Instability of the expanded (CTG)(n) repeats in the myotonin protein kinase gene in cultured lymphoblastoid cell lines from patients with myotonic dystrophy

The mutation associated with myotonic dystrophy (DM) is the expansion of an unstable trinucleotide repeat, (CTG)(n), in the 3'-untranslated region of the myotonin protein kinase gene. Although expanded repeats show both germline and somatic instability, the mechanisms of the instability are poorly understood. To establish a model system in which somatic instability of the DM repeat could be studied in more detail, we established lymphoblastoid cell lines (LBCL) from DM patients. Analysis of the DNA from DM LBCL using Southern blotting showed that the (CTG)(n) repeats were apparently stable up to 29 passages in culture. To study infrequent repeat size mutations that are undetectable due to the size heterogeneity, we established LBCL of single-cell origins by cloning using multiple steps of limiting dilution. After expansion to approximately 10⁶ cells (equivalent to approximately 20 cell cycles), the DNAs of these cell lines were analyzed by the small pool PCR technique using primers flanking the (CTG)(n) repeat region. Two types of mutations of the expanded (CTG)(n) repeat alleles were detected: (1) frequent mutations that show small changes of the (CTG)(n) repeat size, resulting in alleles in a normal distribution around the progenitor allele, and (2) relatively rare mutations with large changes of the (CTG)(n) repeat size, with a bias toward contraction. The former may represent the mechanism responsible for the somatic heterogeneity of the (CTG)(n) repeat size observed in blood cells of DM patients. This in vitro experimental system will be useful for further studies on mechanisms involved in the regulation of the somatic stability of the (CTG)(n) repeats in DM.