Many factors give people a higher risk of cardiovascular disease, including high cholesterol, high blood pressure, smoking, obesity and inactivity. In recent years, genomic research has revealed some of the major genetic risks of cardiovascular disease. The 9p21.3 haplotype is the first common genomic region associated with an increased risk of coronary artery disease, a disease that destroys blood vessels that transport blood into the heart. It also increases the incidence of related diseases such as aneurysms and strokes. It is currently the most influential genetic cause of cardiovascular disease in the world. In the United States, it causes 10% to 15% of cardiovascular disease cases each year.
Although scientists know that the 9p21.3 haplotype is associated with an increased risk of disease, how this happens in the human body is still unknown. One challenge is that this disease risk haplotype is only found in humans and has a poor similarity to genomic regions in mice or other experimental animals. Another challenge is that this genomic region, called the 9p21.3 haplotype, lacks any protein-coding genes in the traditional sense, so it is difficult to predict what it might do.
To overcome these research challenges, in a new study, Professor Kristin Baldwin and his team at the Scripps Research Institute in the United States wanted to produce human vascular cells in culture dishes, which were then genetically studied using genome editing. They collected blood from people carrying a high-risk version of the 9p21.3 haplotype or a low-risk version, and allowed the cells in the blood to be reprogrammed to produce induced pluripotent stem cells (iPS cells). At this stage, the resulting iPS cells are genetically modified using a molecular scissors called transcription activator-like effector nuclease (TALEN) to remove 9p21 from affected donor cells and unaffected donor cells. Next, they induced these genetically edited iPS cells to become vascular smooth muscle cells and studied them in detail using high-resolution gene profiling and bioengineering methods. The results of the study were published online in the Cell Journal on December 6, 2018, under the heading “Unveiling the Role of the Most Impactful Cardiovascular Risk Locus through Haplotype Editing.”
The Baldwin team found that vascular smooth muscle cells derived from high-risk individuals exhibited extremely extensive abnormalities, and in these high-risk versions of 9p21.3 haplotypes, vascular smooth muscle cells (hereinafter referred to as high-risk vascular smooth muscle cells), more than 3,000 genes are affected — nearly 10% of the total number of human genes. Computer-based studies of these genes suggest that these high-risk vascular smooth muscle cells may be defective in critical functions associated with the disease. When tested in the laboratory, the Baldwin team found that mature high-risk vascular smooth muscle cells were more vulnerable than vascular smooth muscle cells carrying low-risk versions of 9p21.3 haplotypes (hereafter referred to as low-risk vascular smooth muscle cells). The contraction forces are smaller and less able to adhere to their surroundings.
Next, the Baldwin team wanted to know if the 3,000 or so genes might help uncover the effects of the recently discovered about 100 other genes associated with the risk of coronary artery disease. Unexpectedly, these high-risk vascular smooth muscle cells change over more than one-third of the genes (38 genes), suggesting that the 9p21.3 haplotype interacts with the gene network in some way or even control this genetic network.
Through more in-depth research, the Baldwin team identified a potential key major regulator (ANRIL), which is itself a member of a mysterious gene that does not produce proteins but produces long-chain non-coding RNA (lncRNA) molecules. They noted that high-risk vascular smooth muscle cells have higher levels of several shorter forms of ANRIL RNA. When they added these shorter ANRIL RNAs to healthy vascular smooth muscle cells, they developed key features of the disease, suggesting that these shorter ANRIL RNAs may be the master conductor of vascular smooth muscle cells converting between healthy and disease-promoting state. In addition, the use of genome editing to eliminate high-risk versions of the 9p21.3 haplotype in high-risk vascular smooth muscle cells would save their stability.
This study demonstrates the power of genome editing of pluripotent stem cells to study the genetic risks of human disease, especially where genetic risk is unique in the human genome region or in junk DNA. These findings not only provide new insights into how this high-risk version of the 9p21.3 haplotype disrupts vascular health, but also provide a new way to study and target gene regulatory networks that are widely involved in coronary artery disease.
It is worth noting that the 9p21.3 haplotype has such a significant effect on the functional and genetic characteristics of vascular smooth muscle cells. Although it does not encode a protein, its impact on the disease is significant. Given that this study reveals its role in destroying blood vessel walls, it is possible to develop a better way to stop it.