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 as sociated with expansion of a polymorphic (CTG)(n) repeat in the 3'-untransl ated region of the DM protein kinase (DMPK) gene. The repeat expansion resu lts in decreased levels of DMPK mRNA and protein, but the mechanism for thi s decreased expression is unknown. Loss of a nuclease-hypersensitive site i n the region of the repeat expansion has been observed in muscle and skin f ibroblasts from DM patients, indicating a change in local chromatin structu re. This change in chromatin structure has been proposed as a mechanism whe reby the expression of DMPK and neighboring genes, sine oculis homeobox (Dr osophila) homolog 5 (SIX5) and dystrophia myotonica-containing WD repeat mo tif (DMWD), might be affected. We have developed a polymerase chain reactio n (PCR)-based method to assay the chromatin sensitivity of the region adjac ent to the repeat expansion in somatic cell hybrids carrying either normal or affected DMPK alleles and show that hybrids carrying expanded alleles ex hibit decreased sensitivity to PvuII digestion in this region. Semiquantita tive 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 c hromosome, and no reduction of DMWD mRNA. Nested RT/PCR analysis of DMPK mR NA from somatic cell hybrids carrying the repeat expansions revealed that m ost of the DMPK transcripts expressed from the expanded alleles lacked exon s 13 and 14, whereas full-length transcripts were expressed predominantly f rom 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' en d of the DMPK pre-mRNA transcript. (C) 2001 Academic Press.