Investigating MT-TL1 gene mutations in patients with suspected mitochondrial cytopathy

Point mutations in mitochondrial tRNA and rRNA genes cause oxidative phosphorylation defects by negatively affecting protein synthesis function. The majority of "mitochondrial cytopathies" are caused by point mutations in the tRNALeu (UUR) (MTTL1) gene. m.3243A>G mutation in the MT-TL1 gene is described in 80% of MELAS patients. However, other rare mutations such as m.3256 C>T, m.3260 A>G, m.3271 T>C, m. 3291 T>C and m.3303 C>T are also identified in the same gene and associated with mitochondrial cytopathies. Within the scope of this study, a novel ARMS-PCR assay has been standardized to screen for the six described point mutations in the MTTL1 gene. The descriptive sensitivity of this approach was aimed to be improved by combining it with HRM technology. Detection limits and the precision of this novel method is verified by using recombinant mtDNA standards carrying heteroplasmic mutations at different rates which is created by PCR-based mutagenesis. Peripheral blood or skeletal muscle tissue samples of 219 patients, prediagnosed as mitochondrial cytopathies at the Departments of Neurology and Child Neurology Units of Hacettepe University Hospitals were screened using the developed method. The results of the screening procedure revealed two patients to have m.3243 A> G mutation, one patient with m.3290 T> C variation, and one patient carrying m.3303 C>T mutation. These mutations were further confirmed by DNA sequence analysis. This novel ARMS-PCR assay study and its results provide a new and technological "proof of concept" for future screening of other rarely observed mtDNA variations in patients.