RNA methylation and the potential role of folate supplementation in preventing Hirschsprung disease

To determine whether folate supplementation may reduce the incidence of Hirschsprung disease (HSCR) by regulating the methylation level of growth arrest-specific 5 (GAS5).

The m6A methylation deficiency model and folate supplementation model were created using the m⁶A inhibitor 3-Deazaadenosine (DAZ) and folate, respectively. The effects of folate on the morphology, mortality, and number of HuC/D positive neurons in zebrafish were observed. The methylation level and proliferation ability of mouse enteric neural crest stem cells (ENCSCs) were detected by m⁶A ELISA and EdU assay respectively. Human HSCR cases and control tissues were collected. The expression level of GAS5 in intestinal tissues was detected by qRT-PCR. M⁶A ELISA, actinomycin D, and MeRIP qRT-PCR were used to detect the effect of folate on m⁶A methylation levels, GAS5 stability, and the content of m⁶A-modified GAS5 in N2a cells. The RIP assay was used to detect the content of GAS5 bound to YTH N⁶-methyladenosine RNA binding protein F2 (YTHDF2) under different folate concentrations.

Supplementation of folate can ameliorate the decreased ENCSC proliferation ability caused by low m⁶A methylation level, reduce embryonic mortality of zebrafish and increase the number of HuC/D positive neurons at 96 hpf. Furthermore, folate may reduce the expression of the demethylase alkB homologue 5 (ALKBH5), weaken ALKBH5's demethylation effect on GAS5, promote the degradation of GAS5 by YTHDF2, and subsequently reduce the expression of GAS5.

Additional folate supplementation during early embryonic development may serve as a strategy for preventing HSCR.