Journal of Human Hypertension (2005) 19, 615–622 & 2005 Nature Publishing Group All rights reserved 0950-9240/05 $30.00 www.nature.com/jhh ORIGINAL ARTICLE Association of HLA-DRB3*0202 and serum IgG antibodies to Chlamydia pneumoniae with essential hypertension in a highly homogeneous population from Majorca (Balearic Islands, Spain) JM Zabay1, J Marco2, J Soler3, L Contu4, L Cappai4, C Carcassi4,GGo´mez2, JM Mulet3, MA Munar2 and C Viader1 1Fundacio´n Balear Transplant, Palma de Mallorca, Spain; 2Servicio de Nefrologı´a, Hospital Universitario Son Dureta, Palma de Mallorca, Spain; 3Laboratorio Inmunogen SL, Palma de Mallorca, Spain; 4Cattedra di Genetica Medica, Universita di Cagliari, Cagliari, Italy Separate studies investigating the relationship of ml in the group of individuals with sIgGa-Cp430 BU/ml essential hypertension (EH) with the HLA system and (OR (95% CI) adjusted for obesity, familial history of with Chlamydia pneumoniae (C. pneumoniae) infection EH and diabetes ¼ 2.06 (1.07–3.97), P ¼ 0.03, and ¼ 4.60 have given conflicting results. Our aim was to clarify (1.06–19.90), P ¼ 0.04, respectively). The association these relationships and determine whether the HLA between EH and sIgGa-Cp was observed in the system and C. pneumoniae infection interact with DRB3*0202( þ ) individuals, but not in the DRB3*0202(À) respect to the risk for EH. An association study (110 subgroup (OR (95% CI) ¼ 11.14 (1.92–64.54), P ¼ 0.004, essential hypertensives and 107 controls) was con- and ¼ 0.98 (0.22–4.43), P ¼ 0.64, respectively (P of the ducted in a highly homogeneous population in the Mantel–Haenszel test for homogeneity of OR ¼ 0.06)). In Balearic Island of Majorca (Spain). Molecular typing of our population, EH was positively associated with HLA- HLA-B and HLA-DRB and quantification of serum levels DRB3*0202 and with high levels of sIgGa-Cp. Moreover, of IgG antibodies to C. pneumoniae (sIgGa-Cp) were a significant interaction of DRB3*0202 on the effect determined. Student’s t-test, v2-statistics, logistic re- of sIgGa-Cp was observed, as the association of EH gression analysis, and general linear model ANOVA with these antibodies depended on the presence of were used for statistical analysis. The results showed DRB3*0202. that EH was related with HLA-DRB3*0202 in the whole Journal of Human Hypertension (2005) 19, 615–622. study population, and with levels of sIgGa-Cp463.5 BU/ doi:10.1038/sj.jhh.1001872; published online 12 May 2005 Keywords: MHC class II; Chlamydia pneumoniae; essential hypertension; genetics; infection; Majorca (Balearic Islands) Introduction been documented in linkage studies.2,3 However, studies to analyse the possible association between Essential hypertension (EH) is a complex genetic 1 EH and antigens or alleles of the HLA system, trait as blood pressure (BP) in each individual is mainly HLA-B and HLA-DR,3–8 have failed to yield due to the combined effects of variation at an consistent results. indeterminate number of BP-determining genetic Although not confirmed by other authors,9 a loci and environmental factors. One of the genetic positive association between Chlamydia pneumo- systems for which a possible relationship with EH niae (C. pneumoniae) infection and EH has recently has been considered is the HLA system and this has been reported.10–12 This observation expands the spectrum of cardiovascular diseases in which C. pneumoniae infection, through immune and Correspondence: Dr C Viader, Fundacio´n Balear Transplant, Avda inflammatory mechanisms, might play a role.13,14 Joan Miro´, 168. 07015 Palma de Mallorca, Spain. E-mail: [email protected] Based on these observations and given the para- Received 13 October 2004; revised 26 January 2005; accepted 3 mount importance of the HLA system in the March 2005; published online 12 May 2005 immune response, it is of great interest to clarify HLA class II, C. pneumoniae and essential hypertension JM Zabay et al 616 the possible relationship of EH with HLA alleles and known or suspected immunodeficiency, hyper- C. pneumoniae infection, as well as to investigate gammaglobulinaemia and connective or autoim- possible interactions between HLA alleles and mune disease. Since neither hypertensive patients C. pneumoniae infection with respect to the EH nor controls were acutely ill when their blood risk. We carried out a case–control study in which samples were collected, we did not expect to include we typed HLA-B, -DRB1, -DRB3, -DRB4 and -DRB5 a significant number of cases with acute C. pneumo- alleles and determined the serum levels of IgG niae infection. As a minimum, both parents and all antibodies to C. pneumoniae (sIgGa-Cp) in a highly four grandparents of patients and controls were homogeneous population in the Balearic Island required to have been born in Majorca. Furthermore, of Majorca (Spain). Our results showed a positive for each participant, both family names (people born association of EH with the allele HLA-DRB3*0202 in Spain officially have two family names, that of and with high levels of sIgGa-Cp, but no association their father, followed by that of their mother, and between sIgGa-Cp and EH was observed unless this women maintain these family names when they are allele was present. married) had to be characteristic of Majorca, and an effort was made to ensure that the patients and controls selected from each village had at least one of Materials and methods the most typical family names from that village. Study subjects Similar numbers of cases and controls were selected from each outpatient clinic. Cases and controls were The research protocol of this case–control study was age- and sex-matched as far as possible. approved by the Institutional Review Committees of the ‘Hospital Universitario Son Dureta’ and the ‘Gerencia Atencio´n Primaria de Mallorca (IBSA- Genetic analysis LUT)’. All participants (110 cases and 107 controls) gave their written, informed consent. Generic typing of HLA-DRB1, -DRB3, -DRB4, -DRB5 Participants were identified from medical records and -B loci, and high-resolution subtyping of HLA- at seven public outpatient clinics in villages located DRB1, -DRB3, -DRB4 and -DRB5 loci were per- formed by molecular techniques, as previously throughout Majorca, where over 75% of the at- 16 tended population were born on the island. The described. preselected individuals were asked to attend a specific appointment during which a questionnaire containing demographic, lifestyle and health-related Serological analysis issues was filled in, a medical examination includ- sIgGa-Cp levels were determined by a commercial ing anthropometry and BP measurement was per- quantitative ELISA kit (SeroCP Quant IgG, Savyon formed, and blood samples for genetic and Diagnostics Ltd., Israel) that uses as antigen purified serological analysis were collected from subjects elementary bodies from C. pneumoniae (TWAR-183) who were definitively enrolled. Laboratory data and gives the results as binding units (BU)/ml. The were gathered from medical records. The BP was assays were performed according to the manufac- measured manually with a calibrated sphygmoman- turer’s instructions. ometer by a skilled health professional; the mean of From the equivalences with a microimmunofluor- two BP measurements, separated by more than escence (MIF) method, these instructions establish 1 min, was taken as the BP for that day. that sera with o10 BU/ml are negative. Only hypertensive individuals without evidence of secondary forms of hypertension were eligible. From among these, those patients with severe Statistical analysis EH (systolic blood pressure (SBP) repeatedly X160 mmHg, or diastolic blood pressure (DBP) The between-groups comparison of quantitative and repeatedly X100 mmHg, or those receiving at least categorical variables was carried out by the unpaired two antihypertensive agents) were selected. We Student’s t-test and by the w2-statistics, respectively. chose individuals with severe EH as several pre- Allele families and alleles of the HLA system, as vious studies had shown that the relationship dichotomous variables, and the level of sIgGa-Cp,as between some genetic markers and EH is enhanced a continuous variable, were tested for association in this group of patients,15 and at least one study with EH by univariate and multivariate logistic reporting a positive association between C. pneu- regression analyses. Crude and adjusted odds ratio moniae infection and EH was also performed with (OR) and 95% confidence interval (CI) were calcu- severe essential hypertensives.10 Normotensive sub- lated from the univariate and multivariate logistic jects (SBP repeatedly o140 mmHg and DBP repeat- regression parameter estimates, respectively. The edly o90 mmHg) were selected as controls from P-values of the univariate analyses with the HLA among patients who had noncardiovascular symp- system were not corrected by the Bonferroni method toms or from individuals who participated in for multiple comparisons,17 mainly because the vaccination programmes. Exclusion criteria were existence of HLA haplotypes introduced considerable Journal of Human Hypertension HLA class II, C. pneumoniae and essential hypertension JM Zabay et al 617 uncertainty about the number of independent tests (BMI430 kg/m2) and frequency of family history of performed or implied. hypertension than controls. Diabetes was also more The effects of the explanatory variables on BP frequent in the group of patients than in the control level were investigated with the general linear group, but the difference did not reach statistical model ANOVA with adjustment for treatment, significance. BMI, age and sex. The association between EH and sIgGa-Cp was also tested by 2 Â 2 tables using Fisher’s exact test, Genetic data after transforming the level of antibodies in a Allele families or alleles that were identified in X20 dichotomous variable. The homogeneity of OR study participants were evaluated for their statisti- obtained from these tables was analysed by the cal association with EH.