*5.4. Insights and Limitations from Functional Genomics at PHACTR1 Locus*

Although rs9349379 is seemingly positioned in a *PHACTR1* intron, it is actually located about 54 kbp upstream of *PHACTR1* transcription start site used in endothelial and smooth muscle cells of the arteries, and about 265 kbp upstream of another *PHACTR1* transcription start site used in macrophages [52]. The homozygous A allele at rs9349379 was associated with higher *PHACTR1* expression in skin fibroblasts and macrophages in healthy donors [46,53] (Figure 2). Publicly available datasets show an enrichment of histone acetylation marks (H3K27ac) close to rs9349379 in arteries, but not in other analysed tissues, suggesting that the region may serve as a tissue-specific enhancer [51]. The rs9349379[G] allele is predicted to disrupt a bona fide binding site for myocyte enhancer factor 2 (*MEF2*) transcription factors, and binding of MEF2s to this site was indeed shown in vitro [54]. However, knockdown of either *MEF2A* or *MEF2C* in human umbilical vein endothelial cells did not result in a decrease of *PHACTR1* expression.

A recent study used induced pluripotent stem cells and CRISPR-Cas9 modification to study the effect of homozygous rs9349379[A] or [G] on gene expression [51]. Modified cells were differentiated into vascular endothelial or vascular smooth muscle cell lineages, and gene expression was analysed using microarrays and qPCR. The results suggested that the rs9349379[G] genotype increases the expression of endothelin-1 (ET-1), whose precursor is encoded by the *EDN1* gene, located about 600 kbp upstream of rs9349379. This observation was corroborated by the finding of higher levels of Big ET-1, a precursor of endothelin-1, in the plasma of healthy subjects harbouring G allele, at least at the homozygous state. ET-1 is a potent vasoconstrictor, which acts on smooth muscle cells to favour their contraction, proliferation and migration. It also plays a role in vascular relaxation through endothelial cells [36]. Accordingly, endothelin-1 imbalance may play a role in the association of rs9349379 to various cardiovascular diseases, although further exploration of the mechanisms specifically involved in FMD is still required.
