Genetic causes of atherosclerosis and their molecular and cellular implications at the susceptibility locus on chromosome 21

Kallweit, Thomas (2022): Genetic causes of atherosclerosis and their molecular and cellular implications at the susceptibility locus on chromosome 21. Dissertation, LMU München: Medizinische Fakultät

Abstract

Atherosclerosis is a chronic inflammatory disease of large and medium-sized vessels. It is induced and maintained by various cellular and molecular mechanisms. Besides environmental factors, the genetic background of an individual contributes to the predisposition for the development of atherosclerosis. With the help of genome-wide association studies (GWAS), many genetic variants associated with atherosclerosis have already been unraveled. However, for most of the variants, their causative role in atherogenesis is still widely unclear, not least because many GWAS hits lie in non-protein-coding parts of the genome which are, thus far, not so well understood. It was the aim of this thesis to create a comprehensive overview of currently known atherosclerosis susceptibility loci from published GWAS and, subsequently, to focus on one non-protein-coding risk locus to explore its genetic, molecular and cellular implications for atherogenesis. Therefore, in a first step, we compiled all GWAS (n = 124) associated with atherosclerosis and its risk factors published up to May 2016. The resulting polymorphisms (SNPs) (n = 216) were used to define haplotype blocks (n = 120), as a tool for examining candidate genetic effector mechanisms. 11 out of 120 atherosclerosis risk-associated haplotype blocks solely contained noncoding RNAs. Focusing on these, and after ranking by effect size, we identified a prominent (OR 1.14) and so far uncharacterized atherosclerosis risk locus on chromosome 21q22.11, and marked it for further examination. It contained two lead SNPs with robust atherosclerosis association, rs9982601 (p-value 1.33x10-13) and rs28451064 (p-value 1.33x10-15). Since both SNPs resided within a lncRNA (AP000318.2) but were in linkage disequilibrium with lower ranking atherosclerosis associations in a relatively large noncoding region (> 100 kilobases), we generated two knockouts of differing sizes in human induced pluripotent stem cells (iPSCs) by CRISPR/CAS9 technology, in order to address potential effector mechanisms. Knockout and control wild type hiPSCs were differentiated into cells relevant for atherosclerosis, namely vascular smooth muscle (SMCs) and endothelial cells (ECs). We detected enhanced expression of neighbouring genes when regions encompassing parts of the lncRNA-encoding core locus were deleted. This effect was most prominently seen in SMCs and pertained to genes 3’ and 5’ to the risk locus, including protein-coding genes, over a genomic distance of at least 700 kilobases. In terms of function, we observed altered apoptosis as well as decreased proliferation and migration, and increased adhesion for the knockout comprising the atherosclerosis risk interval. These are features observed in atherosclerotic lesions, hence underlining the atherogenic character of the knockout. Collectively, our data from knockout analyses narrowed down the effector element to reside between chr21:35589815-35653842 and gave rise to the hypothesis that the atherosclerosis-causing mutation on chr21q22.11 deactivates a noncoding, gene-regulatory element, thereby leading to proatherogenic gene expression nearby, and downstream altered proatherogenic mechanisms in cells that constitute the vascular wall.

• Dokumententyp: Dissertationen (Dissertation, LMU München)
• Keywords: atherosclerosis, chromosome 21, CRISPR, iPSC, genetics
• Themengebiete: 600 Technik, Medizin, angewandte Wissenschaften ; 600 Technik, Medizin, angewandte Wissenschaften > 610 Medizin und Gesundheit
• Fakultäten: Medizinische Fakultät
• Sprache der Hochschulschrift: Englisch
• Datum der mündlichen Prüfung: 20. Oktober 2022
• 1. Bericht­erstatter:in: Holdt, Lesca