Detection of Slipped-DNAs at the Trinucleotide Repeats of the Myotonic Dystrophy Type I Disease Locus in Patient Tissues

Slipped-strand dnas,formed by out-of-register mispairing of repeat units on complementary strands,were proposed over 55 years ago as transient intermediates in repeat length mutations,hypothesized to cause at least 40 neurodegenerative diseases. while slipped-dnas have been characterized in vitro,evidence of slipped-dnas at an endogenous locus in biologically relevant tissues,where instability varies widely,is lacking. here,using an anti-dna junction antibody and immunoprecipitation,we identify slipped-dnas at the unstable trinucleotide repeats (ctg)n•(cag)n of the myotonic dystrophy disease locus in patient brain,heart,muscle and other tissues,where the largest expansions arise in non-mitotic tissues such as cortex and heart,and are smallest in the cerebellum. slipped-dnas are shown to be present on the expanded allele and in chromatinized dna. slipped-dnas are present as clusters of slip-outs along a dna,with each slip-out having 1-100 extrahelical repeats. the allelic levels of slipped-dna containing molecules were significantly greater in the heart over the cerebellum (relative to genomic equivalents of pre-ip input dna) of a dm1 individual; an enrichment consistent with increased allelic levels of slipped-dna structures in tissues having greater levels of ctg instability. surprisingly,this supports the formation of slipped-dnas as persistent mutation products of repeat instability,and not merely as transient mutagenic intermediates. these findings further our understanding of the processes of mutation and genetic variation. © 2013 axford et al.