Accurate Detection and Quantitation of Heteroplasmic Mitochondrial Point Mutations by Pyrosequencing

Abstract

Disease-causing mutations in mitochondrial DNA (mtDNA) are typically heteroplasmic and therefore interpretation of genetic tests for mitochondrial disorders can be problematic. Detection of low level heteroplasmy is technically demanding and it is often difficult to discriminate between the absence of a mutation or the failure of a technique to detect the mutation in a particular tissue. The reliable measurement of heteroplasmy in different tissues may help identify individuals who are at risk of developing specific complications and allow improved prognostic advice for patients and family members. We have evaluated Pyrosequencing technology for the detection and estimation of heteroplasmy for six mitochondrial point mutations associated with the following diseases: Leber's hereditary optical neuropathy (LHON), G3460A, G11778A, and T14484C; mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS), A3243G; myoclonus epilepsy with ragged red fibers (MERRF), A8344G, and neurogenic muscle weakness, ataxia, and retinitis pigmentosa (NARP)/Leighs: T8993G/C. Results obtained from the Pyrosequencing assays for 50 patients with presumptive mitochondrial disease were compared to those obtained using the commonly used diagnostic technique of polymerase chain reaction (PCR) and restriction enzyme digestion. The Pyrosequencing assays provided accurate genotyping and quantitative determination of mutational load with a sensitivity and specificity of 100%. The MELAS A3243G mutation was detected reliably at a level of 1% heteroplasmy. We conclude that Pyrosequencing is a rapid and robust method for detecting heteroplasmic mitochondrial point mutations. 190

Extensive and rapid screening for major mitochondrial DNA point mutations in patients with hereditary hearing loss

“…Over the past 5 years, new technologies have been developed to screen for mtDNA mutations, including denaturing high-performance liquid chromatography; 50,51 a detection system using a mismatchspecific DNA endonuclease; 52 the Biplex Invader assay by hybridization of two overlapping oligonucleotides to the target sequence; 53 the matrix-associated laser desorption/ionization time of flight mass spectrometry assay; 54 the pyrosequncing technology to detect and estimate heteroplasmic mtDNA point mutations; 55 and an entire mtDNA resequencing chip: Mitochip. 56,57 The Mitochip uses hybridization for detecting mutations or polymorphisms, so that the probes mtDNA mutation screening for hereditary HL T Kato et al should be designed.…”

Comprehensive next-generation sequence analyses of the entire mitochondrial genome reveal new insights into the molecular diagnosis of mitochondrial DNA disorders

“…21 Although specific primers or probes can be designed for the quantification of target positions, 7,22 it is laborious to validate the method for every novel mtDNA point mutation. In addition, primers may contain modifications and probes that are specific for either the wild-type or mutant allele, [23][24][25] thus, a difference in PCR amplification efficiency for these two alleles is expected, leading to inherent inaccuracies with the measurement of the degree of heteroplasmy. Our group has recently demonstrated that, for mtDNA analysis at 20,000× coverage, the experimental error rate of the MPS using the Illumina HiSeq2000 platform was 0.326 ± 0.335%, with a limit of detection of 1.33%.…”

The severity of FIP1L1–PDGFRA-positive chronic eosinophilic leukaemia is associated with polymorphic variation at the IL5RA locus

“…For IL5, only a single informative SNP has been identified; rs2069812 (also known as IL5 746 or var929). For each SNP, we designed a pyrosequencing assay as described 14 using the primers and dispensation order detailed in Table 1. Markers were quantified using the Allele Frequency Quantification function in the SNP Software (Biotage AB, Uppsala, Sweden) and called as homozygous when one allele gave reading of 490% and heterozygous when both alleles read 40-60%.…”