Scientific Business Abstracts of the 113th Annual Meeting of the Association of Physicians of Great Britain and Ireland: STX18-AS1 is an lncRNA governing in vitro cardiomyocyte differentiation and predisposing to atrial septal defect via downregulation of NKX2-5

Objectives: To identify the gene and mechanism responsible for the Genome-wide association studies (GWAS) signal (OR = 1.46; P = 2.61 × 10−10) which we previously identified at chromosome 4p16 for atrial septal defect (ASD). To date, while a number of risk variants have been identified from GWAS of congenital heart disease (CHD), none has been functionally confirmed.

Methods and Results: The linkage disequilibrium in the region indicated association was restricted to the long noncoding RNA STX18-AS1. Since STX18-AS1 is not conserved beyond primates, all experiments were conducted in human tissues and cell lines. In 108 RNA samples from right atrial appendages and corresponding DNA, we first confirmed the risk SNPs of ASD were eQTLs for STX18-AS1 in cardiac tissues. Using qPCR, the transcription of STX18-AS1 in embryonic hearts was detected to be the highest at CS14-CS18, the critical time for atrial septation. Furthermore, using in situ hybridization on whole embryonic hearts at CS16-CS19, we detected substantial expression of STX18-AS1 in the atrial septum. We next identified STX18-AS1 as a regulator of the key cardiac transcriptional factor NKX2-5 using CRISPR/Cas9 knockdown in HepG2 cells. Reduced STX18-AS1 transcription inhibited the expression of NKX2-5, mutations in which cause septal defects in humans. Using in vitro 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.

Conclusions: STX18-AS1 is the first long noncoding RNA influencing CHD risk identified from GWAS. The mechanism involves downregulation of the NKX2-5 gene.