Identification and functional analysis of variations in ENG promoter from patients of isolated ventricular septal defects associated with pulmonary hypertension

Abstract

Objective Ventricular septal defect (VSD) is one of the most common types of congenital heart disease (CHD) and pulmonary hypertension (PAH) is a serious complication of VSD. As a transmembrane glycoprotein, Endoglin (ENG) is mainly expressed in endothelial cells, and plays a crucial role in development of cardiopulmonary diseases. Studies have shown that mutations in ENG coding sequence can lead to PAH, but the dysfunction of ENG promoter and its correlation with formation of VSD-PAH are still unclear. We hypothesized that variations in ENG promoter may be related to the pathogenesis of VSD-PAH. Methods Blood samples from 362 patients with isolated VSD, including 184 with PAH and 178 without PAH as control were studied. Total DNA from all subjects was extracted and the PCR combined with Sanger sequencing was used to identify variations from ENG promoter. The luciferase activities of pGL3 basic-ENG promoter with/without variation were detected by dual-luciferase assays at the cellular level. Electrophoretic mobility shift assay (EMSA) was used to test the effect of variations in ENG promoter on binding sites of transcription factors. Results Five novel heterozygous variations were identified in ENG promoter from 4 VSD-PAH patients and 2 from VSD patients. Compared with the wild type, the transcriptional activities of ENG promoter with variation were significantly changed, either up-regulated (g.127854409 C>A, g.127854342C>T, g.127854480 C>T, g.127854697 G>T) or down-regulated (g.127854345 G>A). The results of EMSA showed that variations in ENG promoter affected the number and affinity of binding sites of transcription factors. Conclusion Variations identified from ENG promoter sequence in VSD-PAH patients affect the transcriptional activity and therefore are possibly involved in the development of PAH. These findings may provide a new perspective into the molecular pathogenesis and potential therapeutic insights for patients with VSD-PAH.