As a nutritional factor, folic acid can prevent cardiac and neural defects during embryo development. Our previous study showed that arsenic impairs embryo development by down-regulating Dvr1/GDF1 expression in zebrafish. Here, we investigated whether folic acid could protect against arsenic-mediated embryo toxicity. We found that folic acid supplementation increases hatching and survival rates, decreases malformation rate and ameliorates abnormal cardiac and neural development of zebrafish embryos exposed to arsenite. Both real-time PCR analysis and whole in-mount hybridization showed that folic acid significantly rescued the decrease in Dvr1 expression caused by arsenite. Subsequently, our data demonstrated that arsenite significantly decreased cell viability and GDF1 mRNA and protein levels in HEK293ET cells, while folic acid reversed these effects. Folic acid attenuated the increase in subcellular reactive oxygen species (ROS) levels and oxidative adaptor p66Shc protein expression in parallel with the changes in GDF1 expression and cell viability. P66Shc knockdown significantly inhibited the production of ROS and the down-regulation of GDF1 induced by arsenite. Our data demonstrated that folic acid supplementation protected against arsenic-mediated embryo toxicity by up-regulating the expression of Dvr1/GDF1 and folic acid enhanced the expression of GDF1 by decreasing p66Shc expression and subcellular ROS levels.

Genetic variation in specific transcription factors during heart formation may lead to congenital heart disease (CHD) or even miscarriage. The aim of the present study was to identify CHD-associated genes using next generation sequencing (NGS). The whole exome DNA sequence was obtained from a stillborn fetus diagnosed with tricuspid atresia and complete transposition of the great arteries using high-throughput sequencing methods. Subsequently, genetic variants of CHD-associated genes were selected and verified in 215 non-syndromic CHD patients and 249 healthy control subjects using polymerase chain reaction combined with Sanger sequencing. Genetic variants of previously reported CHD-inducing genes, such as cysteine rich with EGF like domains 1 and cbp/p300-interacting transactivator with Glu/Asp rich carboxy-terminal domain 2, were discovered through the NGS analysis. In addition, a novel non-synonymous mutation of the iroquois homeobox 1 (IRX1) gene (p.Gln240Glu) was identified. A total of three non-synonymous mutations (p.Gln240Glu, p.Ser298Asn and p.Ala381Glu) of the IRX1 gene were verified in 215 non-syndromic CHD patients, but not in 249 healthy volunteers. The results demonstrated that NGS is a powerful tool to study the etiology of CHD. In addition, the results suggest that genetic variants of the IRX1 gene may contribute to the pathogenesis of CHD.

GDF1 plays an important role in left–right patterning and genetic mutations in the coding region of GDF1 are associated with congenital heart disease (CHD). However, the genetic variation in the promoter of GDF1 with sporadic CHD and its expression regulation is little known. The association of the genetic variation in GDF1 promoter with CHD was examined in two case–control studies, including 1084 cases and 1198 controls in the first study and 582 cases and 615 controls in the second study. We identified one single nucleotide polymorphism (SNP) rs181317402 and two novel genetic mutations located in the promoter region of GDF1. Analysis of combined samples revealed a significant association in genotype and allele frequencies of rs181317402 T/G polymorphism between CHD cases in overall or ventricular septal defects or Tetralogy of Fallot and the control group. rs181317402 allele G polymorphism was significantly associated with a decreased risk of CHD. Furthermore, luciferase assay, chromatin immunoprecipitation and DNA pulldown assay indicated that Nkx2.5 transactivated the expression of GDF1 by binding to the promoter of GDF1. Luciferase activity assay showed that rs181317402 allele G significantly increased the basal and Nkx2.5-mediated activity of GDF1 promoter, while the two genetic mutations had the opposite effect. rs181317402 TG genotype was associated with significantly increased mRNA level of GDF1 compared with TT genotype in 18 CHD individuals. Our results demonstrate for the first time that Nkx2.5 acts upstream of GDF1 and the genetic variants in GDF1 promoter may confer genetic susceptibility to sporadic CHD potentially by altering its expression.