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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Project partially supported by the “Qianjiang Research Talent”grant from the Science and Technology Department of Zhejiang Province, China
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Wang, Jy., Gu, Ys., Wang, J. et al. MGB probe assay for rapid detection of mtDNA11778 mutation in the Chinese LHON patients by real-time PCR. J. Zhejiang Univ. Sci. B 9, 610–615 (2008). https://doi.org/10.1631/jzus.B0820058
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DOI: https://doi.org/10.1631/jzus.B0820058
Key words
- Leber’s hereditary optic neuropathy (LHON)
- Mitochondrial DNA (mtDNA)
- MtDNA11778 mutation
- Minor groove binder (MGB) probe
- Real-time polymerase chain reaction (PCR)