Genetic analysis of the CITED2 gene promoter in isolated and sporadic congenital ventricular septal defects

Abstract

Ventricular septal defect (VSD) is the most common congenital heart defect. Previous studies have reported genetic variations in the encoding region of CITED2 highly associated with cardiac malformation but the role of CITED2 gene promoter variations in VSD patients has not yet been explored. We investigated the variation of CITED2 gene promoter and its impacts on gene promoter activity in the DNA of paediatric VSD patients. A total of seven variations were identified by Sanger sequencing in the CITED2 gene promoter region in 400 subjects, including 200 isolated and sporadic VSD patients and 200 healthy controls. Using dual-luciferase reporter assay, we found four of the 7 variations identified significantly decreased the transcriptional activity of the CITED2 gene promoter in HEK-293 cells (P < .05). Further, a bioinformatic analysis with the JASPAR databases was performed and a cluster of putative binding sites for transcription factors was created or disrupted by these variations, leading to low expression of CITED2 protein and development of VSD. Our study for the first time demonstrates genetic variations in the CITED2 gene promoter in the Han Chinese population and the role of these variations in the development of VSD, providing new insights into the aetiology of CHD.

Keywords: congenital heart disease; gene; genetic; ventricular septal defect.

FIGURE 1

A, Study flow diagram; B, Locations of the identified variations within the CITED2 gene promoter. The genetic variations are named according to the genomic DNA sequence of the human CITED2 gene (Genbank accession number NG_016169.1). The transcription start site is at the position of 5001 in the first exon

FIGURE 2

A, Sequencing chromatograms of the variations within the CITED2 gene promoter. For all the heterozygous variations [g.4078A>C (rs1165649373), g.4255C>T (rs1583069506), g.4698A>G (rs529363037), g.4778G>T (rs9385859) and g.4933 C>A], top panels show wild‐type and bottom panels show heterozygous DNA sequences, marked with arrows; B, Relative transcriptional activity of wild‐type and variant CITED2 gene promoters in HEK‐293 cells. Wild‐type and variant CITED2 gene promoters were cloned into reporter gene vector pGL3 and transfected into HEK‐293 cells. The transfected cells were collected, and dual‐luciferase activities were assayed. Empty vector pGL3‐basic was used as a negative control. The transcriptional activity of the wild‐type CITED2 gene promoter was designed as 100%. The relative activities of CITED2 gene promoters were calculated. Lanes 1, pGL3‐basic; WT, pGL3‐wild‐type; V1, pGL3‐4078A>C; V2, pGL3‐4255C>T; V3, pGL3‐4698A>G; V4, pGL‐4778G>T; V5, pGL3‐4933 C>A; *, P < .05; **, P < .01

FIGURE 3

Schema describes the role of CITED2 gene promoter region variations found from the present study and analysed by the JASPAR database in combination with previous studies. The low CITED2 promoter activity caused by the variations contributes to the low expression of CITED2. Consequently, the low expression of CITED2 may be directly involved in the development of VSD. In addition, it may decrease the activity of VSD‐relevant genes and pathways, such as Isl1, Nkx2.5, Gata4, Tbx5, Lefty2, Pitx2 and the Nodal pathway. The low expression of CITED2 may also lead to overexpression of certain cardiac‐related genes such as HIF1α and the VEGF pathway. Further, the low level of CITED2 protein may weaken the combination of CITED2 and ISL1, leading to VSD and may overexpress TFAP2 family, causing low expression of Pitx2 that hinders the formation of a normal heart and increases the risk of VSD