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Editing Out Cardiovascular Disease Risk Locus Exposes How it May Affect Health

NEW YORK (GenomeWeb) – By editing a cardiovascular disease risk locus out of cells' genomes, researchers have begun to tease out how it effects health.

Cardiovascular disease is the most common cause of death worldwide, killing about 17.9 million people in 2016, according to the World Health Organization. While more than 100 genetic variants are known to affect cardiovascular disease risk, the 9p21.3 locus was the first one to be discovered and accounts for between 10 percent and 15 percent of the disease in non-African patients.

A Scripps Research-led team developed induced pluripotent stem cells generated from people homozygous for either the high- or low-risk allele and coaxed those iPSCs to develop into vascular smooth muscle cells. As they reported today in Cell, the researchers found vascular smooth muscle cells with the risk haplotype had altered transcriptional networks.

But using a gene-editing approach to eliminate the 9p21.3 risk haplotype from the muscle cells rescued them. At the same time, the expression of a long non-coding RNA linked to the haplotype induces the phenotype in muscle cells from low-risk individuals, the researchers reported.

"Now, with strong evidence suggesting the 9p21.3 haplotype undermines the stability and function of vascular muscle cells, we may have opened a new route to interventions that could impact many millions of people worldwide," senior author Kristin Baldwin, a professor at Scripps Research, said in a statement.

The researchers generated iPSCs from cardiovascular disease patients who were homozygous for the risk haplotype and from healthy people who were homozygous for the non-risk haplotype. They also generated sets of TALENs to edit out the 9p21.3 locus in a subset of these iPSCs. They differentiated these iPSCs into vascular smooth muscle cells, some with the risk haplotype and some with the risk haplotype edited out as well as some with the non-risk haplotype and some with the non-risk haplotype edited out.

These generated vascular smooth muscle cells that exhibited differences in gene expression, the researchers noted. The expression of some 3,000 genes varied between the vascular smooth muscle cells with the risk haplotype versus those with the risk haplotype edited out. This gene set was enriched for genes involved in cell cycle and DNA replication, but also in cell adhesion and muscle contraction.

The muscle cells with the risk haplotype also acted differently from the other cells. They proliferated more quickly, had decreased adhesion, and decreased contractile force.

Deletion of the risk haplotype, though, reverts the muscle cells to a transcriptional and functional state similar to those of cells with the non-risk haplotype or with the non-risk haplotype edited out.

As the 9p21.3 locus contains the terminal exons of the lncRNA ANRIL, the researchers also examined ANRIL levels within these different vascular smooth muscle cells using RNA-seq and real-time PCR data. The risk haplotype, they found, affects ANRIL expression, as shorter isoforms are present.

When they added exogenous short ANRIL isoforms to non-risk vascular smooth muscle cells, it led the cells to exhibit phenotypes similar to those of risk cells. This indicated to the researchers that ANRIL RNAs could mediate the switch from healthy to disease-promoting cells, but that additional work is needed to figure out how they have that effect.

The researchers then confirmed these findings in iPSC lines generated from a second cohort of individuals.

"It's remarkable that one region of our genome could have such a significant impact on both the functional and genetic characteristics of these blood vessel cells," co-author Eric Topol, executive vice president of Scripps Research, said in a statement. "Now that we know its role in damaging the vascular wall, we are in a better place to find novel ways to prevent it."