The innate immune system elicits an inflammatory process which is

believed to be involved in the development of vascular damage in hypertension. The NOD2

gene (CARD15) is involved in the control of the innate immune system. The aim of this study

was to assess the occurrence of the 3020insC mutation in the NOD2 gene in relation to

vascular damage in hypertensives

Szumczyk-Muszytowska, Grażyna

Manitius, Jacek

Tretyn, Andrzej

Jarkiewicz-Tretyn, Joanna

Muszytowski, Marek

English

Repository of Nicolaus Copernicus University

371www.nt.viamedica.plPRACA ORYGINALNAAdres do korespondencji: prof. dr hab. n. med. Jacek ManitiusDepartment of Nephrology, Hypertension, and Internal MedicineUniversity Hospital no. 1Skłodowskiej-Curie 9, 85–094 BydgoszczTel/fax: (+48) 52 585 4030e-mail: nerka@nerka.cpro.plCopyright © 2009 Via Medica, ISSN 1428–5851Grażyna Szumczyk-Muszytowska1, Jacek Manitius2, Andrzej Tretyn3,Joanna Jarkiewicz-Tretyn4, Marek Muszytowski51Cardiology Department, Outpatient Clinic Lecznice Citomed, Toruń2Department of Nephrology, Hypertension, and Internal Disease, Collegium Medicum, Nicolaus Copernicus University, Bydgoszcz3Department of Biotechnology, Institute of General and Molecular Biology, Nicolaus Copernicus University, Toruń4Cancer Genetics Laboratory, Toruń5Department of Nephrology and Internal Medicine, Ludwik Rydygier Hospital, ToruńThe association between 3020INSC mutation inNOD2 gene and arterial stiffness in hypertensivesSummaryBackground The innate immune system elicits an inflam-matory process which is believed to be involved in the de-velopment of vascular damage in hypertension.The NOD2 gene (CARD15) is involved in the control ofthe innate immune system. The aim of this study was toassess the occurrence of the 3020insC mutation in theNOD2 gene in relation to vascular damage inhypertensives.Materials and methods In 466 hypertensives, genotypingfor 3020insC mutation, cholesterol, serum creatinine, andechocardiography were carried out. Systolic (SBP) anddiastolic (DBP) blood pressure and pulse pressure (PP)were taken at 2–3 month intervals for one year and treatedaccording to ESC/ESH guidelines.Results The 3020insC mutation was found in 7.08% ofhypertensives and was associated with earlier onset of hy-pertension 48.2 ± 12.1 vs. 42.8 ± 10.9, p < 0.01 (age ofpts). After one year of treatment, patients with the muta-tion had significantly higher SBP (138.48 ± 18.35 vs.131.74 ± 14.97, p < 0.04) and PP (57.27 ± 14.04 vs. 52.70± 12.96, p < 0.04) than patients without the mutation,whereas the number of drugs was the same in both groups.Conclusions The results suggest that the 3020insC muta-tion is associated with earlier onset of hypertension andincreased arterial stiffnesskey words: arterial stiffness, innate immunity, NOD2mutationArterial Hypertension 2009, vol. 13, no 6, pages 371–375.IntroductionThe structural and functional changes of vascularvessels characterize arterial hypertension. Remodel-ling of vessels results in thickening of the artery wall,increased pulse pressure, and atherosclerosis [1, 2].Many factors are believed to be involved in the deve-lopment of vascular damage in hypertension, andrecently, innate and adaptive immune mechanismsare recognized as a cause of atherosclerosis and re-modelling of vessels [3–6]. The innate immune sys-tem elicits inflammatory response to various patho-gens via various receptors [7–9]. The NOD2 gene(also known as CARD15), discovered in 2001, is be-lieved to be involved in the control of the innate im-mune system activating nuclear factor kB and cau-sing the inflammatory reaction and apoptosis [10–16].Apoptosis and inflammation play crucial roles inthe process of the of remodelling of arteries in hy-pertension [17]. Several mutations have been iden-tified in the NOD2 gene, which are linked to inflam-mation and apoptosis. One of them, frameshift mu-tation 3020insC (1007fs), occurs in the European po-pulation relatively often and has been proven to beassociated with the inflammatory process [18–20]. In2005 the NOD2 gene was discovered in human en-dothelium [21, 22]. Recently, it was shown that alle-lic variants of NOD2 were associated with preec-lampsia syndrome with susceptibility to severe hy-pertension [23]. In the face of the relation betweenNOD2 mutation and blood pressure control in pre-eclampsia, it appears very likely that mutation in theNOD2 gene is associated with vascular vessel func-tion and structure. We tested only the 1007fs muta-tion in the NOD2 gene because our previous studyshowed that frequency NOD2 gene variants(1007fs,G908R and R702W and P268S polymor-nadciśnienie tętnicze rok 2009, tom 13, nr 6372 www.nt.viamedica.plphism) in the Polish population of patients with rec-tal cancer was found in the 1007fs mutation and the-re is an association between frequency of cancer andage [23–25].The aim of the study was to see whether the oc-currence of the 3020insC mutation in the NOD2gene is associated with the clinical manifestation ofcardiovascular damage in hypertensives.Materials and methodsThe study included 466 hypertensives treated inthe Cardiology Outpatient Clinic in Lecznice Cito-med in Toruń. The study was in accordance withthe Declaration of Helsinki and was approved by theCommission of Bioethics at Collegium Medicum ofNicolaus Copernicus University in Toruń. Informedconsent was obtained from all subjects.The enrolment examination included medical hi-story, current therapy, previously diagnosed cardio-vascular diseases, physical examination, and BMI.Fasting blood was sampled in tubes with EDTA forgenotyping, total cholesterol, and creatinine and sto-red at –80°C. eGFR was estimated according toMDRD formula. Total cholesterol was determinedenzymatically, and creatinine according to Jaffe’smethod. Left ventricular mass (LVM) was estimatedusing a Vivid 3 GE echocardiograph with a 2.5 MHzultrasound probe according to Penn convention andDevereux formula and expressed as left ventricularmass index (LVMI) calculated as LVM/body surfacearea [16].Blood pressure measurements (BP) were taken at2–3 month intervals over one year in accordance withESC/ESH 2003 guidelines. The mean of two conse-cutive measurements were reported. The target blo-od pressure values were < 140 mm Hg systolic (SBP)and < 90 mm Hg diastolic (DBP) for nondiabetics.Pulse pressure (PP) was calculated according to theequation PP = SDP-DBP. All patients were subjec-ted to antihypertensive therapy according to ESC//ESH guidelines.GenotypingThe 3020insC mutation was detected using PCRreaction [4]. For the PCR reaction, four different pri-mers were used: “3020sens”, “3020anty”, “3020wild”,and “3020mut”, forming three different pairs. The“3020wild” and “3020mut” primers have a wild se-quence and a 1007fs mutation equivalent, respecti-vely, on their 3’ ends. The thermal conditions of thePCR reaction and the concentrations of the substra-tes were adjusted so the highest specificity could beobtained. If the 3020insC mutation appeared, the“3020mut” primer bound to the DNA on the tem-plate while the “3020insC” primer did not. After-wards, the product of the reaction was subjected toelectrophoresis. An exemplary photogram of electro-phoresis with marked 3020insC mutation is shownin figure 1.Statistical analysisStatistical analysis was performed with SPSS 12.0PL software and Microsoft Excel calculation sheet.The patients included in the study were divided intotwo groups: one group consisted of patients withidentified 3020insC mutation in NOD2 (NOD2(+)),and the second group comprised patients with nega-tive results (NOD2(–). For each variable, mean va-lue and standard deviation were calculated using t-Student or Mann-Whitney test. Shapiro-Wilk testwas used to estimate normality of distribution.p < 0.05 was considered significant. The ANCOVAtest was applied to compare mean values betweenthe groups. Linear regression analysis was used toevaluate the correlation between variables.Results3020insC mutation in the NOD2/CARD15 genewas found in 33 hypertensives (7.08%) (16 femalesand 17 males). The NOD2(+) was associated withearlier onset of hypertension (tab. I), and higher sy-stolic blood pressure and pulse pressure in the face ofthe same number of taken antihypertensive drugs(tab. II, fig. 2). LVMI and eGFR mean values didFigure 1. Electrophoretic separation of PCR reaction product.The 3020insC mutation is marked by arrows. Single arrow —heterozygote, double arrow — homozygote, K1 — control test:heterozygote, K2 — control test: homozygoteGrażyna Szumczyk-Muszytowska et al. NOD2 gene mutation and vascular stiffness in hypertensives373www.nt.viamedica.plnot differ significantly in respect to the appearance ofthe 3020insC mutation (tab. I). In multivariate ana-lysis gender, SBP and PP were all predictive of LVMI(p = 0.00; p = 0.03; p = 0.04, respectively), whileGFR was influenced only by gender (p = 0.02).None of these parameters appeared to be influencedby the presence of the 3020insC mutation.DiscussionThis cross-sectional study suggests that the3020insC mutation in the NOD2/CARD15 gene pre-disposes to earlier onset of hypertension and worseblood pressure control. It is known that the frequen-cy of these mutations is related to some inflammato-Table I. Clinical characteristics of patients in NOD2(–) and NOD2(+) groups (mean ± SD)Parameter NOD2(–) NOD2(+) pn = 433  n = 33Age (years) 59.4 ± 9.5 56.4 ± 10.4 NSOnset of hypertension (patient age)  48.2 ± 12.1  42.8 ± 10.9  0.01BMI [kg/m2] 29.5 ± 4.3 30.00 ± 4.8 NSTotal cholesterol [mg/dl] 223.1 ± 47.6 216.9 ± 48.1 NSGFR [ml/min/1.73 m2] 86.9 ± 23.5 94.1 ± 27.1 NSLVMI [g/m2] 117.3 ± 24.5 124.2 ± 28.3 NSFigure 2. Number of antihypertensive drugs given to NOD2(+) and NOD2(–) patientsTable II. Comparisons of systolic and diastolic blood pressure and pulse pressure among NOD2(–) and NOD2(+)(mean ± SD) patientsBlood pressure [mm Hg] NOD2(–) NOD2(+) pSBP 1 151.00 ± 22.12 149.09 ± 22.69 NSDBP 1 90.25 ± 44.78 91.36 ± 15.68 NSPP 1 62.39 ± 14.90 57.73 ± 13.41 NSSBP 2 131.74 ± 14.97 138.48 ± 18.35 0.04DBP 2 78.90 ± 7.40 81.52 ± 9.40 NSPP 2 52.70 ± 12.96 57.27 ± 14.04 0.04SBP — systolic blood pressure, DBP — diastolic blood pressure, PP — pulse pressure, 1 — at the beginning of observation, 2 — after one year of observationnadciśnienie tętnicze rok 2009, tom 13, nr 6374 www.nt.viamedica.plry diseases and cancer, but there is no available stu-dy concerning the relation between the mutationand the clinical course of hypertension [25–27].The mechanism/s underlying the association be-tween NOD2 gene mutation and the pathogenesisof hypertension is/are still matter of speculation.NOD2 has been identified as playing a role in inna-te immune response and consequently initiates theinflammatory process leading to activation of mo-nocytes and macrophages. Activated cells bind toendothelium to initiate the inflammatory process invascular vessels, which is associated with reducedartery compliance [28, 29]. Patients with the3020insC mutation are characterized with highersystolic blood pressure and pulse pressure than sub-jects without mutation. This suggests that the ap-pearance of the mutation could have been associa-ted with more advanced inflammatory processes inthe vessels, causing higher vascular stiffness. Ourstudy also revealed that hypertension was diagno-sed earlier in subjects with mutation than in sub-jects without. In several studies, the role of geneticfactors in the early development of hypertension hasbeen shown [30, 31]. Our findings are in agreementwith a study suggesting a genetic background in thepathogenesis of hypertension. However, it is diffi-cult to exclude the role of environmental fator/swhich may be more relevant than the NOD2 muta-tion. Hence question arises whether mutation3020insC predisposes to the development of hyper-tension or not. Taking into account the results ofthe previous study on preeclampsia and the role ofinnate immunity in vascular damage, it appearsvery likely [3–6, 9, 23]. Unfortunately, our studyhas not solved this problem. Moreover, our studyhas some limitations. Firstly, the sample of subjectswith NOD2 mutation was too small to drawn firmconclusions. Secondly, we do not know the frequ-ency of the 3020insC mutation in the populationwithout hypertension, and in the face of this fact, itis difficult to prove whether this mutation plays a rolein the pathogenesis of hypertension.ConclusionsThe 3020insC mutation in the NOD2 gene occursamong hypertensives. It suggests also that the3020insC mutation may play a role in arterial remo-delling in hypertensive patients.References1. Barenbrock M., Hausberg M., Kosch M. et al. 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The association between 3020INSC mutation in NOD2 gene and arterial stiffness in hypertensives.

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The association between 3020INSC mutation in NOD2 gene and arterial stiffness in hypertensives.

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