A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G.A (IVS25+1G.A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor Alexandre Persu 1,2 *, Michel Lambert 3 , Jaap Deinum 4 , Marta Cossu 5,6 , Nathalie de Visscher 3 , Leonid Irenge 7 , Jero ˆ me Ambroise 7 , Jean-Marc Minon 8 , Andrew B. Nesterovitch 9 , Alexander Churbanov 10 , Isolda A. Popova 11 , Sergei M. Danilov 12,13 , A. H. Jan Danser 14 , Jean-Luc Gala 7 1 Pole of Cardiovascular Research, Institut de Recherche Expe ´ rimentale et Clinique, Universite ´ Catholique de Louvain, Brussels, Belgium, 2 Division of Cardiology, Cliniques Universitaires Saint-Luc, Universite ´ Catholique de Louvain, Brussels, Belgium, 3 Division of Internal Medicine, Cliniques Universitaires Saint-Luc, Universite ´ catholique de Louvain, Brussels, Belgium, 4 Department of Internal Medicine, Radboud University Medical Center, Nijmegen, The Netherlands, 5 Department of Rheumatology, Radboud University Medical Center, Nijmegen, The Netherlands, 6 Department of Rheumatology, University Medical Center, Utrecht, The Netherlands, 7 Center of Applied Molecular Technologies, Institut de Recherche Expe ´rimentale et Clinique, Universite ´ catholique de Louvain, Brussels, Belgium, 8 Department of Laboratory Medicine, Transfusion and Thrombosis-Haemostasis Unit, CHR de la Citadelle, Lie ` ge, Belgium, 9 Department of Dermatology, Rush University, Chicago, Illinois, United States of America, 10 Biology Department, New Mexico State University, Las Cruces, New Mexico, United States of America, 11 Institute for Chemistry of Life, Northwestern University, Evanston, Illinois, United States of America, 12 Institute for Personalized Respiratory Medicine, University of Illinois at Chicago, Chicago, Illinois, United States of America, 13 Department of Anesthesiology, University of Illinois at Chicago, Chicago, Illinois, United States of America, 14 Division of Pharmacology and Vascular Medicine, Department of Internal Medicine, Erasmus University, Rotterdam, The Netherlands Abstract Background: Angiotensin-converting enzyme (ACE) (EC 4.15.1) metabolizes many biologically active peptides and plays a key role in blood pressure regulation and vascular remodeling. Elevated ACE levels are associated with different cardiovascular and respiratory diseases. Methods and Results: Two Belgian families with a 8-16-fold increase in blood ACE level were incidentally identified. A novel heterozygous splice site mutation of intron 25 - IVS25+1G.A (c.3691+1G.A) - cosegregating with elevated plasma ACE was identified in both pedigrees. Messenger RNA analysis revealed that the mutation led to the retention of intron 25 and Premature Termination Codon generation. Subjects harboring the mutation were mostly normotensive, had no left ventricular hypertrophy or cardiovascular disease. The levels of renin-angiotensin-aldosterone system components in the mutated cases and wild-type controls were similar, both at baseline and after 50 mg captopril. Compared with non-affected members, quantification of ACE surface expression and shedding using flow cytometry assay of dendritic cells derived from peripheral blood monocytes of affected members, demonstrated a 50% decrease and 3-fold increase, respectively. Together with a dramatic increase in circulating ACE levels, these findings argue in favor of deletion of transmembrane anchor, leading to direct secretion of ACE out of cells. Conclusions: We describe a novel mutation of the ACE gene associated with a major familial elevation of circulating ACE, without evidence of activation of the renin-angiotensin system, target organ damage or cardiovascular complications. These data are consistent with the hypothesis that membrane-bound ACE, rather than circulating ACE, is responsible for Angiotensin II generation and its cardiovascular consequences. Citation: Persu A, Lambert M, Deinum J, Cossu M, de Visscher N, et al. (2013) A Novel Splice-Site Mutation in Angiotensin I-Converting Enzyme (ACE) Gene, c.3691+1G.A (IVS25+1G.A), Causes a Dramatic Increase in Circulating ACE through Deletion of the Transmembrane Anchor. PLoS ONE 8(4): e59537. doi:10.1371/ journal.pone.0059537 Editor: Michael Bader, Max-Delbru ¨ ck Center for Molecular Medicine (MDC), Germany Received January 7, 2013; Accepted February 15, 2013; Published April 1, 2013 Copyright: ß 2013 Persu et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: These studies were partially supported by the Fonds de la Recherche Scientifique Me ´dicale (F.R.S.M.) convention number 3.4.587.08 F (to A.P.). The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. No additional external funding received for this study. Competing Interests: The authors have declared that no competing interests exist. * E-mail: alexandre.persu@uclouvain.be Introduction Angiotensin I-converting enzyme (ACE) is a Zn 2+ carboxydi- peptidase enzyme which transforms angiotensin I in angiotensin II, a potent vasoconstrictor, as well as other biologically active peptides. Through this metabolic process, ACE plays a key role in the regulation of blood pressure and also in the development of vascular pathology and remodeling [1–3]. In man, the gene coding ACE is mapped on chromosome 17 (17q23). It is 21 kilobases long and comprises 26 exons and 25 introns [4]. PLOS ONE | www.plosone.org 1 April 2013 | Volume 8 | Issue 4 | e59537