(689.2) Variants and Cellular Functional Verification of CITED2 Gene Promoter Region in Patients with Atrial Septal Defect

Poster Board Number: D59

Zhuo Chen (The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences, The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences), Huan-Xin Chen (The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences), Xiu-Yun Yin (The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences, The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences), Jun Han (Drug Research and Development Center, Wannan Medical College), Qin Yang (The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences), Guo-Wei He (The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences, The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences, The Institute of Cardiovascular Diseases amp; Department of Cardiovascular Surgery, TEDA International Cardiovascular Hospital, Tianjin University amp; Chinese Academy of Medical Sciences)

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.4933Cgt;A) and three SNPs [1 case of g.4078 Agt;C(rs1165649373), 4 cases of g.4935Cgt;T(rs111470468), 2 cases of g.5027Cgt;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 (Plt;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.

lt;igt;Supported by the National Natural Science Foundation of China [82170353 amp;amp; 81870288]; the Non-profit Central Research Institute Fund of Chinese Academy of Medical Sciences [2020-PT310-007]; Tianjin Municipal Health Commission and Tianjin Binhai New Area Health Commission [lt;igt;KJ20071 amp;amp; lt;/igt;2019BWKY010]; Tianjin Science and Technology Project [18PTZWHZ00060];TEDA International Cardiovascular Hospital[2021-ZX-002 amp;amp; 2019-TD-013].lt;/igt;