Methodological Possibilities for the Determination of the Number of CTG/CAGRepeats in Triplet Repeat Units of the Human Genome

Summary

Background.
Human genome dynamic mutations are a new class of gene mutations represented by an unstablenumber of trinucleotide repeats and causing severe human hereditary neuromuscular and neurodegenerative diseases.The identification of pathological expanded alleles on the molecular level is important for clinical diagnostics.Methods and Results. For the molecular diagnostics of expanded tandem repeat trinucleotide sequences we haveintroduced a fast and efficient TP-PCR fluorescent method according toWarner et al. (1996).We have modified thisTP-PCR method for a rapid detection of expanded CTG alleles of the DMPK gene (myotonic dystrophy, MD) intoa two-level protocol; first, the heterozygote sample DNAs were selected using P1/P2 primers flanking repeat tractsand, second, the TP-PCR protocol used was focused above all on the identification of a pathological allele.A fluorescent-labelled specific primer in TP-PCR was used for the exact determination of the number of CAG repeatsof the gene IT-15 (Huntington’s disease – HD) in the diagnostically important region of the grey zone (35 to 39CAG). The reproducibility of the PCR results was demonstrated on control DNA samples with the known genotypeand, in the case of MD, also by Southern blot analysis. We have especially shown the possibility of a cheaperPCR-P1/P2 and TP-PCR protocol, which can be used, with silver staining of separated PCR products on polyacrylamidegels.Conclusions. Our experience with introducing the above-mentioned PCR methods into laboratory practice clearlydocuments the possibilities of their general applicability in the molecular diagnostics of hereditary diseasescharacterised by instability of the trinucleotide repeat tracts.

Key words:
Neurodegenerative disease, myotonic dystrophy, Huntington's disease, DMPK gene, IT-15gene, trinucleotide repeats, expanded trinucleotide, Triplet Primed PCR.