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TaqMan-MGB probe quantitative PCR assays to genotype and quantify three mtDNA mutations of Leber hereditary optic neuropathy.

ABSTRACT: Leber hereditary optic neuropathy (LHON) is a degenerative disease of the optic nerve associated with one of three mitochondrial DNA (mtDNA) m.3460G>A, m.11778G>A and m.14484T>C mutations. Although several procedures are available to genotype these mutations, quantitative approaches with rapid, low-cost and easy to handle advantages for three LHON mtDNA mutations are rarely reported. Here, we firstly developed a "one-step" tetra-primer amplification-refractory mutation system (T-ARMS) PCR for qualitative genotyping of three LHON mtDNA mutations. Subsequently, we established single, duplex and triplex TaqMan MGB probe-based fluorescence quantitative PCR (qPCR) assays to perform both qualitative and quantitative analyses of three LHON mtDNA mutations. Standard curves based on tenfold diluted plasmid standard exhibited high specificity and sensitivity, stable repeatability and reliable detectable ability of TaqMan probe qPCR assays without cross-reactivity upon probes combination. Moreover, by comparing with SYBR Green qPCR, we further validated the feasibility of the triplex-probe qPCR assay for the quantitative detection of mtDNA copy number in blood samples. In conclusion, our study describes a rapid, low-cost, easy to-handle, and high-throughput TaqMan-MGB probe qPCR assay to perform both qualitative and quantitative analysis of three primary LHON mtDNA mutations, offering a promising approach for genetic screening and testing of LHON mutations.

SUBMITTER: Xue B

PROVIDER: S-EPMC7378831 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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TaqMan-MGB probe quantitative PCR assays to genotype and quantify three mtDNA mutations of Leber hereditary optic neuropathy.

Xue Bingqian B Li Yang Y Wang Xin X Li Rui R Zeng Xin X Yang Meihua M Xu Xiaohui X Ye Tingbo T Bao Liming L Huang Yi Y

Scientific reports 20200723 1

Leber hereditary optic neuropathy (LHON) is a degenerative disease of the optic nerve associated with one of three mitochondrial DNA (mtDNA) m.3460G>A, m.11778G>A and m.14484T>C mutations. Although several procedures are available to genotype these mutations, quantitative approaches with rapid, low-cost and easy to handle advantages for three LHON mtDNA mutations are rarely reported. Here, we firstly developed a "one-step" tetra-primer amplification-refractory mutation system (T-ARMS) PCR for qu ...[more]

PMID: 32704028

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Project description:PurposeLeber hereditary optic neuropathy (LHON) is a disorder characterized by severe and rapidly progressive visual loss when caused by a mutation in the mitochondrial gene encoding NADH:ubiquinone oxidoreductase subunit 4 (ND4). We have initiated a gene therapy trial to determine the safety and tolerability of escalated doses of an adeno-associated virus vector (AAV) expressing a normal ND4 complementary DNA in patients with a G to A mutation at nucleotide 11778 of the mitochondrial genome.DesignIn this prospective open-label trial (NCT02161380), the study drug (self-complementary AAV [scAAV]2(Y444,500,730F)-P1ND4v2) was intravitreally injected unilaterally into the eyes of 5 blind participants with G11778A LHON. Four participants with visual loss for more than 12 months were treated. The fifth participant had visual loss for less than 12 months. The first 3 participants were treated at the low dose of vector (5 × 10(9) vg), and the fourth participant was treated at the medium dose (2.46 × 10(10) vg). The fifth participant with visual loss for less than 12 months received the low dose. Treated participants were followed for 90 to 180 days and underwent ocular and systemic safety assessments along with visual structure and function examinations.ParticipantsFive legally blind patients with G11778A LHON.Main outcome measuresLoss of visual acuity.ResultsVisual acuity as measured by the Early Treatment Diabetic Retinopathy Study (ETDRS) eye chart remained unchanged from baseline to 3 months in the first 3 participants. For 2 participants with 90-day follow-up, acuity increased from hand movements to 7 letters in 1 and by 15 letters in 1, representing an improvement equivalent to 3 lines. No one lost vision, and no serious adverse events were observed. Minor adverse events included a transient increase of intraocular pressure (IOP), exposure keratitis, subconjunctival hemorrhage, a sore throat, and a transient increase in neutralizing antibodies (NAbs) against AAV2 in 1 participant. All blood samples were negative for vector DNA.ConclusionsNo serious safety problems were observed in the first 5 participants enrolled in this phase I trial of virus-based gene transfer in this mitochondrial disorder. Additional study follow-up of these and additional participants planned for the next 4 years is needed to confirm these preliminary observations.

Dataset Information

TaqMan-MGB probe quantitative PCR assays to genotype and quantify three mtDNA mutations of Leber hereditary optic neuropathy.

Publications

TaqMan-MGB probe quantitative PCR assays to genotype and quantify three mtDNA mutations of Leber hereditary optic neuropathy.

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