DHPLC Analysis of Mitochondrial DNA Mutations
A project funded by the United Mitochondrial Disease Foundation (UMDF) and Rita and Steven Achard

Denaturing HPLC(DHPLC) has proven to be a useful diagnostic tool in an expanding list of nuclear gene disorders including cystic fibrosis, breast cancer, acute lymphoblastic leukemia, hemophilia and Rett syndrome. In addition to its specificity for point mutation detection, this technique has the advantage of the ability to detect low percentages of DNA mutations. A major problem in the diagnosis of mitochondrial DNA (mtDNA) disease results from heteroplasmy and polymorphisms represent another difficulty. DHPLC is relatively blind to polymorphisms being particularly suited to heteroplasmy detection. Mutant mtDNA which has marked diagnostic and familial genetic import may often be present in small amounts in the most easily accessible and commonly tested tissue – blood, with larger disease mutation concentrations in other tissues. An example is the common A3243G MELAS mutation estimated to affect 200,000 diabetics in the US with only a tiny number aware of the cause of their disease. When attempted, diagnosis is often difficult because of low levels of mutation in the blood.

DHPLC has recently been used by several research groups to study mtDNA mutations (1-6). Despite its speed, cost effectiveness, sensitivity and specificity, DHPLC analysis is not routinely in use for diagnosis of mtDNA mutations in most laboratories handling patient samples. Reasons for this include the previous un-availability of commercially available primers and reagents designed for mtDNA analysis and lack of information about the DHPLC conditions for specific mutation detection. In this project we use commercially available primers and reagents available in a kit, MitoScreen to characterize the DHPLC conditions and detection sensitivity for heteroplasmic pathogenic mtDNA mutations in order to bring DHPLC into the mainstream of mtDNA mutation diagnosis.

We characterize the DHPLC signatures of mtDNA mutations associated with common clinical phenotypes, including Leigh’s disease, Mitochondrial Encephalomyopathy, Lactic Acidosis, and Stroke-like episodes (MELAS), cardiomyopathy, mitochondrial myopathy and progressive external opthalmoplegia (PEO), and Leber’s hereditary optic neuropathy (LHON) by generating different mutants and analyzing them using DHPLC. Information on the signatures of DHPLC mutations will be posted here along with the analysis conditions as we develop this information. The data for each of the mtDNA mutations are classified according to clinical phenotypes or DNA locations.

 

 

References:

  1. Bayat, A., Walter, J., Lamb, H., Marino, M., Ferguson, M.W. and Ollier, W.E. (2005) Mitochondrial mutation detection using enhanced multiplex denaturing high-performance liquid chromatography. Int J Immunogenet, 32, 199-205.
  2. Biggin, A., Henke, R., Bennetts, B., Thorburn, D.R. and Christodoulou, J. (2005) Mutation screening of the mitochondrial genome using denaturing high-performance liquid chromatography. Mol. Genet. Metab., 84, 61-74.
  3. Liu, M.R., Pan, K.F., Li, Z.F., Wang, Y., Deng, D.J., Zhang, L. and Lu, Y.Y. (2002) Rapid screening mitochondrial DNA mutation by using denaturing high-performance liquid chromatography. World J Gastroenterol, 8, 426-430.
  4. van Den Bosch, B.J., de Coo, R.F., Scholte, H.R., Nijland, J.G., van Den Bogaard, R., de Visser, M., de Die-Smulders, C.E. and Smeets, H.J. (2000) Mutation analysis of the entire mitochondrial genome using denaturing high performance liquid chromatography.
    Nucleic Acids Res, 28, E89.
  5. Meierhofer, D., Mayr, J.A., Ebner, S., Sperl, W. and Kofler, B. (2005) Rapid screening of the entire mitochondrial DNA for low-level heteroplasmic mutations. Mitochondrion, 5, 282-296.
  6. Wulfert, M., Tapprich, C. and Gattermann, N. (2006) Optimized PCR fragments for heteroduplex analysis of the whole human mitochondrial genome with denaturing HPLC.
    J. Chromatogr. B Analyt. Technol. Biomed. Life Sci., 831, 236-247.