Rare loss-of-function mutations of PTGIR are enriched in fibromuscular dysplasia

Abstract

Aims: Fibromuscular dysplasia (FMD) and spontaneous coronary artery dissection (SCAD) are related, non-atherosclerotic arterial diseases mainly affecting middle-aged women. Little is known about their physiopathological mechanisms. We aimed to identify rare genetic causes to elucidate molecular mechanisms implicated in FMD and SCAD.

Methods and results: We analysed 29 exomes that included familial and sporadic FMD. We identified one rare loss-of-function variant (LoF) (frequencygnomAD = 0.000075) shared by two FMD sisters in the prostaglandin I2 receptor gene (PTGIR), a key player in vascular remodelling. Follow-up was conducted by targeted or Sanger sequencing (1071 FMD and 363 SCAD patients) or lookups in exome (264 FMD) or genome sequences (480 SCAD), all independent and unrelated. It revealed four additional LoF allele carriers, in addition to several rare missense variants, among FMD patients, and two LoF allele carriers among SCAD patients, including one carrying a rare splicing mutation (c.768 + 1C>G). We used burden test to test for enrichment in patients compared to gnomAD controls, which detected a putative enrichment in FMD (PTRAPD = 8 × 10-4), but not a significant enrichment (PTRAPD = 0.12) in SCAD. The biological effects of variants on human prostaclycin receptor (hIP) signalling and protein expression were characterized using transient overexpression in human cells. We confirmed the LoFs (Q163X and P17RfsX6) and one missense (L67P), identified in one FMD and one SCAD patient, to severely impair hIP function in vitro.

Conclusions: Our study shows that rare genetic mutations in PTGIR are enriched among FMD patients and found in SCAD patients, suggesting a role for prostacyclin signalling in non-atherosclerotic stenosis and dissection.

Keywords: Fibromuscular dysplasia; Prostacyclin signalling; Rare loss-of-function variants; Spontaneous coronary artery dissection.

Graphical abstract

Figure 1

Flowchart illustrating the genetic approach undertaken in this study. FMD, fibromuscular dysplasia; LoF, loss-of-function; SCAD, spontaneous coronary artery dissection.

Figure 2

Analysis of cAMP synthesis in response to iloprost in HEK293 cells overexpressing WT or mutant prostacyclin receptors. (A and B) Concentration of cAMP in HEK293 cells overexpressing WT prostacyclin receptor (hIP), mutant hIP (A: P17RfsX6 or Q163X, B: A2T, L67P, M107V, or R137C) or transfected with mock plasmid (pcDNA-FLAG-HA). Error bars represent the standard deviation of three technical replicates. (C) Measurement of 50% response concentration to iloprost (EC50) in HEK293 cells overexpressing WT or mutant hIP. N represents the number of independent experiments. Error bars represent the standard error of the mean. Student’s t-test P-value (bilateral test with homoscedastic variance) ***P < 10⁻³. cAMP, cyclic adenosine monophosphate; WT, wild-type.

Figure 3

Evaluation of the expression and localization of the prostacyclin receptor mutated proteins. (A) WT and mutant protein expression. SDS-PAGE/western blot assay on whole cells extracts of HEK293 cells overexpressing WT or mutant prostacyclin receptor (hIP) included a FLAG-HA N-terminal tag and the transfection control (mCherry). Proteins (hIP, mCherry, and β-actin) were detected using specific primary antibodies. We found that hIP is detected as a mix of bands and higher molecular weight smear due to its extensive post-translational modification. Full size blots are presented in Supplementary material online, Figure S7. (B) Protein quantification. We performed five independent experiments (hIP-A2T and M107V were assayed in four experiments), and the average is shown with standard error of the mean as error bars. We indicate Student’s t-test P-value (paired test) when P < 0.05 as *. (C) Immunofluorescence visualization of HEK293 cells overexpressing wild-type or L67P hIP. Protein localization was assayed using hIP specific antibody (purple signal). Fixed cells were incubated with Alexa488-conjugated WGA (green signal), and with 4′,6-diamidino-2-phenylindole (DAPI) (blue signal). Images were taken with a 100× objective on a Zeiss ApoTome system. hIP, human prostacyclin receptor; WGA; Wheat Germ Agglutinin; WT, wild-type.