Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy.

Myotonic dystrophy (DM), an autosomal dominant neuromuscular disease, is associated with expansion of a polymorphic (CTG)n repeat in the 3'-untranslated region of the DM protein kinase (DMPK) gene. The repeat expansion results in decreased levels of DMPK mRNA and protein, but the mechanism for this decreased expression is unknown. Loss of a nuclease-hypersensitive site in the region of the repeat expansion has been observed in muscle and skin fibroblasts from DM patients, indicating a change in local chromatin structure. This change in chromatin structure has been proposed as a mechanism whereby the expression of DMPK and neighboring genes, sine oculis homeobox (Drosophila) homolog 5 (SIX5) and dystrophia myotonica-containing WD repeat motif (DMWD), might be affected. We have developed a polymerase chain reaction (PCR)-based method to assay the chromatin sensitivity of the region adjacent to the repeat expansion in somatic cell hybrids carrying either normal or affected DMPK alleles and show that hybrids carrying expanded alleles exhibit decreased sensitivity to PvuII digestion in this region. Semiquantitative multiplex reverse transcriptase PCR (RT/PCR) assays of gene expression from the chromosomes carrying the expanded alleles showed marked reduction of DMPK mRNA, partial inhibition of SIX5 expression from a congenital DM chromosome, and no reduction of DMWD mRNA. Nested RT/PCR analysis of DMPK mRNA from somatic cell hybrids carrying the repeat expansions revealed that most of the DMPK transcripts expressed from the expanded alleles lacked exons 13 and 14, whereas full-length transcripts were expressed predominantly from the normal alleles. These results suggest that the CTG repeat expansion leads to a decrease in DMPK mRNA levels by affecting splicing at the 3' end of the DMPK pre-mRNA transcript.