Functional Investigation of STX18-AS1, a Long Noncoding RNA Associated with Atrial Septal Defect

Genome-wide association studies (GWAS) have identified a number of associations between common genetic variants and Congenital Heart Disease (CHD). However, responsible genes and mechanisms of action have not as yet been reported for any locus. We carried out gene identification and functional investigation at the previously identified 4p16 risk locus for Atrial Septal Defect (top SNP rs870142, OR=1.46; p= ). Limited linkage disequilibrium in the region strongly suggested the long noncoding RNA STX18-AS1 as the responsible gene in the region. STX18-AS1 is not conserved beyond primates, therefore, all experiments were conducted in human tissues and cell lines. The risk SNPs of ASD were confirmed as eqtls for STX18-AS1 with Right Atrial Appendage samples; the risk allele was associated with lower expression of STX18-AS1. During embryonic heart development, the cardiac expression of STX18-AS1 was highest at CS14-CS18. At these critical stages of atrial septation, the expression of STX18-AS1 was identified in the myocardium of Atrial Septum with in situ hybridization. Using CRISPR/Cas9 knockdown in HepG2 cells, the key cardiac transcriptional factor NKX2-5 was identified as a downstream target of STX18-AS1. Mutations in NKX2.5 cause septal defects in human families. Reduced STX18- AS1 transcription inhibited the expression of NKX2-5, by interacting with the MLL complex and changing the histone modification of H3K4me3 around the promoter region of NKX2.5. Furthermore, using the in vitro model of cardiomyocyte differentiation from human embryonic stem cells (hescs), we demonstrated that the knockdown of STX18-AS1 depleted the potential of hescs in differentiating into cardiomyocytes without changes in cell viability and pluripotency. Our results indicate that STX18-AS1 is the first GWAS identified lncrna that influences cardiac development