Application of real-time quantitative PCR in testing repeat expansion in dystrophia myotonica 1

Abstract

Objective To identify the pathogenic mutation for a Chinese family with dystrophy myotonica ( DM) and explore the potency of real-time quantitative PCR in detecting the trinucleotide repeat expansions causing DM1. Methods Genomic DNA was extracted from peripheral blood samples of family members including 3 patients. Routine PCR were used to amplify the CTG repeats in the DMPK gene and the CCTG repeats in the CNBP gene, the expansion of which can cause DM1 and DM2 respectively. Real-time quantitative PCR were used to determine the relative copy number of these two repeat regions. Furthermore, microsatellite markers from DM1 and DM2 loci were analyzed to verify the diagnosis. Results when subjected to agarose gel elec-trophoresis, the routine PCR product of the DM1 CTG repeats showed that all three patients have a 250 bp band and a smear over 2 kb, while the normal family members had only the 250 bp band. However, with the PCR products of the DM2 CCTG repeat, all family members, including three patients, showed just a band of about 250 bp. The quantitative PCR analysis for the CTG repeat showed that all three patients had a relative copy number (RCN) of about 0.5, but all normal family members have a RCN of about 1. For the CCTG repeat, the RCN is about 1 for both the patients and the normal members. Allele sharing was detected for all markers spanning the DM1 locus, but not for most markers from the DM2 locus, suggesting that the DM1 be the disease locus in this family. Conclusion The pathogenic mutation in the DM family is the CTG repeat expansions in DM1 locus. Real-time quantitative PCR can help to establish the DM1 diagnosis by detecting the copy number losing, which is caused by the amplification failure of large CTG repeat allele. Key words: Real-time quantitative PCR; Myotonic dystrophy; Trinucleotide repeat expansion