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Myotonic dystrophy type 1 (DM1) is an autosomal dominant muscular dystrophy that results from a CTG expansion (50–4000 copies) in the 3′ UTR of the DMPK gene. The disease is classified into four or five somewhat overlapping forms, which incompletely correlate with expansion size in somatic cells of patients. With rare exception, it is affected mothers who transmit the congenital (CDM1) and most severe form of the disease. Why CDM1 is hardly ever transmitted by fathers remains unknown. One model to explain the almost exclusive transmission of CDM1 by affected mothers suggests a selection against hypermethylated large expansions in the germline of male patients. By assessing DNA methylation upstream to the CTG expansion in motile sperm cells of four DM1 patients, together with availability of human embryonic stem cell (hESCs) lines with paternally inherited hypermethylated expansions, we exclude the possibility that DMPK hypermethylation leads to selection against viable sperm cells (as indicated by motility) in DM1 patients.

Pathological mutations involving noncoding microsatellite repeats are typically located near promoters in CpG islands and are coupled with extensive repeat instability when sufficiently long. What causes these regions to be prone to repeat instability is not fully understood. There is a general consensus that instability results from the induction of unusual structures in the DNA by the repeats as a consequence of mispairing between complementary strands. In addition, there is some evidence that repeat instability is mediated by RNA transcription through the formation of three-stranded nucleic structures composed of persistent DNA:RNA hybrids, concomitant with single-strand DNA displacements (R-loops). Using human embryonic stem cells with wild-type and repeat expanded alleles in the FMR1 (CGGs) and C9orf72 (GGGGCCs) genes, we show that these loci constitute preferential sites (hotspots) for DNA unpairing. When R-loops are formed, DNA unpairing is more extensive, and is coupled with the interruptions of double-strand structures by the nontranscribing (G-rich) DNA strand. These interruptions are likely to reflect unusual structures in the DNA that drive repeat instability when the G-rich repeats considerably expand. Further, we demonstrate that when the CGGs in FMR1 are hyper-methylated and transcriptionally inactive, local DNA unpairing is abolished. Our study thus takes one more step toward the identification of dynamic, unconventional DNA structures across the G-rich repeats at FMR1 and C9orf72 disease-associated loci.

Pathological mutations involving noncoding microsatellite repeats are typically located near promoters in CpG islands and are coupled with extensive repeat instability when sufficiently long. What causes these regions to be prone to repeat instability is not fully understood. There is a general consensus that instability results from the induction of unusual structures in the DNA by the repeats as a consequence of mispairing between complementary strands. In addition, there is some evidence that repeat instability is mediated by RNA transcription through the formation of three-stranded nucleic structures composed of persistent DNA:RNA hybrids, concomitant with single-strand DNA displacements (R-loops). Using human embryonic stem cells with wild-type and repeat expanded alleles in the FMR1 (CGGs) and C9orf72 (GGGGCCs) genes, we show that these loci constitute preferential sites (hotspots) for DNA unpairing. When R-loops are formed, DNA unpairing is more extensive, and is coupled with the interruptions of double-strand structures by the nontranscribing (G-rich) DNA strand. These interruptions are likely to reflect unusual structures in the DNA that drive repeat instability when the G-rich repeats considerably expand. Further, we demonstrate that when the CGGs in FMR1 are hyper-methylated and transcriptionally inactive, local DNA unpairing is abolished. Our study thus takes one more step toward the identification of dynamic, unconventional DNA structures across the G-rich repeats at FMR1 and C9orf72 disease-associated loci.