmids were determined by substitution of quantitative coefficient to the standard curve. The amount of mutant type was expressed as % heteroplasmy against total mitochondrial DNA<BR>While results from PCR-RFLP were 5% and 95% heteroplasmy, the quantitative Invader Assay presented 2% and 91% heteroplasmy. Accuracy and reproducibility has been verified. This quantitative Invader Assay may become useful tool in determining the relationship between the amount of heteroplasmy and clinical manifestation.
- Otology Japan
Otology Japan 17(5), 691-696, 2007-12-25
THE JAPAN OTOLOGICAL SOCIETY