Short Communication

Cardiology 2011;119:5456 DOI: 10.1159/000329919

Received: February 18, 2011 Accepted after revision: June 9, 2011 Published online: August 12, 2011

Atherosclerosis and PTPN22: A Study in Coronary Artery Disease

P. Saccucci a M. Banci b E. Cozzoli a A. Neri a A. Magrini a E. Bottini a F. Gloria-Bottini a
Department of Biopathology and Imaging Diagnostics, University of Rome Tor Vergata, and b Department of Cardiology, Valmontone Hospital, Rome, Italy
a

Key Words PTPN22 Coronary artery disease Atherosclerosis

Abstract Objectives: Recently, it has been shown that PTPN22 genetic polymorphism is associated with phenotypes related to the risk of atherosclerosis. In the present note, we have searched for a possible association of PTPN22 polymorphism with coronary artery disease (CAD). Methods: One hundred and thirty-four non-diabetic subjects admitted to hospital for CAD and 174 healthy subjects (blood donors) were studied. PTPN22 genotypes were determined by DNA analysis. Statistical analyses were performed by SPSS programs. Results: In CAD patients, the proportion of carriers of the *T allele of PTPN22 is significantly higher compared to healthy controls (OR 2.66; 95% CI 1.076.72). Conclusions: The present observation confirms the association of PTPN22 phenotype with atherosclerosis and suggests a role of immune mechanism in the pathogenesis of CAD.
Copyright 2011 S. Karger AG, Basel

Lyp is a protein-tyrosine-phosphatase codified by PTPN22 and involved in the regulation of T-cell receptor signaling. The gene shows a single nucleotide polymorphism C/T at +1858 resulting in the W620 variant that is associated with autoimmune diseases. The variant is a gain of function of the enzyme that more strongly inhibits T-cell receptor-mediated signals, and it has been suggested that the increased susceptibility to autoimmune disorders is due to failure to delete autoreactive T cells during intrathymic selection [2, 3]. In the present note, we have searched for a possible association of PTPN22 polymorphism with coronary artery disease (CAD). Since diabetes is strongly associated with CAD and may overshadow the effects of other factors, the study was carried out in patients without diabetes.
Subjects and Methods
One hundred and thirty-four non-diabetic subjects admitted to hospital for CAD and 174 healthy subjects (blood donors) were studied. Informed written consent was obtained from all subjects to participate in the study, approved by the ethical committee of the hospital. The PTPN22 polymorphism has two alleles, *C1858 (encoding the R620 variant, here simply called *C) and *T1858 (encoding the W620 variant, here simply called *T), and has three genotypes, *C/*C,*C/*T and *T/*T. The *T/*T genotype is very rare. Patients were genotyped as previously described [2]. A DNA fragment was

Introduction

Recently, it has been shown that PTPN22 genetic polymorphism is associated with phenotypes related to the risk of atherosclerosis [1].
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Fulvia Gloria-Bottini, MD Division of Biopathology of Human Population and Environmental Pathology Department of Biopathology and Imaging Diagnostics, University of Rome Tor Vergata, School of Medicine, Via Montpellier 1, IT00133 Rome (Italy) Tel. +39 06 7259 6030, E-Mail gloria@med.uniroma2.it

Table 1. Clinical data in non-diabetic subjects admitted to the Valmontone hospital for CAD

Results

Parameter Infarction Major coronary lesions Bypass Angioplastic surgery Females High total cholesterol (>200 mg/dl) High triglycerides (>150 mg/dl) Hypertension Smoking habit Age, years Body mass index

Patients 38.0 56.0 29.1 29.8 47.8 65.7 40.3 81.3 43.3 64.981.09 27.580.44

Data are given as percentages or the mean 8 SE.

Table 2. PTPN22 distribution in non-diabetic subjects admitted for CAD and in controls

Carriers of the *T allele, % CAD Controls 12.6 5.2

Total 134 174

2 = 4.601; degrees of freedom = 1; p = 0.032; OR 2.66; 95% CI 1.086.72.

amplified by polymerase chain reaction in a 25-l total-volume reaction, containing 100 ng of genomic DNA, 2.5 nM MgCl2, 1 ! buffer Gold (Applied Biosystems, Foster City, Calif., USA), 10 pmol of each primer, 0.2 mM deoxyribonucleotide triphosphate and 0.5 units of AmpliTaq Gold (Applied Biosystems). Thirty cycles (30 s at 95 C, 30 s at 60 C and 30 s at 72 C) were performed with DNA Thermal Cycler (Perkin Elmer). Sense and antisense primers for the polymerase chain reaction were 5-TCA CCA GCT TCC TCA ACC ACA-3 and 5-GTA ATT GTT GCT TCA ACG GAA TTT-3, respectively. The C]T transition at codon 620 (NCBI refSNP ID:rs2476601) creates a restriction site for XcmI in the *T allele. The polymorphism was identified by XcmI restriction endonuclear (NEB, Beverly, Mass., USA) digestion of the polymerase chain reaction-amplified fragment. Each digestion was resolved on 3% agarose gel. After electrophoresis, the gel was stained with ethidium bromide, and the fragment was visualized by UV. The 2 test of independence and odds ratio analyses were performed by the SPSS programs [4]. A three-way contingency table analysis to detect interaction was carried out by a log linear model according to Sokal and Rohlf [5]. Most of the patients included in the present paper have been previously studied for association between CAD and acid phosphatase locus 1 (ACP1) [6].

Table1 reports clinical data of the sample study. Table2 shows PTPN22 genotype distribution in non-diabetic patients admitted to hospital for CAD and in controls. In CAD patients, the proportion of carriers of the *T allele is significantly higher compared to healthy controls (OR 2.66; 95% CI 1.086.72; power test 0.93). We have also studied 103 CAD patients with diabetes and 155 patients admitted to hospital for cardiovascular diseases without CAD. Among CAD diabetics, 1.9% were carriers of the *T allele, while among non-CAD patients, 5.8% were carriers of the *T allele. Therefore, the association with PTPN22 is present in non-diabetic CAD patients only. We have searched for possible effects of clinical variables and of some genetic factors on the association between PTPN22 and CAD. Obesity, dyslipidemia, hypertension, smoking habit, age and sex did not show a statistically significant effect on the association between PTPN22 and CAD. ACP1 and adenosine deaminase locus 1 genetic polymorphisms previously shown to be associated with CAD [68] have been considered. A statistically significant effect has been observed for ACP1 only. In CAD patients, the proportion of *T allele carriers is significantly higher in low activity *A/*A and *A/*B genotypes of ACP1 (19.35 vs. 5.75% in controls; p = 0.021; OR 3.036; 95% CI 1.19113.731) but not significantly different from controls in medium to high activity *B/*B, *A/*C and *B/*C ACP1 genotypes (7.00 vs. 4.60% in controls; p = 0.753). Possible effects of the presence of the *T allele of PTPN22 on ejection fraction, previous history of infarction and presence of arrhythmia have also been considered. No statistically significant effect was observed.

Discussion

The present observation confirms the association of PTPN22 phenotype with atherosclerosis and suggests a role of immune mechanisms in the pathogenesis of CAD. PTPN22 polymorphism has been associated with susceptibility to chronic autoimmune inflammatory diseases associated with accelerated atherosclerosis [9]. However, PTPN22 polymorphism has not been found to be associated with an increased incidence of cardiovascular events or with endothelial dysfunction in a patient with rheumatoid arthritis, the prototype of inflammatory disease associated with increased cardiovascular burden
Cardiology 2011;119:5456

CAD and PTPN22

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[10]. Therefore, it is possible that mechanisms leading to atherosclerosis may differ from one condition to another. The lack of association between CAD and PTPN22 in diabetic subjects with CAD points to differences between non-diabetic and diabetic subjects in the pathogenetic mechanisms leading to CAD. ACP1 or cLMWPTP (cytosolic low molecular weight protein tyrosine phosphatase) is a polymorphic enzyme showing strong quantitative variations of total enzymatic activity among genotypes. In the Caucasian population, there are six genotypes attributed to the presence of three codominant alleles ACP1*A, ACP1*B and ACP1*C, at an autosomal locus. The enzyme is composed of two isoforms, F and S, which have different molecular and catalytic properties [11]. The three ACP1 alleles have been sequenced and found to be based on three single nucleotide polymorphisms (SNPs) that affect both the total enzymatic activity and the ratio between F and S isoforms. A fixed combination of these SNPs defines the common alleles *A,*B and *C. In the *A allele, amino acid 105 is an arginine residue (codon CGA), while it is a glutamine (codon CAA) in *B and *C. The two other SNPs do not change the encoded amino acid residues but strongly affect the alternative mRNA splicing and, as a result, the ratio between the two iso-

forms: F:S is 2:1 in *A, 4:1 in *B and 1:4 in *C. The total enzymatic activity measured with p-nitrophenylphosphate as a substrate is in the order of *A/*A ! *A/*B ! (*B/*B,*A/*C) ! *B/*C ! *C/*C [11]. Two separate functions have been attributed to ACP1: phosphoprotein tyrosine phosphatase and flavin mononucleotide phosphatase [11]. ACP1 dephosphorylates a negative phosphorylation site in the ZAP-70 tyrosine kinase in T cells [12]. This event leads to increased activation of this kinase and enhanced signaling from T-cell antigen receptor. Our data show an interaction between ACP1 and PTPN22 resulting in an increased susceptibility to CAD in the presence of the *T allele of PTPN22 and low activity ACP1 genotypes *A/*A and *A/*B. Failure to delete autoreactive T cells during intrathymic selection due to the W620 variant of PTPN22 and decreased activity of ZAP-70 tyrosine kinase in T cells due to low ACP1 activity could cooperate to increase the risk of atherosclerosis. Several studies point to an important role of immune mechanisms in the pathogenesis of atherosclerosis [13 15]; therefore, the cooperation of two genetic systems involved in T-cell activity appears biologically plausible. The possibility of similar interaction in other immune disorders is worthy of further investigations.

References
1 Pertovaara M, Raitala A, Juonala M, Kahonen M, Lehtimaki T, Viikari JSA, Raitaka ri OT, Hurme M: Autoimmunity and atherosclerosis: functional polymorphism of PTPN22 is associated with phenotypes related to the risk of atherosclerosis. The Cardiovascular Risk in Young Finns Study. Clin Exp Immunol 2006;147:265269. 2 Bottini N, Musumeci L, Alonso A, Rahmouni S, Nika K, Rostamkhani M, MacMurray J, Meloni GF, Lucarelli P, Pellecchia M, Eisenbarth GS, Comings D, Mustelin T: A functional variant of lymphoid tyrosine phosphatase is associated with type I diabetes. Nat Genet 2004;36:337338. 3 Vang T, Congia M, Macis MD, Musumeci L, Orr V, Zavattari P, Nika K, Tautz L, Taskn K, Cucca F, Mustelin T, Bottini N: Autoimmune-associated lymphoid tyrosine phosphatase is a gain-of-function variant. Nat Genet 2005;37:13171319. 4 Statistical Package for the Social Sciences, SPSS/PC+ Version 5.0. Chicago, SPSS, 1992. 5 Sokal J, Rohlf F: Biometry. New York, Freeman, 1981. 6 Banci M, Saccucci P, DAnnibale F, Dofcaci A, Trionfera G, Magrini A, Bottini N, Bottini E, Gloria-Bottini F: ACP1 genetic polymorphism and coronary artery disease: an association study. Cardiology 2009;113:236242. Safranow K, Rzeuski R, Binczak-Kuleta A, Czyzycka E, Skowronek J, Jakubowska K, Wojtarowicz A, Loniewska B, Ciechanowicz A, Kornacewicz-Jach Z, Chlubek D: ADA*2 allele of the adenosine deaminase gene may protect against coronary artery disease. Cardiology 2007;108:275281. Banci M, Saccucci P, DAnnibale F, Dofcaci A, Trionfera G, Magrini A, Bottini N, Bottini E, Gloria-Bottini F: Adenosine deaminase genetic polymorphism and coronary artery disease. Cardiology 2009; 112:7475. Orozco G, Snchez E, Gonzlez-Gay MA, Lpez-Nevot MA, Torres B, Cliz R, OrtegoCenteno N, Jimnez-Alonso J, Pascual-Salcedo D, Balsa A, de Pablo R, Nuez-Roldan A, Gonzlez-Escribano MF, Martn J: Association of a functional single-nucleotide polymorphism of PTPN22, encoding lymphoid protein phosphatase, with rheumatoid arthritis and systemic lupus erythematosus. Arthritis Rheum 2005;52:219224. Palomino-Morales R, Gonzalez-Juanatey C, Vazquez-Rodriguez TR, Rodriguez L, Miranda-Filloy JA, Pascual-Salcedo D, Balsa A, Fernandez-Gutierrez B, Llorca J, Martin J, Gonzalez-Gay MA: Lack of association of PTPN22, STAT4 and TRAF1/C5 gene polymorphisms with cardiovascular risk in rheumatoid arthritis. Clin Exp Rheumatol 2010; 28:695701. Bottini N, Bottini E, Gloria-Bottini F, Mustelin T: Low-molecular-weight protein tyrosine phosphatase and human disease: in search of biochemical mechanisms. Arch Immunol Ther Exp 2002;50:95104. Bottini N, Stefanini L, Williams S, Alonso A, Jascur T, Abraham RT, Couture C, Mustelin T: Activation of ZAP-70 through specific dephosphorylation at the inhibitory Tyr-292 by the low molecular weight phosphotyrosine phosphatase (LMPTP). J Biol Chem 2002; 277:2422024224. Hansson GK: Inflammation and immune response in atherosclerosis. Curr Atheroscler Rep 1999;1:150155. Ostos MA, Recalde D, Zakin MM, Scott-Algara D: Implication of natural killer T cells in atherosclerosis development during a LPS-induced chronic inflammation. FEBS Lett 2002;519:2329. Frostegrd J: Atherosclerosis in patients with autoimmune disorders. Arterioscler Thromb Vasc Biol 2005; 25:17761785.

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Saccucci /Banci /Cozzoli /Neri /Magrini / Bottini /Gloria-Bottini

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