Double whammy: the genetic variants in CECR2 and high Hcy on the development of neural tube defects

Abstract

Introduction: Neural tube defects (NTDs) are serious congenital malformations. The etiology of NTDs involves both genetic and environmental factors. Loss of CECR2 in mice has been shown to result in NTDs. Our previous study indicated that high homocysteine (HHcy) levels could further reduced the expression level of CECR2. This investigation aims to explore the genetic influence of the chromatin remodeling gene, CECR2, in humans and determine if HHcy can have a synergistic effect on protein expression. Methods: We conducted Next-Generation Sequencing (NGS) of the CECR2 gene in 373 NTD cases and 222 healthy controls, followed by functional assay application to select and evaluate CECR2 missense variants and subsequent Western blotting to identify protein expression levels. Results: From the analysis, we identified nine rare, NTD-specific mutations within the CECR2 gene. Significantly, four missense variants (p.E327V, p.T521S, p.G701R, and p.G868R) were selected via functional screening. The E9.5 mouse ectodermal stem cell line NE-4C, transfected with plasmids expressing p.E327V, p.T521S, p.G868R variants or a recombinant harboring all four (named as 4Mut), exhibited notable reductions in CECR2 protein expression. Furthermore, exposure to homocysteine thiolactone (HTL), an extremely reactive homocysteine metabolite, amplified the reduction in CECR2 expression, accompanied by a significant increase in the apoptotic molecule Caspase3 activity, a potential NTD inducer. Importantly, folic acid (FA) supplementation effectively counteracted the CECR2 expression decline induced by CECR2 mutation and HTL treatment, leading to reduced apoptosis. Discussion: Our observations underscore a synergistic relationship between HHcy and genetic variations in CECR2 concerning NTDs, thereby reinforcing the concept of gene-environment interaction phenomena in NTD etiology.

Keywords: CECR2; apoptosis; homocysteine; mutation; neural tube defects.

FIGURE 1

Rare missense variants identified in CECR2 gene. (A)Structure of the CECR2 transcript (NM_031413.3) and proteins (NP_113601.2), positions of 9 identified missense variants were marked in the gene sequence. Heterozygous variants of p.Glu327Val (c.980A>T), p.Thr521Ser (c.1562C>G), p.Gly701Arg (c.2101G>A) and p.Gly868Arg (c.2602G>A) were further confirmed via sequencing. (B) Alignment of human CECR2 protein sequence with other orthologues sequences, including zebrafish: A0A8M1RL92 (Uniprot); bovin: F1MRJ9; chicken: A0A8V0YBW6; pig: A0A287AW08; mouse: E9Q2Z1; rat: F1LYI0; and monkey: A0A2K5EPY0 by T-Coffee. Solid black arrow indicate a variant.

FIGURE 2

Effects of CECR2 missense variants on CECR2 protein expression. NE-4C cells were transfected with wild-type (WT), empty vector plasmid, or the 4 selected CECR2 variants. WB was performed with whole-cell proteins lysate and analyzed proteins were immunoblotted with anti-Cecr2 antibody (Upper), and anti-Gapdh antibody (lower), which was served as loading control.

FIGURE 3

The HHcy and CECR2 missense variants synergistically reduced the expression of CECR2 protein expression. (A) NE-4C were treated with different concentrations of HTL, and the expression level of CECR2 was detected by WB. (B) Transfected the mutant p.T521S into NE-4C cells with or without 1 mM HTL treatment, and detect the effect on the expression of Cecr2 protein by WB. (C) NE-4C cells were treated with 1 mM HTL and transfected with 4 CECR2 variants, respectively. WB was further employed to detect CECR2 protein expression.

FIGURE 4

The p.T521S variant induce cells apoptosis. (A) After transfection with p.T521S mutant or 4Mut plasmids, the expression level of apoptotic molecules: cleaved-caspase3 was detected by WB. (B) Cell apoptosis was analyzed by flow cytometry after cells double staining with FITC and PI.

FIGURE 5

Folic acid can rescue decreased of CECR2 protein expression caused by CECR2 missense variants or high hcy. The cells treated with HTL were transfected with p.T521S variant (A) or 4-mixed variant (B), respectively, and supplemented with or without FA, the expression of Cecr2 protein under different treatment conditions was detected by WB method. (C,D) IF assays were performed to detect the effect of FA on the expression and distribution of Cecr2 protein in p.T521S variant transfected cells. (E) The rescue effect of FA on apoptosis was detected by WB in p.T521S or 4Mut transfected cells.