ESPE Abstracts

Background: Makorin RING finger protein 3 (MKRN3) is an important factor located on chromosome 15 in the imprinting Prader-Willi syndrome - associated region. Imprinted MKRN3 expressed in hypothalamic regions essential for puberty initiation and mutations have been found in patients with central precocious puberty (CPP). CPP caused by the early activation of pulsatile Gonadotropin releasing hormone (GnRH) secretion is clinically defined by the early maturation of the entire hypothalamic-pituitary gonadal axis and the development of secondary sexual characteristics before the age of 8 years in girls and 9 years in boys. DNA methylation is an epigenetic process associated with the control of expression of several genes. The aim of this study is to investigate the promoter methylation status of MKRN3 in CPP patients and in the hypothalamus of mouse prior, during and after puberty.

Methods: This study was performed on 26 girls with CPP tested negative for mutations in the coding region of MKRN3 and DLK1 compared to healthy controls. Genomic DNA was extracted from peripheral blood samples. Imprinting status was evaluated in all samples by multiplex ligation-dependent probe amplification (MLPA). In addition, promoter methylation was studied by bisulfite conversion followed by Sanger sequencing (bisulfite-sequencing). Also, promoter methylation status of Mkrn3 was examined by bisulfite-sequencing both in hypothalamus and peripheral blood collected from mice at three developmental stages; pre-pubertal, during puberty and post-pubertal.

Results and Conclusion: The imprinting status of the PWS locus in the 26 CPP patients was normal. In addition, the CpG sites of the promoter region of MKRN3 was hypermethylated both in CPP patients and healthy controls indicating that MKRN3 gene is silenced in peripheral blood cells. Hypermethylation of Mkrn3 promoter was also observed in mouse genomic DNA from peripheral blood samples in all three developmental stages; pre-pubertal, during puberty and post-pubertal confirming our observation in human peripheral blood samples. However, the methylation status of Mkrn3 promoter CpG sites in mouse hypothalamic samples was significantly differentiated when compared to peripheral blood samples as well between the three developmental stages. Our results suggested that Mkrn3 promoter methylation in hypothalamus may be considered as an important mechanism in the epigenetic regulation during puberty.