Advantages and considerations in the confirmation of mitochondrial DNA mutations by denaturing HPLC and pyrosequencing

Abstract

Human mitochondrial DNA (mtDNA) encodes 13 polypeptides essential for oxidative phosphorylation. Because of the unique features of "replicative segregation" and "threshold expression" of mtDNA genetics, identification of homoplasmy versus heteroplasmy status is critical. Results from various detection methods may lead to different interpretations on formation or outcome of mtDNA mutations, such as the conclusion of somatic mutation versus genetic drift in cancers. Denaturing high-performance liquid chromatography (DHPLC) and pyrosequencing (PSQ) have recently been employed to confirm the presence of heteroplasmy of mtDNA because of their high sensitivity in detecting heteroplasmic mutations compared with direct sequencing. Moreover, PSQ has superior ability in quantifying percentage of heteroplasmy. However, there could be disagreement between these two techniques and several issues specific for mtDNA should be taken into consideration. Particularly, DHPLC analysis should be more prone to be interfered by nuclear mitochondrial sequences (Numts), if it is coamplified with mtDNA, than PSQ analysis.

Interference of Co-Amplified Nuclear Mitochondrial DNA Sequences on the Determination of Human mtDNA Heteroplasmy by Using the SURVEYOR Nuclease and the WAVE HS System

“…By conducting analysis on a serial of samples with different percentages of mutant, Janne et al reported that the sensitivity of SN/WAVE-HS method for the EFRF gene reached 1% of mutant [10], which was similar to 2% observed in this study for plasmids with an SNP. We identified that DHPLC was suitable to detect mutant at 5% or greater in this study, which was consistent with that reported in our previous study [4] and in other studies [8], [27] for mtDNA. Because testing on the optimal temperatures required for DHPLC analysis is not conducted for SN/WAVE-HS analysis, and the WAVE HS System is superior to gel electrophoresis for the identification of cleaved DNA fragments with their approximate sizes, theoretically, the SN/WAVE-HS platform should provide a considerably more efficient and convenient method than DHPLC for the screening of mtDNA mutations and heteroplasmy.…”

“…The mtDNA-specific primers that did not amplify DNA from 143B-ρ 0 cells for the amplicon of nt 3374–3894 have been previously described [4]. The forward primer 5′-GAAACTTAGATCCAGGTGTCGC-3′ and reverse primers 5′-AGCAGCAATTTGTAAGTGTCCC-3′ were designed as nDNA-specific primers, which amplified a fragment of human SOD2 gene across an exon and an intron with the length of 293 bp.…”

“…As previously described in our publication [4], to examine how primers designed for human mtDNA matched with NUMTs, we routinely conducted searches by using the Basic Local Alignment Search Tool (BLAST) not only against the nucleotide database in National Center for Biotechnology Information (NCBI), but also against a local database containing the rCRS and the 228 accession numbers currently available on GenBank from the 247 accession numbers that were identified to contain NUMTs sequences by Mishmar et al [13], which were also available on the MITOMAP (http://www.mitomap.org/MITOMAP/PseudogeneList). In this study, we used the BioEdit online tool (http://www.mbio.ncsu.edu/bioedit/bioedit.html) to create the local database.…”

“…Somatic mtDNA mutations in aging-related diseases and cancers in humans have also been investigated extensively [2], [3]. Because decision-making on homoplasmy or heteroplasmy status of mtDNA mutations can have implications, such as the determination of maternal inheritance or the differentiation between somatic mutations and genetic drift in cancer patients, detection of low-percentage heteroplasmy of mtDNA by high-sensitivity techniques, such as denaturing high-performance liquid chromatography (DHPLC) and pyrosequencing (PSQ), has become an increasing research interest, as we discussed previously [4]. PSQ is a powerful technique for the quantification of mtDNA heteroplasmy for a known single nucleotide polymorphism (SNP) or mutation without the need of establishing a standard curve [4], [5], whereas allele-refractory mutation system (AMRS)-based quantitative polymerase chain reaction (qPCR) represents a highly sensitive technique that requires a standard curve made by mixing 2 DNA samples containing different alleles [6].…”