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

Abstract

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.

Identifying sequence variants in the human mitochondrial genome using high-resolution melt (HRM) profiling

“…The mitochondrial genome has been assessed employing a variety of different strategies [Wang et al, 2008a;Wong and Bai, 2006;Wong and Senadheera, 1997]. In this study, fragments from the mtDNA scanning panel identified heteroplasmic variants ranging from 1 to 100% mutant allele fraction (Table 1; Fig.…”

“…Identification of specific variants has been performed by allele-specific PCR, a variety of real-time PCR procedures, and real-time amplification refractory mutation system quantitative PCR [Wang et al, 2008a;Wong and Bai, 2006;Wong and Senadheera, 1997]. Mitochondrial DNA sequence variants have been screened for by indirect means such as denaturing high-performance liquid chromatography, denaturing gradient gel electrophoresis, temperature gradient gel electrophoresis, and other methods [van Den Bosch et al, 2000;van Eijsden et al, 2006;White et al, 2005;Wong et al, 2004Wong et al, , 2002Xiu-Cheng Fan et al, 2008].…”

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“…Nhi u nghi n ứu ho r ng d ng kh ng đồng nhất ủ đột biến ó thể ảnh hưởng một phần đến s biểu hi n v di truy n ủ b nh LHON [9][10]. V v y vi xá định á nhân t góp phần l m th y đ i kiểu h nh ủ đột biến v những hiểu biết v ơ hế phát sinh b nh do đột biến G11778A l ần thiết.…”

Development and validation of a novel PCR-RFLP based method for the detection of 3 primary mitochondrial mutations in Leber's hereditary optic neuropathy patients

“…Current diagnostic strategies for the 3 most common mutations causing LHON include individual endpoint PCR-RFLP [ 29 ], allele specific PCR [ 30 ], real time PCR [ 31 ] and PCR followed by Sanger / pyrosequencing [ 32 ]. In this study, we successfully designed a simple multiplex PCR-RFLP to detect the 3 primary mitochondrial LHON mutations and also describe the synthesis of a series of LHON control sequences that act as a robust and patient-free resource for LHON test controls and assay development.…”