Detection of mutations and heteroplasmy in the mitochondrial genome using denaturing high performance liquid chromatography. P.E. McAndrew¹, J.M. Devaney¹, E.L. Pettit¹, E.I. Schwartz¹, D.A. Stephan², T.W. Prior³, M.A. Marino¹. 1) Transgenomic Inc, Gaithersburg, MD; 2) Research Center for Genetic Medicine, Children's National Medical Center, Washington, DC; 3) Department of Pathology, The Ohio State University, Columbus, OH.
   The high mutation rate of mtDNA is due to the combined effects of the reactive oxygen species generated during oxidative phosphorylation, the lack of protective histones, and the lack of an efficient DNA repair mechanism. Defects in the human mitochondrial genome have been reported for several degenerative diseases, cancer, and aging. The normal sequence coexists with the mtDNA mutation for many degenerative diseases, and the proportion and distribution of this heteroplasmy often determines the severity of clinical symptoms. Several mtDNA mutations have been identified in cancer cell lines and primary tumors, and it has been suggested that mtDNA hypervariable regions represent somatic mutation hot spots which may be useful for cancer detection. An efficient method to rapidly screen the mtDNA regions implicated in human diseases and to scan the mitochondrial genome for somatic mutations in tumor samples would be a powerful tool for molecular diagnostic testing. We are currently using denaturing high performance liquid chromatography (DHPLC) as a method to detect mutations and heteroplasmy in mtDNA. DHPLC uses an ion-pair reversed-phase column to separate heteroduplex molecules from homoduplex fragments. We designed a multiplex assay and determined the optimal elution temperature and gradient conditions for several regions of the mtDNA genome, including the hypervariable regions. These conditions are being applied to evaluate several common mtDNA diseases including LHON, MERFF, and MELAS, and can be used to scan the mtDNA genome for somatic mutations in tumor samples. An SRM developed by NIST was used to evaluate heteroplasmy. We were able to detect as little as 1% heteroplasmy in a constructed sample with a silent mutation at position 6371 within the coding region. DHPLC is a rapid, versatile technology which will enable us to detect mutations in mtDNA and determine the degree of heteroplasmy in clinical samples.