Novel Mitochondrial DNA Variant That May Give a False Positive Diagnosis for the T8993C Mutation

Background: Two of the most common mutations in the mitochondrial DNA (mtDNA) of children occur at nucleotide 8993 (nt8993). The base substitutions of T to G (T8993G) and T to C (T8993C) are known to cause neurologic disorders and are routinely screened for in patients suspected of having a mitochondrial disorder. Methods and Results: Both mutations at nt8993 create a novel HpaII restriction endonuclease site and are usually detected by polymerase chain reaction (PCR) amplification of a section of the mtDNA containing nt8993, followed by HpaII digestion. The resulting fragment sizes are then analyzed by agarose gel electropho resis. Initial testing on a child referred for analysis suggested that the proband and his maternal relatives all had the common mtDNA mutation T8993C; however, subsequent restriction endonuclease and DNA sequencing analysis showed that the proband and his maternal relatives were homoplasmic for a novel variant at nt8856. This variant also creates an Hpa II restriction endonuclease site, and the fragments generated by the site are almost identical in size to those generated as a result of the nt8993 mutation when commonly used primers amplify the PCR product. Conclusions: A novel mutation in the mtDNA at nt8856 creates an HpaII restriction endonuclease site that has the potential to generate false positives when PCR products are tested for mutations at nt8993. This emphasizes the need for restriction endonuclease-based diagnostic tests for mtDNA mutations to account for the highly polymorphic nature of the mtDNA sequence and the importance of confirming a mutation by a second method.