Expansion size and presence of CCG/CTC/CGG sequence interruptions in the expanded CTG array are independently associated to hypermethylation at the DMPK locus in myotonic dystrophy type 1 (DM1)

• CTG expansions modify the methylation status of the DMPK locus in DM1 patients.
• Pure and interrupted CTG expansions are associated with a polarized pattern of hypermethylation.
• Hypermethylation of the DMPK locus is found in early-onset DM1 patients.
• Hypermethylation of DMPK locus does not affect DMPK or SIX5 expression.

ABSTRACTA differential CpG methylation profile upstream of the expanded CTG array at the DMPK locus has been reported in patients with myotonic dystrophy type 1 (DM1), suggesting that hypermethylation might modulate DM1 phenotype, possibly affecting expression levels of DMPK and/or flanking genes. To clarify this issue, we characterized by methylation sensitive high resolution melting (MS-HRM) the CpG methylation pattern of DNA sequences flanking the pathological CTG expansion in 13 childhood-onset, 37 juvenile/adult-onset, 7 congenital DM1 patients carrying uninterrupted CTG expansions and in 9 DM1 patients carrying variant expansions vs 30 controls. Association of methylation status with disease features (nCTG, age, sex, MIRS, disease duration) was also assessed. Finally, DMPK and SIX5 expression levels were evaluated in leukocytes from controls, methylated and unmethylated DM1 patients.We found hypermethylation involving upstream sequences of DM1 locus in patients with uninterrupted CTG expansions > 1000 CTG and affected by a congenital or childhood onset form. Besides the n(CTG) and early disease onset, hypermethylation was also significantly associated with maternal transmission.On the other hand, hypermethylation involved the 3′ of the CTG array in DM1 patients carrying variant expansions. DMPK and SIX5 expression did not significantly differ in methylated vs unmethylated DM1 patients. Our results suggest that either the inherited size of the expanded allele and the presence of interruptions at the 3′ end are associated with a highly polarized pattern of CpG methylation at the DM1 locus and that, at least in leukocytes, DM1 locus hypermethylation would not significantly affect DMPK or SIX5 expression.