Population and Molecular Genetic Studies of Congenital Heart Disease

UoM administered thesis: Phd


Introduction: Congenital heart defect (CHD) is the most common type of birth defect. Many investigations have been conducted on CHD prevalence. However, a summary of prevalence data on CHDs at the global level is lacking. Several risk regions for CHDs have been picked up by genome-wide association studies (GWAS). However, none of them has been reported with experimental proof of their biological functions during heart development. Methods and Results: This thesis comprises two studies: (1) Meta-analysis and system review on CHD prevalence and (2) investigation of the role of STX18-AS1 in heart development. In the first study, in order to summarise CHD prevalence data across the world, meta-analyses were conducted for live births and school children. Compared with Van de Linde’s report on live births, my meta-analysis included up-to-date publications after 2009, more subjects, and more CHD subtypes. Prevalence increasing trends (especially for mild lesions) were suggested for both live births and school children. Region differences were observed between Africa and other continents. Income was an important factor relative to CHD prevalence. Lower income was linked to lower reported birth prevalence, more unrecognised CHDs, and a higher percentage of unrepaired CHDs till school ages. STX18-AS1, the gene investigated in the second study is a long noncoding RNA (lncRNA) containing risk Single-nucleotide polymorphisms (SNPs) for ASD (GWAS). With eQTL analysis, STX18-AS1 was confirmed to be associated with risk SNPs (rs870142 and rs16835979) in heart tissues. With qPCR and in situ hybridisation, the expression of STX18-AS1 was detected in human embryonic hearts with temporal specificity at CS14-18 and at the location of the atrial septum. GapmeR and CRISPR were successively applied to knockdown STX18-AS1 in HepG2 cells to explore its target genes. CRISPR generated deletions at the first two exons of STX18-AS1 and significantly reduced STX18-AS1 transcription, while GapmeR showed unsatisfactory knocking down efficiency and off-target effects. NKX2-5 was identified as a specific target for STX18-AS1 with CRISPR at both RNA and protein levels. With CHIP, H3K4me3 was shown to be reduced along with NKX2-5. With the application of the in vitro cardiomyocyte differentiation model, the differentiation potential of human embryonic stem cells (H9) was found to be partially depleted with intact pluripotency and inhibited expression of core cardiac genes. Conclusion: CHD prevalence, especially mild lesions, is increasing in recent decades. Few valid studies are available for underdeveloped regions like Africa. The management of CHDs is still a problem in lower income countries. STX18-AS1 is a lncRNA expressed in the heart with spatial-temporal specificity. It was able to regulate the core cardiac gene NKX2-5 through histone modification and changed the cardiomyocyte differentiation potential of H9 cells. Therefore, STX18-AS1 is a necessary regulative lncRNA for human cardiac development.


Original languageEnglish
Awarding Institution
Award date31 Dec 2018