Novel and Recurrent Gain-of-Function Mutation in PRKG1 Causes Thoracic Aortic Aneurysms and Acute Aortic Dissections. D. Guo1, E. Regalado1, D. E. Casteel2, R. L. Santos-Cortez3, L. Gong1, J. J. Kim4, S. Dyack5, S. G. Horne6, G. Chang1, G. Jondeau7, C. Boileau7, J. S. Collelli8, Z. Li9, S. M. Leal3, J. Shendure10, M. J. Rieder10, M. J. Bamshad10, D. A. Nickerson10, C. Kim11, D. M. Milewicz1,12, GenTAC Registry Consortium; NHLBI-Go Exome Sequencing Project 1) Dept Internal Med, Univ Texas/Houston Med Sch, Houston, TX; 2) Dept Medicine and Cancer Center, Univ California at San Diego, San Diego, CA; 3) Dept Molecular and Human Genetics, Baylor College of Medicine, Houston, TX; 4) Dept Pharmacology, Baylor College of Medicine, Houston, TX; 5) Dept Pediatrics, Dalhousie University, Halifax, Nova Scotia, Canada; 6) Dept Medicine, Dalhousie University, Halifax, Nova Scotia, Canada; 7) Hopital Bichat, Paris, France; 8) Michael E. DeBakey Dept Surgery, Baylor College of Medicine, Houston, TX; 9) Dept Internal Med, Univ Kentucky, Lexington, KY; 10) Dept Genome Sciences, Univ Washington, Seattle, WA; 11) Dept Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX; 12) Texas Heart Institute at St. Lukes Episcopal Hospital, Houston, TX.

   Ascending thoracic aortic aneurysms can lead to life-threatening acute aortic dissections (TAAD). Previous studies have identified gene mutations that lead to decreased contraction of vascular smooth muscle cells (SMCs) causing familial TAAD (FTAAD), such as loss-of-function mutations in myosin light chain kinase (MLCK), which are predicted to decrease phosphorylation of the regulatory light chain (pRLC) and decrease SMC contraction. To identify further genes that cause FTAAD, exome sequencing of distant relatives from large families, followed by Sanger sequencing of unrelated probands with FTAAD, identified the same rare variant, PRKG1 c.530G>A (p.Arg177Gln), that segregated with TAAD in four families with a combined two-point LOD score of 7.88. Majority of affected individuals had aortic dissections (63%) at relatively young ages (mean 31 years, range 17 - 51 years) and TAAD was fully penetrant in both men and women. PRKG1 encodes a type I cGMP-dependent protein kinase (PKG I) that is activated upon binding of cGMP. PKG I exists as two splice variants ( and ), each with a unique set of substrates. PKG I phosphorylates regulatory myosin-binding subunit leading to de-phosphorylation of RLC and SMC relaxation. Protein structure analysis showed that Arg-177 is located at the cGMP binding domain A and critical for cGMP binding. Fluorescence polarization assay demonstrated that p.Arg177Gln abolished cGMP binding activity. However, kinase activity assay showed the mutant PKG I was highly active even without cGMP, while WT PKG I required cGMP for activation. These results suggest that the p.Arg177Gln mutation structurally perturbed PKG I and abolished allosteric inhibition of kinase activity resulting in a kinase whose activity is no longer modulated by cGMP. Immunoblot assays also showed reduced levels of pRLC in mutant fibroblasts from patients when exposed to low doses of 8-Fluo-cGMP compared with control fibroblasts. Thus, we have identified loss-of-function mutations in MLCK controlling SMC contraction and gain-of-function mutations in the kinase controlling relaxation. Finally, these data illustrate the complexity of associating rare variants with complex, adult onset diseases. Association studies based on a burden test of rare variants in PRKG1 with TAAD would not demonstrate an association, and studies with the specific PRKG1 p.Arg177Gln variant would only show an association in a large cohort or if family-based studies were pursued.

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