The Impact of GATA2 Expression and Methylation on Patients with Hirschsprung's Disease

Hirschsprung's (HIRSH-sproongz) disease is a multifactorial disorder characterized by the failure of enteric nervous system (ENS) development and is associated with loss-of-function variants, primarily in the RET gene. Rearranged during transfection (RET) expression is tightly regulated within a complex gene regulatory network (GRN) involving many transcription factors, such as GATA2. Aberrant DNA methylation in GATA2 may indicate a reduced enhancer activity, resulting in the silencing of RET and contributing to the failure of ENS development in HIRSH. However, the epigenetic mechanisms underlying these processes have yet to be established.

We analyzed the GATA2 mRNA expression and DNA methylation levels in colonic tissues from the HIRSH patients and controls using quantitative polymerase chain reaction (qPCR) and methylation-specific quantitative PCR (MSP-qPCR). HIRSH tissues, including aganglionic and ganglionic segments, were obtained during pull-through surgery. In addition, control tissues were collected from patients with anorectal malformation (ARM) undergoing definitive surgery, and no clinical evidence of ENS issues was found.

Our study contained 27 unrelated HIRSH patients (20 males and seven females) and 20 controls (12 males and eight females). GATA2 expression was significantly increased in HIRSH than in control colon (ganglionic: 11.78 ± 0.97 vs. 12.59 ± 0.46; p=0.049; aganglionic: 11.94 ± 0.63 vs. 12.59 ± 0.46; p=0.030). In addition, the percentage of methylation was higher in the HIRSH patients than in the control (60.71 ± 2.94% [ganglionic] vs. 79.55 ± 1.79% [aganglionic] vs. 40.43 ± 3.67% [control]).

Overexpression of GATA2 in our subjects may indicate a compensatory mechanism for the loss of other TFs within RET-EDNRB or other HIRSH-associated GRN, as an in vitro study has shown that loss of GATA1 in mouse embryonic stem cells results in a marked increase of GATA2 expression, implying potential interchangeability or compensatory mechanism of GATA TFs in RET GRN. Several mechanisms on how specific DNA hypermethylation pathways can lead to upregulated gene expression have been proposed, including facilitating transcription in gene bodies by suppressing cryptic promoters and protecting the borders of promoters or enhancers against unwanted expansion or contraction.

We demonstrated aberrant expression and methylation of GATA2 in the HIRSH patients. The findings of this study also indicate that GATA2 hypermethylation is associated with increased GATA2 expression, contradicting the established inverse association between DNA methylation and transcriptional activity. This may indicate a distinctive interplay between the epigenetics of GATA2 and RET GRN in the context of HIRSH.