title: MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR.
abstract: OBJECTIVE
  Leber's hereditary optic neuropathy (LHON) is a maternally inherited degeneration of the optic nerve caused by point mutations of mitochondrial DNA (mtDNA). Many unsolved questions regarding the penetrance and pathophysiological mechanism of LHON demand efficient and reliable mutation testing. This study aims to develop a minor groove binder (MGB) probe assay for rapid detection of mtDNA11778 mutation and heteroplasmy in Chinese LHON patients by real-time polymerase chain reaction (PCR).
  
  
  METHODS
  Forty-eight patients suspected of having LHON and their maternal relatives underwent a molecular genetic evaluation, with 20 normal individuals as a control group at the same time. A real-time PCR involving two MGB probes was used to detect the mtDNA11778 mutation and heteroplasmy. A linear standard curve was obtained by pUCmLHONG and pUCmLHONA clones.
  
  
  RESULTS
  All 48 LHON patients and their maternal relatives were positive for mtDNA11778 mutation in our assay, 27 heteroplasmic and 21 homoplasmic. Eighteen cases did not show an occurrence of the disease, while 9 developed the disease among the 27 heteroplasmic mutation cases. Eleven did not show an occurrence of the disease, while 10 cases developed the disease among 21 homoplasmic mutation cases. There was a significant difference in the incidence between the heteroplasmic and the homoplasmic mutation types. The time needed for running a real-time PCR assay was only 80 min.
  
  
  CONCLUSIONS
  This real-time PCR assay is a rapid, reliable method for mtDNA mutation detection as well as heteroplasmy quantification. Detecting this ratio is very important for predicting phenotypic expression of unaffected carriers.
authors.affiliation: Department of Ophthalmology, the First Affiliated Hospital, School of Medicine, Zhejiang University, Hangzhou 310003, China.
authors.forename: Jian-yong
authors.initials: JY
authors.lastname: Wang
authors.forename: Yang-shun
authors.initials: YS
authors.lastname: Gu
authors.forename: Jing
authors.initials: J
authors.lastname: Wang
authors.forename: Yi
authors.initials: Y
authors.lastname: Tong
authors.forename: Ying
authors.initials: Y
authors.lastname: Wang
authors.forename: Jun-bing
authors.initials: JB
authors.lastname: Shao
authors.forename: Ming
authors.initials: M
authors.lastname: Qi