Major Histocompatibility Complex HLA Region Largely Explains the Genetic Variance Exercised on Neutrophil Membrane Proteinase 3 Expression : Journal of the American Society of Nephrology

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Major Histocompatibility Complex HLA Region Largely Explains the Genetic Variance Exercised on Neutrophil Membrane Proteinase 3 Expression

von Vietinghoff, Sibylle*; Busjahn, Andreas; Schönemann, Constanze; Massenkeil, Gero§; Otto, Björn*; Luft, Friedrich C.*; Kettritz, Ralph*

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Journal of the American Society of Nephrology 17(11):p 3185-3191, November 2006. | DOI: 10.1681/ASN.2006050522
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ANCA-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 increased by cytokines such as TNF-α, the percentage of positive cells is stable in any given individual and remains so during a variety of inflammatory diseases and immunosuppressive therapies (14). A high percentage of neutrophils that express PR3 on their cell surface is a risk factor for ANCA-associated 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 (610). 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 (11,1420) or found no significant correlation (2123), as reviewed elsewhere (2427).

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.

Materials and Methods

Probands and Patients

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, Charité 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 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 (13). 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.

HLA Analysis

In patients who were admitted for stem cell transplantation, HLA analysis was performed according to the German Consensus Conferences (Deutsche Gesellschaft für 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 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 (Charité 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 FITC-conjugated 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 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.


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 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.


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 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 (2123), 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 β and TNF genes are in the HLA region. However, their influence is inconsistent in various studies (3640). 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.

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).
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 (r 2 = 0.28, P < 0.001). (B) Backward regression analysis (r 2 = 0.64, P = 0.001). This correlation is similarly robust as that of MZ twins.
Figure 3:
Prediction of the presence of Wegener’s granulomatosis by discriminate function analysis on the basis of HLA antigens. □, correctly predicted individuals; ▪, 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).
Table 1:
HLA antigen frequencies in patients with Wegener’s granulomatosis and control subjects
Table 2:
Clinical parameters, correlation with neutrophil membrane PR3 percentage reported previously (19), and significance of regression between these clinical parameters and HLA antigensa

This study was supported by a grant-in-aid from the Bundesministerium für 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).

Published online ahead of print. Publication date available at


1. Rarok AA, Stegeman CA, Limburg PC, Kallenberg CG: Neutrophil membrane expression of proteinase 3 (PR3) is related to relapse in PR3-ANCA-associated vasculitis. J Am Soc Nephrol 13: 2232–2238, 2002
2. Schreiber A, Busjahn A, Luft FC, Kettritz R: Membrane expression of proteinase 3 is genetically determined. J Am Soc Nephrol 14: 68–75, 2003
3. Witko-Sarsat V, Lesavre P, Lopez S, Bessou G, Hieblot C, Prum B, Noel LH, Guillevin L, Ravaud P, Sermet-Gaudelus I, Timsit J, Grunfeld JP, Halbwachs-Mecarelli L: A large subset of neutrophils expressing membrane proteinase 3 is a risk factor for vasculitis and rheumatoid arthritis. J Am Soc Nephrol 10: 1224–1233, 1999
4. Abdgawad M, Hellmark T, Gunnarsson L, Westman KW, Segelmark M: Increased neutrophil membrane expression and plasma level of proteinase 3 in systemic vasculitis are not a consequence of the −564 A/G promotor polymorphism. Clin Exp Immunol 145: 63–70, 2006
5. Buhaescu I, Covic A, Levy J: Systemic vasculitis: Still a challenging disease. Am J Kidney Dis 46: 173–185, 2005
6. Hay EM, Beaman M, Ralston AJ, Ackrill P, Bernstein RM, Holt PJ: Wegener’s granulomatosis occurring in siblings. Br J Rheumatol 30: 144–145, 1991
7. Knudsen BB, Joergensen T, Munch-Jensen B: Wegener’s granulomatosis in a family. A short report. Scand J Rheumatol 17: 225–227, 1988
    8. Muniain MA, Moreno JC, Gonzalez Campora R: Wegener’s granulomatosis in two sisters. Ann Rheum Dis 45: 417–421, 1986
      9. Nowack R, Lehmann H, Flores-Suarez LF, Nanhou A, van der Woude FJ: Familial occurrence of systemic vasculitis and rapidly progressive glomerulonephritis. Am J Kidney Dis 34: 364–373, 1999
        10. Manganelli P, Giacosa R, Fietta P, Zanetti A, Neri TM: Familial vasculitides: Churg-Strauss syndrome and Wegener’s granulomatosis in 2 first-degree relatives. J Rheumatol 30: 618–621, 2003
        11. Spencer SJ, Burns A, Gaskin G, Pusey CD, Rees AJ: HLA class II specificities in vasculitis with antibodies to neutrophil cytoplasmic antigens. Kidney Int 41: 1059–1063, 1992
        12. Katz P, Alling DW, Haynes BF, Fauci AS: Association of Wegener’s granulomatosis with HLA-B8. Clin Immunol Immunopathol 14: 268–270, 1979
        13. Shankarkumar U, Ghosh K, Pradhan VD, Badakere SS, Mohanty D: Immunogenetic association in patients with antineutrophil cytoplasmic antibodies (ANCA) from Mumbai, Maharashtra, India. J Autoimmun 24: 227–233, 2005
        14. Boki KA, Dafni U, Karpouzas GA, Papasteriades C, Drosos AA, Moutsopoulos HM: Necrotizing vasculitis in Greece: Clinical, immunological and immunogenetic aspects. A study of 66 patients. Br J Rheumatol 36: 1059–1066, 1997
        15. Elkon KB, Sutherland DC, Rees AJ, Hughes GR, Batchelor JR: HLA antigen frequencies in systemic vasculitis: Increase in HLA-DR2 in Wegener’s granulomatosis. Arthritis Rheum 26: 102–105, 1983
          16. Gencik M, Meller S, Borgmann S, Fricke H: Proteinase 3 gene polymorphisms and Wegener’s granulomatosis. Kidney Int 58: 2473–2477, 2000
            17. Hagen EC, Stegeman CA, D’Amaro J, Schreuder GM, Lems SP, Tervaert JW, de Jong GM, Hene RJ, Kallenberg CG, Daha MR, et al.: Decreased frequency of HLA-DR13DR6 in Wegener’s granulomatosis. Kidney Int 48: 801–805, 1995
              18. Papiha SS, Murty GE, Ad’Hia A, Mains BT, Venning M: Association of Wegener’s granulomatosis with HLA antigens and other genetic markers. Ann Rheum Dis 51: 246–248, 1992
                19. Thomson JB, Hulse D, Galbraith I, McKay IC, Field M: Autoantibody associations with MHC class II antigens in scleroderma and autoimmune vasculitis. Autoimmunity 19: 265–269, 1994
                  20. Tsuchiya N, Kobayashi S, Kawasaki A, Kyogoku C, Arimura Y, Yoshida M, Tokunaga K, Hashimoto H: Genetic background of Japanese patients with antineutrophil cytoplasmic antibody-associated vasculitis: Association of HLA-DRB1*0901 with microscopic polyangiitis. J Rheumatol 30: 1534–1540, 2003
                  21. Cotch MF, Fauci AS, Hoffman GS: HLA typing in patients with Wegener granulomatosis. Ann Intern Med 122: 635–1540, 1995
                  22. Murty GE, Mains BT, Middleton D, Maxwell AP, Savage DA: HLA antigen frequencies and Wegener’s granulomatosis. Clin Otolaryngol Allied Sci 16: 448–451, 1991
                    23. Strimlan CV, Taswell HF, Kueppers F, DeRemee RA, McDonald TJ: HLA-A antigens of patients with Wegener’s granulomatosis. Tissue Antigens 11: 129–131, 1978
                    24. Griffith ME, Pusey CD: HLA genes in ANCA-associated vasculitides. Exp Clin Immunogenet 14: 196–205, 1997
                    25. Fietta P: Systemic vasculitides: Immunogenetics and familial clustering. Clin Exp Rheumatol 22: 238–251, 2004
                      26. Jagiello P, Aries P, Arning L, Wagenleiter SE, Csernok E, Hellmich B, Gross WL, Epplen JT: The PTPN22 620W allele is a risk factor for Wegener’s granulomatosis. Arthritis Rheum 52: 4039–4043, 2005
                        27. Rihova Z, Honsova E, Zavada J, Vankova Z, Jancova E, Reiterova J, Tesar V: Two familial cases of antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis. Rheumatology (Oxford) 45: 356–357, 2006
                        28. Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC, Hoffman GS, Hunder GG, Kallenberg CG, et al.: Nomenclature of systemic vasculitides. Proposal of an international consensus conference. Arthritis Rheum 37: 187–192, 1994
                        29. Leavitt RY, Fauci AS, Bloch DA, Michel BA, Hunder GG, Arend WP, Calabrese LH, Fries JF, Lie JT, Lightfoot RW Jr, et al.: The American College of Rheumatology 1990 criteria for the classification of Wegener’s granulomatosis. Arthritis Rheum 33: 1101–1107, 1990
                        30. Heymann GA, Lassahn A, Schonemann C, Salama A: A novel HLA-A*680104 variant. Tissue Antigens 67: 169–170, 2006
                        31. Schreiber A, Otto B, Ju X, Zenke M, Goebel U, Luft FC, Kettritz R: Membrane proteinase 3 expression in patients with Wegener’s granulomatosis and in human hematopoietic stem cell-derived neutrophils. J Am Soc Nephrol 16: 2216–2224, 2005
                        32. Lynn DL, Thomas A, Best N, Spiegelhalter D: WinBUGS: A Bayesian modelling framework: Concepts, structure, and extensibility. Stat Comput 10: 325–337, 2000
                        33. Levey AS, Bosch JP, Lewis JB, Greene T, Rogers N, Roth D: A more accurate method to estimate glomerular filtration rate from serum creatinine: A new prediction equation. Modification of Diet in Renal Disease Study Group. Ann Intern Med 130: 461–470, 1999
                        34. Cordell HJ, Clayton DG: Genetic association studies. Lancet 366: 1121–1131, 2005
                        35. Borgmann S, Haubitz M: Genetic impact of pathogenesis and prognosis of ANCA-associated vasculitides. Clin Exp Rheumatol 22[Suppl 36]: S79–S86, 2004
                        36. Jagiello P, Gencik M, Arning L, Wieczorek S, Kunstmann E, Csernok E, Gross WL, Epplen JT: New genomic region for Wegener’s granulomatosis as revealed by an extended association screen with 202 apoptosis-related genes. Hum Genet 114: 468–477, 2004
                        37. Szyld P, Jagiello P, Csernok E, Gross WL, Epplen JT: On the Wegener granulomatosis associated region on chromosome 6p21.3. BMC Med Genet 7: 21–477, 2006
                          38. Spriewald BM, Witzke O, Wassmuth R, Wenzel RR, Arnold ML, Philipp T, Kalden JR: Distinct tumour necrosis factor alpha, interferon gamma, interleukin 10, and cytotoxic T cell antigen 4 gene polymorphisms in disease occurrence and end stage renal disease in Wegener’s granulomatosis. Ann Rheum Dis 64: 457–461, 2005
                            39. Zhou Y, Giscombe R, Huang D, Lefvert AK: Novel genetic association of Wegener’s granulomatosis with the interleukin 10 gene. J Rheumatol 29: 317–320, 2002
                              40. Mascher B, Schmitt W, Csernok E, Tatsis E, Reil A, Gross WL, Seyfarth M: Polymorphisms in the tumor necrosis factor genes in Wegener’s granulomatosis. Exp Clin Immunogenet 14: 226–233, 1997
                              41. Barrett J, Rezvani K: Neutrophil granule proteins as targets of leukemia-specific immune responses. Curr Opin Hematol 13: 15–20, 2006
                              42. Molldrem JJ, Lee PP, Kant S, Wieder E, Jiang W, Lu S, Wang C, Davis MM: Chronic myelogenous leukemia shapes host immunity by selective deletion of high-avidity leukemia-specific T cells. J Clin Invest 111: 639–647, 2003
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