ESPE Abstracts

Background: In the published literature, eating disorders appear as a cause of pubertal delay/arrest in young girls. Furthermore, several studies associate anorexia nervosa (AN) with epigenetic changes that may heighten the risk of the disorder and lead to clinical signs as functional hypothalamic amenorrhea. In the present study we investigated whether AN correlates with a distinct DNA methylation profile compared to normal-eating pubertal girls.

Methods: Using the Infinium Human DNA Methylation EPIC BeadChip array 850K, we analyzed genome-wide DNA methylation patterns in leukocytes collected from 10 female anorexic patients with primary (n = 4) or secondary (n = 6) amenorrhea (age 13.36±1.69; BMI -1.34±1.07) and 13 healthy pubertal girls matched by age (age 13.49±1.48; BMI 0.32± 0.98).

Results: Examination of methylome changes associated with AN revealed 87 differentially methylated regions (DMRs) compared to healthy girls. Most of these DMRs exhibited hypomethylation in the AN group (69%) and harbored genes, as Clusterin, regulating hypothalamic feeding pathways, and MKRN3, involved in pubertal timing regulation. Moreover we showed that methylation levels in members of AN group differed from those in the control group for 2072 CpG sites (62.40% hypomethylated) located within genes previously associated with metabolic processes (e.g., LEPR and NR1H3) and age of menarche (e.g., GHR, MKL2, GAB2, CTBP2, and DLK1). Additionally, some CpGs resided in genes such as CDH7, associated with hypogonadotropic hypogonadism, a condition clinically overlapping with functional hypothalamic hypogonadism in anorexia. Interestingly, a total of 92 differentially methylated genes encoding zinc-finger (ZNF) proteins, including MKRN3, exhibited distinct epigenetic modifications between the two groups. Ingenuity Pathway Analysis of all CpGs highlighted pathways related to metabolism and body mass (e.g., adipogenesis, leptin, and sirtuin signaling), neuronal signaling (e.g., semaphoring and gustation pathways), and several pathways potentially associated with puberty (e.g., estrogen, GABA receptor, and GNRH signaling).

Conclusion: Most of the observed changes consisted of hypomethylation of DMRs or CpG sites, with ZNFs standing out as the population of transcriptional repressors affected by these modifications. Particularly significant was the hypomethylation of MKRN3, which acts as brake on GnRH production, thus playing a crucial role in mammalian puberty regulation. The distinct methylation profiles observed in normal-eater and anorexic girls suggest that epigenetic changes may be a marker for both metabolic dysfunction and pubertal arrest, implying potential mutual influence between these aspects in humans. Indeed, many differentially methylated genes are implicated in signaling pathways essential for neuronal and sexual development.