Major Histocompatibility Complex HLA Region ... - Semantic Scholar

Major Histocompatibility Complex HLA Region Largely Explains the Genetic Variance Exercised on Neutrophil Membrane Proteinase 3 Expression Sibylle von Vietinghoff,* Andreas Busjahn,† Constanze Scho¨nemann,‡ Gero Massenkeil,§ Bjo¨rn Otto,* Friedrich C. Luft,* and Ralph Kettritz* Medical Faculty of the Charite´, *Department of Nephrology and Hypertension, Franz Volhard Clinic, HELIOS Klinikum-Berlin, Max Delbru¨ck Center for Molecular Medicine Campus Buch, ‡Institute of Transfusion Medicine, Campus Virchow Klinikum, and §Department of Hematology and Oncology, Campus Virchow Klinikum, and † HealthTwiSt, Berlin, Germany ANCA-associated vasculitides, a common cause of rapidly progressive glomerulonephritis, are influenced by genetic variance. Neutrophil membrane expression of the ANCA antigen proteinase 3 (PR3) is pathogenically important. A subset of membrane PR3–positive neutrophils can be distinguished from a membrane-negative subset in any given subject. The percentage of membrane PR3–positive neutrophils is genetically determined. In this study, 17 pairs of HLA-matched siblings were typed for their percentage of membrane PR3–positive neutrophils. The HLA-matched siblings showed a high concordance (r ⴝ 0.67, P < 0.05), similar to that seen in monozygotic twins. For testing of whether the HLA system influences membrane PR3 percentage, membrane PR3 typing and HLA typing of 51 unrelated patients with Wegener’s granulomatosis and 49 normal control subjects was performed. Using two independent statistical methods, a group of 34 HLA antigens was found to predict a large fraction of the membrane PR3 phenotype in patients and control subjects. Certain major histocompatibility HLA antigens have been implicated to conflicting degrees in ANCA-associated vasculitides. However, in earlier studies, the contribution of the HLA system to the genetic variance of the disease was unclear. In this cohort, found was an association of Wegener’s granulomatosis with the same group of HLA antigens that predicted for membrane PR3 percentage and a similar correlation with clinical parameters at initial presentation. The disease status in 80% of the patients and 82% of the control subjects could be predicted correctly on the basis of HLA typing by discriminate function analysis (P < 0.001). After removal of the predicted individual from the sample, this association remained significant (64 and 63% correct prediction; P < 0.001). The data suggest that a complex interaction of the entire HLA system is responsible for the genetic influence on membrane PR3 percentage and Wegener’s granulomatosis. J Am Soc Nephrol 17: 3185–3191, 2006. doi: 10.1681/ASN.2006050522

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NCA-associated vasculitides are characterized by necrotizing inflammation of small blood vessels of the upper airway, lower airway, and kidney. Other body sites and organs may be affected, albeit to a variable and lesser degree. A characteristic feature of Wegener’s granulomatosis, the most common form of ANCA-associated vasculitis, is the occurrence of IgG autoantibodies directed against neutrophil proteinase 3 (PR3). By immunofluorescence, the autoantibodies produce a characteristic cytoplasmic staining pattern on permeabilized neutrophils and therefore are named c-ANCA. PR3 also can be found on the cell membrane of living isolated neutrophils. A neutrophil subset that expresses PR3 on the cell membrane can be distinguished from a cell membrane PR3– negative subset. Whereas the amount of surface PR3 can be

Received May 24, 2006. Accepted August 17, 2006. Published online ahead of print. Publication date available at www.jasn.org. Address correspondence to: Dr. Sibylle von Vietinghoff, Wiltbergstrasse 50, 13125 Berlin, Germany. Phone: ⫹49-30-9417-2246; Fax: ⫹49-30-9417-2206; E-mail: [email protected] Copyright © 2006 by the American Society of Nephrology

increased by cytokines such as TNF-␣, the percentage of positive cells is stable in any given individual and remains so during a variety of inflammatory diseases and immunosuppressive therapies (1– 4). A high percentage of neutrophils that express PR3 on their cell surface is a risk factor for ANCAassociated vasculitides and other autoimmune diseases (5). We reported previously that membrane PR3 percentage is genetically determined (2). When comparing membrane PR3 percentage on neutrophils from monozygotic (MZ) and dizygotic (DZ) twins, we found that the intrapair correlation in MZ twins was remarkably robust, whereas no intrapair correlation was seen in DZ twins. However, which genes or loci determine membrane PR3 percentage is currently unknown. ANCA-associated vasculitides occur in siblings, within families, and the disease prevalence varies among populations, suggesting genetic variance (6 –10). Spencer et al. (11) described HLA class II antigen association in patients with ANCA vasculitis. Other investigators made similar, often conflicting, observations with single HLA I (12,13) and HLA II alleles (11,14 –20) or found no significant correlation (21–23), as reviewed elsewhere (24 –27). ISSN: 1046-6673/1711-3185

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In this study, we investigated the membrane PR3 percentage in HLA-matched siblings. These siblings were selected as concordant at the HLA locus but were similar at all other loci only to the degree that any first-degree relatives are concordant. The findings in these siblings were similar to those that we had described for MZ twins earlier and prompted us to perform HLA typing in 100 individuals (51 patients with Wegener’s granulomatosis and 49 normal control subjects) to determine the effect of genetic variance that is exerted by the HLA locus on membrane PR3 percentage. We then tested for associations of HLA antigens with the occurrence of Wegener’s granulomatosis and with markers of disease severity at initial presentation.

recipients and 69 ⫾ 22% in the donors. Fifty-one patients with Wegener’s granulomatosis (mean age 51 yr) were recruited from our inpatient and outpatient department. Wegener’s granulomatosis was diagnosed according to the Chapel Hill Consensus Conference and to the American College of Rheumatology criteria as described previously (28,29). The control cohort of 49 individuals (mean age 36 yr) was recruited from a pool of normal volunteers from our blood bank. The percentage of neutrophils that expressed membrane PR3 was 61 ⫾ 22% in the control subjects and 74 ⫾ 21% in the patients. The percentage of membrane PR3 is stable over time in any given individual; therefore, age matching was not necessary (1–3). The study was carried out according to the principles of the Declaration of Helsinki, and written informed consent was obtained from all participants before the studies after our ethics committee gave approval.

Materials and Methods

HLA Analysis

Probands and Patients

In patients who were admitted for stem cell transplantation, HLA analysis was performed according to the German Consensus Conferences (Deutsche Gesellschaft fu¨r Immungenetik and Deutsche Arbeitsgemeinschaft Knochenmark- und Blutstammzell-Transplantation 2000 and 2005) and the international standards of the European Federation of Immunogenetics and the American Society of Histocompatibility and Immunogenetics. HLA class I molecular typing was performed at the two-digit level by sequence-specific oligonucleotide (SSO) hybridization (Reli, Dynal, Oslo, Norway) and sequence-specific primer (SSP) techniques (Genovision, Vienna, Austria; and Biotest, Dreieich, Germany), and HLA class II alleles were defined at the four-digit level by SSP (Genovision) according to the instructions of the manufacturer as

Fifteen MZ and 12 DZ pairs of twins were recruited by advertisement (HealthTwiSt GmbH) and phenotyped as described previously (2). HLA-matched siblings were 17 stem cell transplant recipients (four women and 13 men) before transplantation and their designated stem cell donors (eight women and nine men; mean aged 42 and 43 yr, respectively) at the Department of Hematology and Oncology, Charite´ Campus Virchow Klinikum (Berlin, Germany). Of the recipients, 10 had a diagnosis of acute myelogenous leukemia, two had acute lymphocytic leukemia, two had chronic myelogenous leukemia, two had multiple myeloma, and one had chronic lymphocytic leukemia. The percentage of neutrophils that expressed membrane PR3 was 73 ⫾ 19% in the

Figure 1. (A) Percentage of membrane proteinase 3 (PR3)–positive neutrophil subset comparing monozygotic (MZ) twin1 with MZ twin2 (r ⫽ 0.99, P ⫽ 0.001; 95% confidence interval) is given. MZ twins were highly concordant. (B) Percentage of membrane PR3–positive neutrophil subset comparing dizygotic (DZ) twin1 with DZ twin2 (r ⫽ 0.06; NS) is given. DZ twins were not concordant. (C) Percentage of membrane PR3–positive neutrophil subset comparing HLA-matched sibling stem cell donors and recipients before transplantation are given. A significant relationship was found (r ⫽ 0.67, P ⬍ 0.05).

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described previously (30). The loci A, B, DRB1, and DQB1 were estimated. Additional testing of DBP 1 and Cw was performed to select the best compatible donor between HLA-A, B, DRB1, and DQB1 identical donors. All stem cell transplant patients and their donors studied were identical for all tested loci, apart from a single DQB1 mismatch (03 versus 06) in one patient. Molecular HLA analysis in patients with Wegener’s granulomatosis and control subjects was performed at the two-digit level in SSP technique (HLA-ready gene kit; InnoTrain, Kronberg, Germany). DNA was extracted from EDTA blood samples with Qiagen DNA isolation kits (Qiagen, Hilden, Germany). HLA typing for stem cell transplantation and retyping of 20 randomly selected control samples was performed in a European Federation of Immunogenetics accredited HLA laboratory (Charite´ Campus Virchow Klinikum). Results are displayed using standard allele filters that eliminated HLA antigen frequencies below 0.01% and were evaluated with the SCORE software (Helmberg, Graz, Austria).

Neutrophil Isolation and Assessment of Membrane PR3 Percentage by Flow Cytometry Neutrophils were isolated from heparinized whole blood by red blood cell sedimentation with dextran 1%, followed by Ficoll-Hypaque density gradient centrifugation and hypotonic erythrocyte lysis. The cell viability was determined in every cell preparation by trypan blue exclusion and exceeded 99%. The neutrophil percentage in the suspension was ⬎95% by Wright-Giemsa staining. FACS was used as described previously to evaluate the membrane PR3 percentage on neutrophils (31). Briefly, membrane PR3 was detected using CLB12.8 mAb (CLB, Amsterdam, Netherlands) and labeled with secondary FITCconjugated F(ab) fragment of goat anti-mouse IgG (DAKO, Hamburg, Germany) using a FACScan (Becton Dickinson, Heidelberg, Germany). A total of 10,000 events per sample were collected and analyzed with Cell-quest Pro software (Becton Dickinson). Data are reported as percentage of the total neutrophil population that was membrane PR3 positive.

Statistical Analyses The presence of specific HLA antigens was coded for an additive model; the frequency of each specific antigen in a participant was given as 0, 1, or 2. Unless otherwise stated, all statistics were calculated using SPSS 12 (SPSS, Chicago, IL) on a Windows PC. The relation between HLA antigens and membrane PR3 percentage was tested by stepwise forward (threshold P ⬍ 0.05) and backward (threshold P ⬍ 0.1) multiple regression. Although the backward approach is likely to result in a better prediction on the basis of a larger number of HLA antigens and is most appropriate in light of the hypothesis that multiple interacting genes cause the observed phenotype, it may be prone to overfitting. A significant regression that is based on a reduced number of antigens in the forward approach reduces the likelihood of such a bias. Furthermore, because this analysis may be biased by the nonindependence of predictor variables, we applied a Bayesian statistics approach using Markov chain Monte Carlo methods for multiple regression to confirm regression coefficients and P values using 10,000 replications of sample estimates. These analyses were calculated using the software WinBUGS 1.4.1 for Windows (32). To test whether HLA antigens can predict the presence of disease (Wegener’s granulomatosis), we used logistic regression and multivariate discriminate function analysis. In logistic regression, a continuous risk value and a threshold for being affected are calculated. In the discriminate function analysis, the presence of disease as a categorical variable is predicted. In this analysis, cross-validation of prediction was applied by a jack-knife procedure in which each participant is classified

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by a function that is derived from all remaining participants in the sample. This analysis was restricted to HLA antigens that were significant in the regression with the percentage of PR3-positive cells. Although statistical inference is based on these multivariate analyses, we provide an additional direct comparison of antigen frequencies together with the P value from Fisher exact test. P ⬍ 0.05 was accepted as significant. We analyzed clinical parameters, hemoglobin, platelets, leukocytes, creatinine, C-reactive protein, total protein, and serum albumin to test for regression between these parameters and HLA antigens. GFR was estimated using the modified Modification of Diet in Renal Disease (MDRD) formula (33) as follows: GFR ⫽ 186 ⫻ (serum creatinine/ 88.4)⫺1.154 ⫻ age⫺0.203 (in women, result multiplied by 0.742). These phenotypes and their concordance with membrane PR3 had been determined in our cohort as reported in a previous study (31). Nominal P values are reported. Potential bias by multiple testing was corrected by a false discovery rate that was calculated in SPSS as indicated.

Results Figure 1 shows membrane PR3 percentage in different pairs of siblings. Figure 1A demonstrates the concordance for membrane PR3–positive neutrophils that we observed in MZ twins and that we reported previously (2). This result documents that the membrane PR3–positive phenotype is robustly influenced by genetic variance in individuals who are concordant at all gene loci. Figure 1B shows the lack of concordance that was observed in DZ twins who are related to the same degree as any siblings. This finding strongly supports an interactive effect of multiple genes on this phenotype, as opposed to simple additive effects. In a new group of HLA-matched siblings, designated stem cell donors and recipients before transplantation (Figure 1C), we found a concordance of membrane PR3 similar to that seen in MZ twins (r ⫽ 0.67, P ⬍ 0.05). These siblings were concordant at important HLA loci but had the same degree of concordance as our DZ twins at all other loci. This result suggests that genetic variance in the HLA region is highly responsible for the percentage of membrane PR3–positive neutrophils. To test whether membrane PR3 percentage can be predicted

Figure 2. Percentage of membrane PR3–positive neutrophil subset in patients with Wegener’s granulomatosis and control subjects is given on the ordinate. On the abscissa are the values predicted by the regression analysis. (A) Forward regression analysis (r2 ⫽ 0.28, P ⬍ 0.001). (B) Backward regression analysis (r2 ⫽ 0.64, P ⫽ 0.001). This correlation is similarly robust as that of MZ twins.

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Table 1. HLA antigen frequencies in patients with Wegener’s granulomatosis and control subjects HLA Locus A*

B*

Cw*

DRB1*

DQB*

DRB3* DRB4* DRB5*a a

Wegener’s Granulomatosis (n 关%兴)

Control (n 关%兴)

Antigen 01 02 03 11 23 24 25 26a 29a 30a 31 32 68a 07 08 13a 14 15a 18 27 35a 37a 38 39a 40a 44a 49a 50a 51 52 53 55a 56 57a 58 01a 02a 03a 04 05a 06a 07a 08a 12a 14 15a 16 01 03a 04a 07 08a 09 10a 11 12 13a 14a 15 16 02 03 04a 05a 06a

Absent

Heterozygous

Homozygous

Absent

Heterozygous

Homozygous

37 (72) 25 (49) 31 (61) 46 (90) 51 (100) 44 (86) 43 (84) 49 (96) 49 (96) 49 (96) 50 (98) 50 (98) 42 (82) 35 (69) 39 (76) 47 (92) 48 (94) 39 (76) 43 (84) 51 (100) 45 (88) 51 (100) 50 (98) 51 (100) 43 (84) 39 (76) 49 (96) 49 (96) 46 (90) 49 (96) 51 (100) 51 (100) 49 (96) 46 (90) 51 (100) 49 (94) 50 (96) 36 (69) 47 (90) 46 (88) 41 (79) 20 (38) 49 (94) 42 (81) 50 (96) 48 (92) 51 (98) 43 (84) 42 (82) 39 (76) 40 (78) 48 (94) 49 (96) 49 (96) 42 (82) 46 (90) 37 (72) 49 (96) 31 (61) 51 (100) 36 (71) 19 (37) 48 (94) 41 (80) 20 (39) 19 (37) 29 (57) 32 (63)

14 (28) 23 (45) 18 (35) 5 (10) 0 (0) 7 (14) 8 (16) 2 (4) 2 (4) 2 (4) 1 (2) 1 (2) 9 (18) 14 (27) 12 (24) 4 (8) 3 (6) 12 (24) 8 (16) 0 (0) 6 (12) 0 (0) 1 (2) 0 (0) 8 (16) 12 (24) 2 (4) 2 (4) 5 (10) 2 (4) 0 (0) 0 (0) 2 (4) 5 (10) 0 (0) 2 (4) 2 (4) 14 (27) 5 (10) 6 (12) 10 (19) 27 (52) 3 (6) 10 (19) 2 (4) 4 (8) 1 (2) 7 (14) 9 (18) 12 (24) 10 (20) 3 (6) 2 (4) 2 (4) 8 (16) 4 (8) 14 (28) 2 (4) 19 (37) 0 (0) 14 (27) 26 (51) 3 (6) 9 (18) 28 (55) 32 (63) 22 (43) 19 (37)

0 (0) 3 (6) 2 (4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (4) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) 2 (4) 0 (0) 0 (0) 1 (2) 5 (10) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0) 1 (2) 1 (2) 0 (0) 0 (0) 1 (2) 0 (0) 1 (2) 6 (12) 0 (0) 1 (2) 3 (6)

36 (71) 24 (47) 38 (74) 41 (80) 47 (92) 47 (92) 47 (92) 47 (92) 48 (94) 50 (98) 48 (94) 47 (92) 48 (94) 36 (71) 47 (92) 46 (90) 49 (96) 43 (84) 41 (80) 44 (86) 44 (86) 48 (94) 50 (98) 50 (98) 46 (90) 47 (92) 50 (98) 48 (94) 43 (84) 50 (98) 50 (98) 46 (90) 51 (100) 49 (96) 47 (92) 47 (92) 44 (86) 39 (76) 41 (80) 48 (94) 41 (80) 22 (43) 49 (96) 46 (90) 48 (94) 45 (88) 50 (98) 38 (74) 45 (88) 43 (84) 37 (72) 48 (94) 48 (94) 51 (100) 44 (86) 50 (98) 35 (69) 50 (98) 37 (72) 48 (94) 34 (68) 28 (56) 48 (96) 33 (66) 22 (44) 26 (51) 27 (53) 33 (65)

15 (29) 22 (43) 13 (26) 9 (18) 4 (8) 4 (8) 4 (8) 3 (6) 3 (6) 1 (2) 3 (6) 4 (8) 3 (6) 13 (25) 4 (8) 5 (10) 2 (4) 7 (14) 10 (20) 7 (14) 7 (14) 3 (6) 1 (2) 1 (2) 5 (10) 4 (8) 1 (2) 3 (6) 8 (16) 1 (2) 1 (2) 4 (8) 0 (0) 2 (4) 4 (8) 4 (8) 7 (14) 10 (20) 10 (20) 3 (6) 8 (16) 24 (47) 2 (4) 5 (10) 3 (6) 6 (12) 1 (2) 11 (22) 6 (12) 8 (16) 13 (26) 3 (6) 3 (6) 0 (0) 6 (12) 1 (2) 12 (23) 1 (2) 9 (18) 3 (6) 14 (28) 18 (36) 2 (4) 15 (30) 21 (42) 25 (49) 24 (47) 18 (35)

0 (0) 5 (10) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (4) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (4) 0 (0) 0 (0) 2 (4) 5 (10) 0 (0) 0 (0) 0 (0) 0 (0) 0 (0) 2 (4) 0 (0) 0 (0) 1 (2) 0 (0) 0 (0) 0 (0) 1 (2) 0 (0) 4 (8) 0 (0) 5 (10) 0 (0) 2 (4) 4 (8) 0 (0) 2 (4) 7 (14)

Contained in best fitting regression model.

P (ANCA versus Control)

0.054

0.013

0.056

0.092

0.045

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by the HLA system, we typed a total of 100 individuals (49 normal control subjects and 51 patients with Wegener’s granulomatosis) for HLA and membrane PR3 percentage. With the use of a multiple forward regression, the minimal set of HLA antigens that is necessary for a significant prediction of membrane PR3 was five and resulted in an r2 of 0.28 (ANOVA model P ⬍ 0.001; Figure 2A). The backward regression approach identified a set of 34 antigens (as indicated in Table 1) and increased the r2 value to 0.64 (ANOVA model P ⬍ 0.001). A scatter plot of measured and predicted values for this regression is given in Figure 2B. The measured and predicted values were concordant. We next tested for associations of HLA antigens with the occurrence of Wegener’s granulomatosis in our cohort. Frequencies of HLA antigens for patients and control subjects are given in Table 1. We found borderline nominal significance for certain antigens; however, when corrected for multiple testing, no significant differences remained. In contrast, the classification of participants on the basis of HLA antigens into patients with Wegener’s granulomatosis and control subjects by logistic regression resulted in 100% correct prediction. To verify prediction power and to test whether prediction would extend to cases outside the classification sample, we applied two distinct approaches of discriminate function analysis. Using the entire sample, 82% of case patients and 80% of control subjects were classified correctly (P ⬍ 0.001; Figure 3A). In this analysis, predictive power within the sample is tested. Because the predictive power may not apply to new cases, a cross-validation approach that was based on jack-knife techniques was used. Every single participant in the sample was classified by a distinct function that was obtained from all remaining participants. In this more stringent manner of classification, 63% of case patients and 64% of control subjects were identified when removed from the classification sample (P ⬍ 0.001; Figure 3B). We then analyzed the correlation between HLA antigens and clinical parameters at the time of initial presentation with active disease (Table 2). We compared the correlations between clinical parameters and HLA antigens with correlations between the same clinical parameters and the neutrophil membrane PR3 phenotype from our group that were reported previously (20). Significant correlations with HLA antigens were observed for hemoglobin, creatinine, and estimated GFR, whereas C-reactive protein and serum albumin show P values that are suggestive of a relationship. No correlation was seen for platelets, leukocyte count, and total protein concentration. The analysis shows that HLA antigens predicted correlations similar to the ones that were predicted by membrane PR3 percentage.

Discussion The important finding in our study was that the HLA region is responsible for a major portion of the genetic effect on the percentage of neutrophils that exhibit membrane PR3 expression in both patients with Wegener’s granulomatosis and normal control subjects. We found that membrane PR3 percentage in HLA-compatible siblings showed a similarly robust intrapair relationship as found in MZ twin pairs, whereas the DZ twin cohort did not demonstrate any concordance for membrane


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PR3 percentage. The observations in the twin cohorts suggested that nonlinear genetic interaction might be responsible for the percentage of membrane PR3–positive neutrophils. In a model of nonlinear interaction, also termed epistasis, association analysis must cover most, if not all, genes involved, because the phenotype depends on the product rather than on the sum of single-gene effects (34). The observation of concordance in HLA-matched sibling pairs generated the hypothesis that HLA antigens were responsible for the phenotype. To test this notion in unrelated individuals, we typed healthy control subjects and patients with Wegener’s granulomatosis for HLA and membrane PR3. We found that a subset of HLA class I and II antigens predicted membrane PR3 percentage with a high degree of precision. Therefore, our data suggest that the HLA antigens determine membrane PR3 percentage through complex interaction of all tested loci. We also showed that we could predict those individuals who had Wegener’s granulomatosis using the same subset of HLA antigens that also determined membrane PR3 expression. Previous studies of HLA associations with Wegener’s granulomatosis are inconsistent, and in our cohort, like in some others (21–23), single allele associations disappeared when appropriate adjustments were made for multiple testing. However, the entire set of HLA antigens had predictive value for Wegener’s granulomatosis disease status in our cohort. This finding also applied when the tested individual was removed from the classification sample, indicating influence of the HLA antigen set on the onset of Wegener’s granulomatosis. Correlations with increased creatinine concentration and other laboratory values at presentation indicate some influence on the severity of the disease. Genetic influence plays a role in ANCA-associated vasculitides; however, which genes are responsible is unclear (24,35). Apart from HLA, a number of other polymorphisms, including cytokines and their receptors, have been studied. Some of these genes, including the retinoid X receptor ␤ and TNF genes are in the HLA region. However, their influence is inconsistent in

Figure 3. Prediction of the presence of Wegener’s granulomatosis by discriminate function analysis on the basis of HLA antigens. u, correctly predicted individuals; f, falsely predicted individuals. (A) Prediction based on the entire sample, with correct classification of 82% of patients and 80% of control subjects (P ⬍ 0.001). (B) Prediction after exclusion of the tested individual by jack-knife technique. This resulted in correct classification of 63% of patients and 64% of control subjects (P ⬍ 0.001).

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Table 2. Clinical parameters, correlation with neutrophil membrane PR3 percentage reported previously (19), and significance of regression between these clinical parameters and HLA antigensa Parameter

Correlation with Membrane PR3 Percentage

P (Correlation with Membrane PR3 Percentage)

P (Regression HLA)

Hemoglobin (g/L) Platelets (␮l3) Leukocytes (␮l3) Creatinine (␮mol/L) CRP (units) Total protein (g/L) Albumin (g/L) Cockroft GFR eGFR eGFR (1 yr)

⫺0.305 0.037 0.124 0.351 0.256 ⫺0.161 ⫺0.397 0.399

0.006b NS NS 0.001b 0.021b NS 0.001b 0.001b

0.014b NS NS 0.022b 0.08 NS 0.03 0.04 0.015b NS

a

For hemoglobin, creatinine, and GFR estimates (Modification of Diet in Renal Disease formula), significant correlations were observed. CRP, C-reactive protein; eGFR, estimated GFR; PR3, proteinase 3. b False discovery rate ⬍5%.

various studies (36 – 40). If nonadditive, epistatic interactions played a major role in the genetics of ANCA-associated vasculitides, then conflicting results would have to be expected. The genetic influence could be determined only by studying all or most of the predisposing genes in each individual. Our study showed the genetic importance of the HLA region for the percentage of membrane PR3– expressing neutrophils; however, the study cannot shed light on the mechanisms involved. HLA I molecules are displayed on all nucleated cells and are not bimodally distributed in neutrophils. Therefore, it is unlikely that the HLA system physically mediates the surface display. Nonetheless, HLA-mediated display of PR3 fragments on the surface of myeloid precursor cells has been studied recently as a target for immunotherapy of myelogenous leukemia (41,42); the immunogenicity seemed to vary with the HLA subtype. One could speculate that the immunogenicity of PR3 and thus susceptibility for ANCA-associated vasculitis at least partially depends on the HLA subtypes in different individuals. Our study showed an interactive effect in the HLA region as the genetic determinant for membrane PR3 percentage and its influence on Wegener’s granulomatosis. We believe that these findings are relevant for further genetic studies of this disease.

3.

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5. 6.

7.

8.

9.

Acknowledgments This study was supported by a grant-in-aid from the Bundesministerium fu¨r Bildung und Forschung (InnoRegio 03 i 4509B). The Deutsche Forschungsgemeinschaft supports R.K. and F.C.L. HLA typing was performed in collaboration with Imtec Immunodiagnostika (Berlin, Germany).

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