Major Histocompatibility Complex HLA Region Largely Explains the Genetic Variance Exercised on Neutrophil Membrane Proteinase 3 Expression
Sibylle von Vietinghoff*,
Andreas Busjahn,
Constanze Schönemann,
Gero Massenkeil,
Björn Otto*,
Friedrich C. Luft* and
Ralph Kettritz*
Medical Faculty of the Charité, * Department of Nephrology and Hypertension, Franz Volhard Clinic, HELIOS Klinikum-Berlin, Max Delbrück Center for Molecular Medicine Campus Buch; Institute of Transfusion Medicine, Campus Virchow Klinikum; Department of Hematology and Oncology, Campus Virchow Klinikum; and HealthTwiSt, Berlin, Germany
Address correspondence to: Dr. Sibylle von Vietinghoff, Wiltbergstrasse 50, 13125 Berlin, Germany. Phone: +49-30-9417-2246; Fax:+49-30-9417-2206; E-mail: sibylle.vietinghoff{at}charite.de
Received for publication May 24, 2006.
Accepted for publication August 17, 2006.
ANCA-associated vasculitides, a common cause of rapidly progressiveglomerulonephritis, are influenced by genetic variance. Neutrophilmembrane expression of the ANCA antigen proteinase 3 (PR3) ispathogenically important. A subset of membrane PR3–positiveneutrophils can be distinguished from a membrane-negative subsetin any given subject. The percentage of membrane PR3–positiveneutrophils is genetically determined. In this study, 17 pairsof HLA-matched siblings were typed for their percentage of membranePR3–positive neutrophils. The HLA-matched siblings showeda high concordance (r = 0.67, P < 0.05), similar to thatseen in monozygotic twins. For testing of whether the HLA systeminfluences membrane PR3 percentage, membrane PR3 typing andHLA typing of 51 unrelated patients with Wegener’s granulomatosisand 49 normal control subjects was performed. Using two independentstatistical methods, a group of 34 HLA antigens was found topredict a large fraction of the membrane PR3 phenotype in patientsand control subjects. Certain major histocompatibility HLA antigenshave been implicated to conflicting degrees in ANCA-associatedvasculitides. However, in earlier studies, the contributionof the HLA system to the genetic variance of the disease wasunclear. In this cohort, found was an association of Wegener’sgranulomatosis with the same group of HLA antigens that predictedfor membrane PR3 percentage and a similar correlation with clinicalparameters at initial presentation. The disease status in 80%of the patients and 82% of the control subjects could be predictedcorrectly on the basis of HLA typing by discriminate functionanalysis (P < 0.001). After removal of the predicted individualfrom the sample, this association remained significant (64 and63% correct prediction; P < 0.001). The data suggest thata complex interaction of the entire HLA system is responsiblefor the genetic influence on membrane PR3 percentage and Wegener’sgranulomatosis.
ANCA-associated vasculitides are characterized by necrotizinginflammation of small blood vessels of the upper airway, lowerairway, and kidney. Other body sites and organs may be affected,albeit to a variable and lesser degree. A characteristic featureof Wegener’s granulomatosis, the most common form of ANCA-associatedvasculitis, is the occurrence of IgG autoantibodies directedagainst neutrophil proteinase 3 (PR3). By immunofluorescence,the autoantibodies produce a characteristic cytoplasmic stainingpattern on permeabilized neutrophils and therefore are namedc-ANCA. PR3 also can be found on the cell membrane of livingisolated neutrophils. A neutrophil subset that expresses PR3on the cell membrane can be distinguished from a cell membranePR3–negative subset. Whereas the amount of surface PR3can be increased by cytokines such as TNF-, the percentage ofpositive cells is stable in any given individual and remainsso during a variety of inflammatory diseases and immunosuppressivetherapies (1–4). A high percentage of neutrophils thatexpress PR3 on their cell surface is a risk factor for ANCA-associatedvasculitides and other autoimmune diseases (5). We reportedpreviously that membrane PR3 percentage is genetically determined(2). When comparing membrane PR3 percentage on neutrophils frommonozygotic (MZ) and dizygotic (DZ) twins, we found that theintrapair correlation in MZ twins was remarkably robust, whereasno intrapair correlation was seen in DZ twins. However, whichgenes or loci determine membrane PR3 percentage is currentlyunknown.
ANCA-associated vasculitides occur in siblings, within families,and the disease prevalence varies among populations, suggestinggenetic variance (6–10). Spencer et al. (11) describedHLA class II antigen association in patients with ANCA vasculitis.Other investigators made similar, often conflicting, observationswith single HLA I (12,13) and HLA II alleles (11,14–20)or found no significant correlation (21–23), as reviewedelsewhere (24–27).
In this study, we investigated the membrane PR3 percentage inHLA-matched siblings. These siblings were selected as concordantat the HLA locus but were similar at all other loci only tothe degree that any first-degree relatives are concordant. Thefindings in these siblings were similar to those that we haddescribed for MZ twins earlier and prompted us to perform HLAtyping in 100 individuals (51 patients with Wegener’sgranulomatosis and 49 normal control subjects) to determinethe effect of genetic variance that is exerted by the HLA locuson membrane PR3 percentage. We then tested for associationsof HLA antigens with the occurrence of Wegener’s granulomatosisand with markers of disease severity at initial presentation.
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 designatedstem cell donors (eight women and nine men; mean aged 42 and43 yr, respectively) at the Department of Hematology and Oncology,Charité Campus Virchow Klinikum (Berlin, Germany). Ofthe recipients, 10 had a diagnosis of acute myelogenous leukemia,two had acute lymphocytic leukemia, two had chronic myelogenousleukemia, two had multiple myeloma, and one had chronic lymphocyticleukemia. The percentage of neutrophils that expressed membranePR3 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 outpatientdepartment. Wegener’s granulomatosis was diagnosed accordingto the Chapel Hill Consensus Conference and to the AmericanCollege of Rheumatology criteria as described previously (28,29).The control cohort of 49 individuals (mean age 36 yr) was recruitedfrom a pool of normal volunteers from our blood bank. The percentageof 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 givenindividual; therefore, age matching was not necessary (1–3).The study was carried out according to the principles of theDeclaration of Helsinki, and written informed consent was obtainedfrom all participants before the studies after our ethics committeegave approval.
HLA Analysis
In patients who were admitted for stem cell transplantation,HLA analysis was performed according to the German ConsensusConferences (Deutsche Gesellschaft für Immungenetik andDeutsche Arbeitsgemeinschaft Knochenmark- und Blutstammzell-Transplantation2000 and 2005) and the international standards of the EuropeanFederation of Immunogenetics and the American Society of Histocompatibilityand Immunogenetics. HLA class I molecular typing was performedat the two-digit level by sequence-specific oligonucleotide(SSO) hybridization (Reli, Dynal, Oslo, Norway) and sequence-specificprimer (SSP) techniques (Genovision, Vienna, Austria; and Biotest,Dreieich, Germany), and HLA class II alleles were defined atthe four-digit level by SSP (Genovision) according to the instructionsof the manufacturer as described previously (30). The loci A,B, DRB1, and DQB1 were estimated. Additional testing of DBP1 and Cw was performed to select the best compatible donor betweenHLA-A, B, DRB1, and DQB1 identical donors. All stem cell transplantpatients and their donors studied were identical for all testedloci, apart from a single DQB1 mismatch (03 versus 06) in onepatient. Molecular HLA analysis in patients with Wegener’sgranulomatosis and control subjects was performed at the two-digitlevel in SSP technique (HLA-ready gene kit; InnoTrain, Kronberg,Germany). DNA was extracted from EDTA blood samples with QiagenDNA isolation kits (Qiagen, Hilden, Germany). HLA typing forstem cell transplantation and retyping of 20 randomly selectedcontrol samples was performed in a European Federation of Immunogeneticsaccredited HLA laboratory (Charité Campus Virchow Klinikum).Results are displayed using standard allele filters that eliminatedHLA antigen frequencies below 0.01% and were evaluated withthe SCORE software (Helmberg, Graz, Austria).
Neutrophil Isolation and Assessment of Membrane PR3 Percentage by Flow Cytometry
Neutrophils were isolated from heparinized whole blood by redblood cell sedimentation with dextran 1%, followed by Ficoll-Hypaquedensity gradient centrifugation and hypotonic erythrocyte lysis.The cell viability was determined in every cell preparationby trypan blue exclusion and exceeded 99%. The neutrophil percentagein the suspension was >95% by Wright-Giemsa staining. FACSwas used as described previously to evaluate the membrane PR3percentage on neutrophils (31). Briefly, membrane PR3 was detectedusing CLB12.8 mAb (CLB, Amsterdam, Netherlands) and labeledwith secondary FITC-conjugated F(ab) fragment of goat anti-mouseIgG (DAKO, Hamburg, Germany) using a FACScan (Becton Dickinson,Heidelberg, Germany). A total of 10,000 events per sample werecollected and analyzed with Cell-quest Pro software (BectonDickinson). Data are reported as percentage of the total neutrophilpopulation that was membrane PR3 positive.
Statistical Analyses
The presence of specific HLA antigens was coded for an additivemodel; the frequency of each specific antigen in a participantwas given as 0, 1, or 2. Unless otherwise stated, all statisticswere calculated using SPSS 12 (SPSS, Chicago, IL) on a WindowsPC. The relation between HLA antigens and membrane PR3 percentagewas tested by stepwise forward (threshold P < 0.05) and backward(threshold P < 0.1) multiple regression. Although the backwardapproach is likely to result in a better prediction on the basisof a larger number of HLA antigens and is most appropriate inlight of the hypothesis that multiple interacting genes causethe observed phenotype, it may be prone to overfitting. A significantregression that is based on a reduced number of antigens inthe forward approach reduces the likelihood of such a bias.Furthermore, because this analysis may be biased by the nonindependenceof predictor variables, we applied a Bayesian statistics approachusing Markov chain Monte Carlo methods for multiple regressionto confirm regression coefficients and P values using 10,000replications of sample estimates. These analyses were calculatedusing 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 regressionand multivariate discriminate function analysis. In logisticregression, a continuous risk value and a threshold for beingaffected 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 appliedby a jack-knife procedure in which each participant is classifiedby a function that is derived from all remaining participantsin the sample. This analysis was restricted to HLA antigensthat were significant in the regression with the percentageof PR3-positive cells. Although statistical inference is basedon these multivariate analyses, we provide an additional directcomparison of antigen frequencies together with the P valuefrom 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 albuminto test for regression between these parameters and HLA antigens.GFR was estimated using the modified Modification of Diet inRenal Disease (MDRD) formula (33) as follows: GFR = 186 x (serumcreatinine/88.4)–1.154 x age–0.203 (in women, resultmultiplied by 0.742). These phenotypes and their concordancewith membrane PR3 had been determined in our cohort as reportedin a previous study (31). Nominal P values are reported. Potentialbias by multiple testing was corrected by a false discoveryrate that was calculated in SPSS as indicated.
Figure 1 shows membrane PR3 percentage in different pairs ofsiblings. Figure 1A demonstrates the concordance for membranePR3–positive neutrophils that we observed in MZ twinsand that we reported previously (2). This result documents thatthe membrane PR3–positive phenotype is robustly influencedby genetic variance in individuals who are concordant at allgene loci. Figure 1B shows the lack of concordance that wasobserved in DZ twins who are related to the same degree as anysiblings. This finding strongly supports an interactive effectof multiple genes on this phenotype, as opposed to simple additiveeffects. In a new group of HLA-matched siblings, designatedstem cell donors and recipients before transplantation (Figure 1C),we found a concordance of membrane PR3 similar to that seenin MZ twins (r = 0.67, P < 0.05). These siblings were concordantat important HLA loci but had the same degree of concordanceas our DZ twins at all other loci. This result suggests thatgenetic variance in the HLA region is highly responsible forthe percentage of membrane PR3–positive neutrophils.
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).
To test whether membrane PR3 percentage can be predicted bythe HLA system, we typed a total of 100 individuals (49 normalcontrol subjects and 51 patients with Wegener’s granulomatosis)for HLA and membrane PR3 percentage. With the use of a multipleforward regression, the minimal set of HLA antigens that isnecessary for a significant prediction of membrane PR3 was fiveand 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 predictedvalues for this regression is given in Figure 2B. The measuredand predicted values were concordant.
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.
Table 1. HLA antigen frequencies in patients with Wegener’s granulomatosis and control subjects
We next tested for associations of HLA antigens with the occurrenceof Wegener’s granulomatosis in our cohort. Frequenciesof HLA antigens for patients and control subjects are givenin Table 1. We found borderline nominal significance for certainantigens; however, when corrected for multiple testing, no significantdifferences remained. In contrast, the classification of participantson the basis of HLA antigens into patients with Wegener’sgranulomatosis and control subjects by logistic regression resultedin 100% correct prediction. To verify prediction power and totest whether prediction would extend to cases outside the classificationsample, we applied two distinct approaches of discriminate functionanalysis. Using the entire sample, 82% of case patients and80% of control subjects were classified correctly (P < 0.001;Figure 3A). In this analysis, predictive power within the sampleis tested. Because the predictive power may not apply to newcases, a cross-validation approach that was based on jack-knifetechniques was used. Every single participant in the samplewas classified by a distinct function that was obtained fromall remaining participants. In this more stringent manner ofclassification, 63% of case patients and 64% of control subjectswere identified when removed from the classification sample(P < 0.001; Figure 3B).
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).
We then analyzed the correlation between HLA antigens and clinicalparameters at the time of initial presentation with active disease(Table 2). We compared the correlations between clinical parametersand HLA antigens with correlations between the same clinicalparameters and the neutrophil membrane PR3 phenotype from ourgroup that were reported previously (20). Significant correlationswith HLA antigens were observed for hemoglobin, creatinine,and estimated GFR, whereas C-reactive protein and serum albuminshow P values that are suggestive of a relationship. No correlationwas seen for platelets, leukocyte count, and total protein concentration.The analysis shows that HLA antigens predicted correlationssimilar to the ones that were predicted by membrane PR3 percentage.
Table 2. Clinical parameters, correlation with neutrophil membrane PR3 percentage reported previously (19), and significance of regression between these clinical parameters and HLA antigensa
The important finding in our study was that the HLA region isresponsible for a major portion of the genetic effect on thepercentage of neutrophils that exhibit membrane PR3 expressionin both patients with Wegener’s granulomatosis and normalcontrol subjects. We found that membrane PR3 percentage in HLA-compatiblesiblings showed a similarly robust intrapair relationship asfound in MZ twin pairs, whereas the DZ twin cohort did not demonstrateany concordance for membrane PR3 percentage. The observationsin the twin cohorts suggested that nonlinear genetic interactionmight be responsible for the percentage of membrane PR3–positiveneutrophils. In a model of nonlinear interaction, also termedepistasis, association analysis must cover most, if not all,genes involved, because the phenotype depends on the productrather than on the sum of single-gene effects (34). The observationof concordance in HLA-matched sibling pairs generated the hypothesisthat HLA antigens were responsible for the phenotype. To testthis notion in unrelated individuals, we typed healthy controlsubjects and patients with Wegener’s granulomatosis forHLA and membrane PR3. We found that a subset of HLA class Iand II antigens predicted membrane PR3 percentage with a highdegree of precision. Therefore, our data suggest that the HLAantigens determine membrane PR3 percentage through complex interactionof all tested loci.
We also showed that we could predict those individuals who hadWegener’s granulomatosis using the same subset of HLAantigens that also determined membrane PR3 expression. Previousstudies of HLA associations with Wegener’s granulomatosisare inconsistent, and in our cohort, like in some others (21–23),single allele associations disappeared when appropriate adjustmentswere made for multiple testing. However, the entire set of HLAantigens had predictive value for Wegener’s granulomatosisdisease status in our cohort. This finding also applied whenthe tested individual was removed from the classification sample,indicating influence of the HLA antigen set on the onset ofWegener’s granulomatosis. Correlations with increasedcreatinine concentration and other laboratory values at presentationindicate 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). Apartfrom HLA, a number of other polymorphisms, including cytokinesand their receptors, have been studied. Some of these genes,including the retinoid X receptor and TNF genes are in theHLA region. However, their influence is inconsistent in variousstudies (36–40). If nonadditive, epistatic interactionsplayed a major role in the genetics of ANCA-associated vasculitides,then conflicting results would have to be expected. The geneticinfluence could be determined only by studying all or most ofthe predisposing genes in each individual.
Our study showed the genetic importance of the HLA region forthe 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 arenot bimodally distributed in neutrophils. Therefore, it is unlikelythat the HLA system physically mediates the surface display.Nonetheless, HLA-mediated display of PR3 fragments on the surfaceof myeloid precursor cells has been studied recently as a targetfor immunotherapy of myelogenous leukemia (41,42); the immunogenicityseemed to vary with the HLA subtype. One could speculate thatthe immunogenicity of PR3 and thus susceptibility for ANCA-associatedvasculitis at least partially depends on the HLA subtypes indifferent individuals. Our study showed an interactive effectin the HLA region as the genetic determinant for membrane PR3percentage and its influence on Wegener’s granulomatosis.We believe that these findings are relevant for further geneticstudies of this disease.
Acknowledgments
This study was supported by a grant-in-aid from the Bundesministeriumfür Bildung und Forschung (InnoRegio 03 i 4509B). The DeutscheForschungsgemeinschaft supports R.K. and F.C.L.
HLA typing was performed in collaboration with Imtec Immunodiagnostika(Berlin, Germany).
Footnotes
Published online ahead of print. Publication date availableat www.jasn.org.
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Abstract
Introduction
, 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 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. 
and TNF genes are in the HLA region. However, their influence is inconsistent in 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. 
, 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).