Recurrent gain-of-function mutation in PRKG1 causes thoracic aortic aneurysms and acute aortic dissections

Abstract

Gene mutations that lead to decreased contraction of vascular smooth-muscle cells (SMCs) can cause inherited thoracic aortic aneurysms and dissections. Exome sequencing of distant relatives affected by thoracic aortic disease and subsequent Sanger sequencing of additional probands with familial thoracic aortic disease identified the same rare variant, PRKG1 c.530G>A (p.Arg177Gln), in four families. This mutation segregated with aortic disease in these families with a combined two-point LOD score of 7.88. The majority of affected individuals presented with acute aortic dissections (63%) at relatively young ages (mean 31 years, range 17-51 years). PRKG1 encodes type I cGMP-dependent protein kinase (PKG-1), which is activated upon binding of cGMP and controls SMC relaxation. Although the p.Arg177Gln alteration disrupts binding to the high-affinity cGMP binding site within the regulatory domain, the altered PKG-1 is constitutively active even in the absence of cGMP. The increased PKG-1 activity leads to decreased phosphorylation of the myosin regulatory light chain in fibroblasts and is predicted to cause decreased contraction of vascular SMCs. Thus, identification of a gain-of-function mutation in PRKG1 as a cause of thoracic aortic disease provides further evidence that proper SMC contractile function is critical for maintaining the integrity of the thoracic aorta throughout a lifetime.

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.

Figures

Figure 1

Identification of PRKG1 c.530G>A (p. Arg177Gln) as the Mutation Responsible for Thoracic Aortic Disease in Families TAA216, TAA292, TAA508, and TAA690 (A) Family pedigrees. The legend indicates the disease and mutation status of the family members. The age at diagnosis of aortic aneurysm or dissection (“dx”) and age at death (“d”) are shown in years. A diagonal line across a symbol indicates that an individual is deceased, an arrow indicates a proband, and a single asterisk indicates an individual whose DNA was used for exome sequencing. Mutation-positive children who are unaffected and two adults who have an unknown disease status are not included in the pedigrees. (B) Ortholog conservation of PKG-1 variants identified in individuals with thoracic aortic disease and surrounding amino acids. (C) Schematic representation of PRKG1. The boxes represent exons 1–18, and the UTRs and the open reading frame are designated. The PRKG1 rare variants identified in this study are on the top of the gene diagram, and the rare variants identified in European Americans in the NHLBI Exome Variant Server are indicated below. Bold type designates variants predicted to be possibly or probably damaging by PolyPhen-2 analysis, and regular type designates variants predicted to be benign.

Figure 2

Effect of PKG-1α p.Arg177Gln on cGMP Binding and Kinase Activity (A) Structural model of the CNB-A domain of PKG-1α. The crystal structure of the PKG-1β (92–227):cGMP complex (Protein Data Bank ID 3OD0) was used for modeling the CNB-A domain of PKG-1α because PKG-1α and PKG-1β have the same protein sequence at this region and this is the only structure available for CNB-A with bound cGMP. The phosphate-binding cassette (PBC) is colored in yellow, the αB helix is in red, and the rest is in cyan. cGMP is colored by atom type with white and black carbons. The secondary-structure elements and both termini are labeled. In the right panel, a zoomed-in view shows the region around Arg177 (corresponding to Arg192 in PKG-1β). Arg177 not only binds the cyclic phosphate of cGMP but also stabilizes the PBC by interacting with Leu139 (corresponding to Leu154 in PKG-1β) at strand β5 and Gly167 (corresponding to Gly181 in PKG-1β) at the base of PBC. (B) Direct fluorescence-polarization assay using 8-Fluo-cGMP. PKG-1α amino acids 79–212 and p.Arg177Gln PKG-1α were cloned into pQTEV, and both protein fragments were purified from E. coli Δcya TP2000, which lacks adenylyl cyclase activity. The proteins were purified as described previously. The direct fluorescence-polarization assay was performed according to the procedure described by Moll. (C) Kinase activities of wild-type (“WT”) and altered (“ALT”) proteins, as well as of a 1:1 mixture of wild-type and altered peptides, were assayed with 35 ng protein in reactions containing 8 μg kemptide. Flag-tagged wild-type and altered PKG-1α proteins were purified from transfected HEK293T cells with the use of anti-Flag M2 agarose (Sigma-Aldrich). Reactions were performed for 5 min at 30°C in 40 mM HEPES (pH 7.0), 8 μg kemptide (Sigma-Aldrich), 10 mM MgCl2, 60 μM ATP, 0.6 μCi 32P-γ-ATP, and variable amounts of cGMP. Reactions were stopped by spotting on P81 phosphocellulose paper, and activity was measured by liquid scintillation counting. The results show that p.Arg177Gln led to constitutive activation of kinase when assayed alone and increased activity of the kinase when purified with the wild-type protein. Assays were performed in triplicate. (D) Fibroblasts explanted from individuals TAA216 III:10 and III:21 and age-matched control fibroblasts were exposed to variable doses of 8-Br-cGMP. The methods of fibroblast cell culture and 8-Br-cGMP treatment followed the procedures previously described. RLC phosphorylation was lower in the mutant fibroblasts when exposed to 8-Br-cGMP than in control fibroblasts (left panel). On the x axis, the percentage of pRLC is based on the pRLC level in each sample over the pRLC level averaged from the two control fibroblast lines in the absence of 8-Br-cGMP (right panel). The concentration unit of 8-Br-cGMP is mM, and assays were performed in triplicate.

Figure 3

Aortic Pathology from Individuals TAA216 III:10 and TAA292 II:4, with the PRKG1 Mutation Resulting in p.Arg177Gln, and an Unaffected Control Compared with the control aorta, aortas from cases showed medial degeneration upon Movat staining, as shown by increased proteoglycan deposition (blue), elastic-fiber fragmentation and loss (black), and decreased cells (red). Immunostaining for Von Willebrand factor (vWF, marker of endothelial cells; brown) showed increased vascularity (arrows) in the medial layer of aortas from individuals with the PRKG1 mutation. In the control aortas, only the vasa vasorum in the adventitial layer or at the boundary (arrowhead) of the medial and adventitial layers stained with vWF. Note that the aortic specimen from TAA292 II:4 is from a dissected segment of the aorta and the images only show the medial layer of aorta.