A Novel Amplification-Refractory Mutation System-PCR Strategy to Screen MT-TL1 Pathogenic Variants in Patient Repositories

Pathogenic variants within mitochondrial tRNA and rRNA genes negatively affect protein synthesis function and cause oxidative phosphorylation defects. The majority of mitochondrial cytopathies are caused by pathogenic point variants within the mitochondrial tRNA gene for leucine ( ). This study was designed to evaluate a novel amplification-refractory mutation system (ARMS)-PCR based assay to screen patient samples with a clinical diagnosis of mitochondrial cytopathies. Tissue DNA samples from 219 affected individuals were screened for the pathogenic variants m.3271T>C, m.3291Ty >C, m.3303C>T, m.3256C>T, and m.3260A>G along with the most frequent m.3243A>G mutation in the gene. The assay included a "High Resolution Melt curve analysis" to enhance detection limits. The precision of the assay was verified using synthetic controls with variant heteroplasmy ratios. The screening identified the second reported m.3303C>T case as well as two patients with m.3243A>G variants and a rare variant exhibiting m.3290T>C. ARMS-PCR is superior to Sanger sequencing for the detection of variations exhibiting low heteroplasmy. These results provide "proof of concepts" for the implementation of this application for future screening of rare mtDNA variations in sample repositories.