Identification and functional analysis of variants in ISL1 promoter from patients with isolated atrial septal defects

Abstract

ObjectiveAtrial septal defect (ASD) is one of the most common types of congenital heart disease (CHD). As a transcription factor, ISL1 is expressed specifically in second heart field during embryogenesis and plays a crucial role in heart development and cardiovascular morphogenesis. Studies have shown association of ISL1 multiple variants with diverse type of CHD including ASD, whereas the variants of ISL1 gene promoter region in ASD patients have not been reported. We hypothesized that variants in ISL1 promoter may be involved in the formation of ASD.MethodsBlood samples from 593 subjects, including 300 patients with isolated ASD and 293 healthy controls were studied. Total DNA from all subjects was extracted and the PCR combined with Sanger sequencing was used to identify ISL1 gene promoter variants. Functional analysis of DNA sequence variants was performed using a dual luciferase reporter assay. Electrophoretic mobility shift assay (EMSA) was selected to examine the influence of the variants in ISL1 gene promoter on binding of transcription factors. All possible binding sites of transcription factor influenced by the identified variants were predicted by the PROMO database.ResultsFour variants in ISL1 gene promoter were found only in patients with ASD by sequencing. Three of the 4 variations [g.4923 G>C (rs541081886), g.5079 A>G (rs1371835943), g.5309 G>A (rs116222082)] significantly decreased the transcriptional activities compared with the wild‐type ISL1 gene promoter (P < 0.05). The EMSA revealed that these variants [g.4923 G>C (rs541081886), g.5079 A>G (rs1371835943), g.5309 G>A (rs116222082)] in ISL1 promoter affected the number and affinity of binding sites of transcription factors. Further analysis with the online PROMO database demonstrated that a cluster of putative binding sites for transcription factors may be altered by these variants.ConclusionThese sequence variants identified from the promoter region of ISL1 gene in ASD patients are likely involved in the development of ASD by affecting the transcriptional activity and altering ISL1 levels. Therefore, these findings may provide new insights into the molecular etiology and potential therapeutic strategy of ASD.