Knowledge of NUMTs and mitochondrial DNA specific primer designing is of utmost importance to avoid misidentification of heteroplasmic mutations

NUMTs (Nuclear DNA sequences of mitochondrial origin) are regions of mitochondrial DNA (mtDNA), integrated into the nuclear genome in course of evolution. Hence, mtDNA-specific primer designing becomes important for allowing the correct detection and quantification of mtDNA heteroplasmy. For this study, through extensive data mining we collected the primer information for mtDNA mutations reported in three mitochondrial diseases (MELAS, LHON, Leigh disease) along with Parkinson’s disease that has a mitochondrial implication. Through in silico PCR and BLASTn, we assessed if those primers have the propensity to anneal within NUMTs and thus co-amplify the concerned mtDNA segment as well as the NUMT(s). BLASTn also revealed if the concerned mutations in mtDNA are the actual bases in the co-amplified NUMTs and ENSEMBL mining detected if they coincide with known polymorphisms in the same. We detected that for ~ 69% (86 out of 125) of the heteroplasmic mutations, non-specific primers have been used that could co-amplify NUMTs and thus, depending upon the status of the base in question within it, may lead to false allele calls or erroneous quantification of heteroplasmic mtDNA mutation. In fact, for two of the reported mutations, the mutant bases in mtDNA were the actual bases in the NUMTs; while in nine cases, polymorphisms in the NUMTs coincided with the precise location of mtDNA mutation and the mutated bases were found to be the same as that of the polymorphic variants in the NUMTs. This study thus raises a note of concern towards primer designing in conventional mtDNA mutation detection studies.