PHACTR1 Is a Genetic Susceptibility Locus for Fibromuscular Dysplasia Supporting Its Complex Genetic Pattern of Inheritance

Abstract

Fibromuscular dysplasia (FMD) is a nonatherosclerotic vascular disease leading to stenosis, dissection and aneurysm affecting mainly the renal and cerebrovascular arteries. FMD is often an underdiagnosed cause of hypertension and stroke, has higher prevalence in females (~80%) but its pathophysiology is unclear. We analyzed ~26K common variants (MAF>0.05) generated by exome-chip arrays in 249 FMD patients and 689 controls. We replicated 13 loci (P<10-4) in 402 cases and 2,537 controls and confirmed an association between FMD and a variant in the phosphatase and actin regulator 1 gene (PHACTR1). Three additional case control cohorts including 512 cases and 669 replicated this result and overall reached the genomic level of significance (OR = 1.39, P = 7.4×10-10, 1,154 cases and 3,895 controls). The top variant, rs9349379, is intronic to PHACTR1, a risk locus for coronary artery disease, migraine, and cervical artery dissection. The analyses of geometrical parameters of carotids from ~2,500 healthy volunteers indicate higher intima media thickness (P = 1.97×10-4) and wall to lumen ratio (P = 0.002) in rs9349379-A carriers, suggesting indices of carotid hypertrophy previously described in carotids of FMD patients. Immunohistochemistry detected PHACTR1 in endothelium and smooth muscle cells of FMD and normal human carotids. The expression of PHACTR1 by genotypes in primary human fibroblasts showed higher expression in rs9349379-A carriers (N = 86, P = 0.003). Phactr1 knockdown in zebrafish resulted in dilated vessels indicating subtle impaired vascular development. We report the first susceptibility locus for FMD and provide evidence for a complex genetic pattern of inheritance and indices of shared pathophysiology between FMD and other cardiovascular and neurovascular diseases.

Fig 1. Association between rs9349379 and artery thickness in healthy controls.

Intima-media thickness and wall to lumen ratio are presented by genotype in all (A,D) women (B,E) and men (C,F) participants. We indicated p-values for the linear regression analyses under the additive model adjusted for age, sex and body surface area.

Fig 2. PHACTR1 mRNA expression and immunostaining.

(A) PHACTR1 mRNA expression in human fibroblasts. PHACTR1 mRNA levels were determined by RT-qPCR in cultured fibroblasts from controls (N = 39) and FMD cases (N = 51). GAPDH expression was used as control for normalization and data are expressed as mean fold change of PHACTR1 relative to GAPDH. There is a non-significant trend toward overexpression of PHACTR1 in FMD cases compared to controls (P = 0.61, Mann-Whitney test) whereas significant differences are uncovered after stratification by genotypes (P = 0.003, Kruskal-Wallis test). (B) Immunostaining of normal and FMD internal carotid using anti-PHACTR1 antibody. PHACTR1 was detected in endothelium and smooth muscle cells in the media. Staining is mostly cytoplasmic with regular alignment in normal carotids and typical disorganized cellular structure in media of FMD carotid.

Fig 3. phactr1 modulation in zebrafish affects vascular dimensions and patterning.

Two-dimensional projections obtained from z-series confocal images in the trunk of control and phactr1 knockdown (KD) zebrafish embryos (two representative images per condition). Green represents the vascular endothelium as marked by EGFP. Greyscale represents the corresponding DIC bright field image of the fish trunk. DA: Dorsal aorta, SV: Segmental vessel, PCV: Posterior cardinal vein. Quantification of inner vascular diameter for the dorsal aorta (DA), posterior cardinal vein (PCV) and caudal artery (CA). (*) represents P<0.05.