Methylation of the G-C rich DMPK gene: causation for the severity difference between adultonset and congenital myotonic dystrophy?

Barnette, Brian
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University of Delaware
Myotonic dystrophy type I is an autosomal dominant genetic disease. The disease arises from an expansion of a trinucleotide repeat, (CTG)n located in the 3’ untranslated (3’ UTR) region of the dystrophica myotonin protein kinase (DMPK) gene on chromosome 19q13.2-q13.3. Located proximal to this region is a CpG island, a region of DNA with a high concentration of CpG sequences that often associate with gene promoters in humans. Changes in these promoter regions, including the methylation status, modify expression of genes that they regulate. The purpose of this study was to test the hypothesis that there are allelic differences in the methylation status of this CpG island in normal, adult-onset and congenital myotonic dystrophy patients by using DNA prepared from somatic cell hybrids that carry either the normal or expanded DMPK allele. In addition, once the methylation status had been ascertained, it was the goal to determine if hypermethylation of the normal allele correlated with a decreased expression of the DMPK gene. This correlation could suggest the reason for severity differences between adult-onset and congenital DM1. The method of bisulfite-conversion was used to ascertain the methylation status of the CpG island. This method converts all unmethylated cytosine residues into uracil residues. The converted DNA of congenital DM1 patients was then amplified by means of High-Fidelity PCR where the converted uracil residues are amplified as thymine residues. This amplified DNA is then cloned and sequenced to compare the methylation status of the CpG island among individuals. After examining the methylation status of this CpG island in one CDM1 patient and a patient presenting with signs and symptoms associated with DM1, it was determined that the non-mutant allele of these patients is not methylated. Since the non-mutant allele was determined to be unmethylated, hypermethylation could not be the cause of its decreased expression.