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Variants and Cellular Functional Verification of CITED2 Gene Promoter Region in Patients with Atrial Septal Defect
Author(s) -
Chen Zhuo,
Chen HuanXin,
Yin XiuYun,
Han Jun,
Yang Qin,
He GuoWei
Publication year - 2022
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2022.36.s1.r2842
Subject(s) - promoter , gene , luciferase , sanger sequencing , biology , electrophoretic mobility shift assay , coding region , genetics , transcription factor , microbiology and biotechnology , reporter gene , pathogenesis , gene expression , dna sequencing , immunology , transfection
Objectives Congenital heart disease (CHD) is a common disease of human birth defects, and atrial septal defect (ASD) is the most common form of ASD. Genetic variants in the coding region of the CITED2 gene is known to be significantly correlated with cardiac malformations, but variants of the CITED2 promoter region and its relationship with the formation of ASD are still unclear. We hypothesize that the variants of the CITED2 promoter may be related to pathogenesis of ASD. The purpose of this study was to screen variants in the promoter region of the CITED2 gene and to verify the effect of the variants on gene expression at the cellular level. Methods The blood samples of 332 ASD patients and 293 unrelated healthy children used as controls were studied. The total DNA of all subjects was extracted, and the CITED2 promoter variants were screened by PCR combined with Sanger sequencing. The luciferase activity of the CITED2 promoter was measured by a dual luciferase reporter at the cellular level. Electrophoretic mobility change assay (EMSA) and bioinformatics analysis were used to test the effect of CITED2 promoter changes on transcription factor binding sites. Results Four variants in the promoter region of the CITED2 gene were found only in the 332 ASD patients with zero occurrence in the 293 control subjects. These included one novel heterozygous variant (g.4933C>A) and three SNPs [1 case of g.4078 A>C(rs1165649373), 4 cases of g.4935C>T(rs111470468), 2 cases of g.5027C>T (rs112831934)]. The cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK‐293 cells (P<0.05). In addition, EMSA results and database analysis showed that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes of the expression of CITED2 protein. Conclusion The variants of CITED2 promoter sequence in ASD patients affect transcriptional activity and are likely involved in the occurrence and development of ASD. These findings may provide new perspectives on the molecular pathogenesis and potential therapeutic insights of ASD patients.