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. 2008 Aug;9(8):610-5.
doi: 10.1631/jzus.B0820058.

MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

Jian-yong Wang  1 , Yang-shun Gu, Jing Wang, Yi Tong, Ying Wang, Jun-bing Shao, Ming Qi
Affiliations

MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR

Jian-yong Wang et al. J Zhejiang Univ Sci B. 2008 Aug.

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.

Conclusion: 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.

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Figures

Fig. 1
Fig. 1
Detection of the mutation type or wild type of mtDNA11778 This mtDNA genotyping is heteroplasmy. The FAM fluorescent signal represented the mutation type and the VIC fluorescent signal represented the wild type. Two fluorescent signals were measured simultaneously
Fig. 2
Fig. 2
Analysis of the C t,FAM/C t,VIC of the LHON patients
See this image and copyright information in PMC

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