Researchers Develop New Molecular Technique for Detection of DNA Deletions in Mitochondrial Diseases

Researchers at Newcastle University in the United Kingdom recently published in the journal Scientific Reports a new technique to determine genetic deletions in mitochondrial DNA that are relevant in the context of mitochondria-related diseases. The study is entitled “Triplex real-time PCR – an improved method to detect a wide spectrum of mitochondrial DNA deletions in single cells.”

Point mutations and large-scale rearrangements (deletions or duplications) in the mitochondrial genome (mtDNA) are associated with mitochondrial diseases. These genetic alterations in mtDNA can accumulate in individual muscle fibers, leading to mitochondrial dysfunction.

The mtDNA “major arc” is the DNA region in the mitochondrial genome where most of the disease-associated deletions have been observed by scientists. While rare, studies have also reported deletions in other genomic regions, namely minor arc deletions.

Only few techniques allow the detection and quantification of both major and minor arc mtDNA deletions in single muscle fibers, and the only reliable quantification technique for mtDNA is based on polymerase chain reaction (PCR), a molecular biology method that can amplify DNA generating thousands to millions of copies.

In the study, researchers revealed a new method based on a triplex real-time PCR assay that allows both the detection and quantification of the majority of mtDNA deletions in single cells. Two specific mitochondrial genes are analyzed through this technique: MT-ND1, which is present in the minor arc and is rarely detected, and MT-ND4, a gene within the major arc that is often deleted. In addition, the mitochondrial non-coding D-Loop region is also assessed as it is within a highly conserved region of the mtDNA.

The team tested its triplex real-time PCR assay in DNA samples from several tissues (homogenate and single cell DNA), including muscle tissue from healthy individuals and patients with mtDNA deletions and found that the assay provided reliable and reproducible results.

The research team concluded that their triplex real-time PCR assay represents a highly sensitive and reliable molecular tool that can accurately detect and quantify several major and minor arc mtDNA deletions in tissues. The authors believe that this tool can help uncover details on the molecular pathogenesis of mitochondrial diseases and on the role of mtDNA deletions in both research and diagnostic settings.