No association of G-463A myeloperoxidase gene polymorphism with MPOANCA-associated vasculitis
Anette Fiebeler1,
Stefan Borgmann3,
Alexander Woywodt2,
Hermann Haller2 and
Marion Haubitz2
1Division of Nephrology, Franz-Volhard-Clinic, Berlin, HELIOS, 2Division of Nephrology, Hannover Medical School, Hannover and 3Division of Medical Microbiology University of Tübingen, Germany
Correspondence and offprint requests to: Marion Haubitz, MD, Department of Nephrology, Medical School Hannover, D-30623 Hannover, Germany. Email: haubitz.marion{at}mh-hannover.de
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Abstract
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Background. The activation of neutrophils and monocytes by ANCA, resulting in the release of reactive oxygen species and proteases like myeloperoxidase (MPO), is essential to the pathogenesis of ANCA-associated vasculitis. As the A allele of the G-463A MPO gene polymorphism is associated with diminished activity of MPO, it is conceivable that the presence of this allele protects against MPOANCA-associated vasculitis.
Methods. Allelic frequencies of the G-463A polymorphism were studied in 119 ANCA-associated vasculitis patients, 48 with MPOANCA and 71 with proteinase 3 (PR3)ANCA.
Results. Allelic frequencies of MPO G-463A promoter polymorphism did not differ between MPOANCA- and PR3ANCA-associated vasculitis patients. Moreover, allelic distribution was similar to that of the normal population.
Conclusions. The data suggest that G-463A polymorphism does not seem to contribute to either MPOANCA- or PR3ANCA-associated vasculitis formation.
Keywords: ANCA; microscopic polyangiitis; myeloperoxidase; polymorphism; vasculitis; Wegener's granulomatosis
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Introduction
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Genetic predisposition has been discussed as contributing to the formation of ANCA-associated vasculitis [1]. Polymorphism within the gene of proteinase 3 (PR3), the main target protein of c-ANCA, has been described [2] and allelic frequencies of PR3 gene polymorphisms were increased in patients with Wegener's granulomatosis (WG). As one of these disease-associated polymorphisms was located within the PR3 promoter region, alteration of PR3 protein expression might contribute to the formation of c-ANCA-associated vasculitis. Interestingly, PR3 membrane expression is genetically controlled [3], and elevated levels of membrane PR3 expression were found in patients with WG [4]. Moreover, within the group of WG patients, elevated levels were associated with an increased risk of relapses [4]. Therefore, promoter polymorphism of ANCA target genes, leading to elevated protein expression rates, might contribute to the formation of ANCA-associated vasculitis.
Conversely, the presence of alleles resulting in a reduced expression of ANCA target proteins might be beneficial in preventing the development of ANCA-associated diseases. Myeloperoxidase (MPO) is the main target antigen of p-ANCA found in the majority of patients with microscopic polyangiitis, and occasionally in patients with WG [5,6]. In the MPO promoter region a G-463A polymorphism has been described, which is characterized by a single base substitution of guanine to adenine. The A allele of this polymorphism is associated with reduced MPO activity and carriers of the less frequent A allele have a lower risk of lung cancer, presumably by virtue of reduced activation of carcinogens in tobacco smoke [7,8].
MPOANCAs have been demonstrated to activate primed granulocytes and monocytes, resulting in respiratory burst with the release of reactive oxygen species and proteases leading to endothelial damage [9,10]. However, MPOANCAs do not induce respiratory burst in leukocytes from MPO-deficient individuals [11]. It therefore appears intriguing to speculate that diminished activity of MPO due to A allele carriage of the G-463A polymorphism confers reduced susceptibility to the development of MPOANCA-associated vasculitis. In the present study we therefore analysed whether allelic frequencies of this polymorphism in MPOANCA-associated vasculitis patients differ from those of healthy controls or PR3ANCA-associated vasculitis patients.
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Subjects and methods
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Allelic frequencies of the G-463A polymorphism within the MPO promoter were analysed in 119 Caucasian ANCA-associated vasculitis patients originating from the northern part of Germany. Forty-eight patients had MPOANCA-associated vasculitis and 71 patients had PR3ANCA-associated vasculitis. Patients characteristics are shown in Table 1. ANCA positivity and specificity had been documented at the time of active disease by indirect immunofluorescence and an ELISA for MPOANCA and PR3ANCA.
A commercially available kit (Qiagen-RNeasy Mini Kit, Qiagen, Hilden Germany) was used to extract DNA from peripheral blood leukocytes. For genotyping procedure, the polymorphic site at position 463 of the MPO gene was amplified as described by Cascorbi et al. [7]. In brief, DNA was extracted from leucocytes with a standard phenolchloroform protocol. The MPO-DNA including the polyphorphism locus 463 was amplified using the following primers: FW: 5'-CGGTATAGGCACACAATGGTGAG, RV: 5;-GCAATGGTTCAAGCGATTCTTC. The PCR product was digested with Acil (New England Biolabs) resulting in four different fragments depending on the genotype. To visualize the results, the fragments were separated with gel electrophoresis and stained with ethidium bromide.
Differences between the groups were compared using
2-test (Bonferroni correction was performed for multiple comparison) and Monte Carlo simulation (empirical error rate for small patient numbers [12]). A P-value <0.05 was considered significant.
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Results
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Frequencies of the G and A allele carriage of MPO G-463A polymorphism were investigated in MPOANCA- and PR3ANCA-associated vasculitis. HardyWeinberg equilibrium was observed in both patient groups. Allelic frequencies of the MPO G-463A polymorphism did not differ significantly between the patient groups. The frequency of the A allele was 15.6% in MPOANCA and 22.5% in PR3ANCA patients. As shown in Table 2 no statistical difference of the genotype frequencies between the two patient groups was observed, either. Moreover, MPO G-463A polymorphism genotype frequencies of both ANCA patients groups were similar to those observed in various Caucasian reference populations. Interestingly, like the patients analysed in the present study, one of the reference populations originated from the northern part of Germany. In addition, no differences were observed after ANCA-associated vasculitis patients had been stratified to gender (Table 2), organ manifestations or diseases (WG or microscopic polyangiitis, data not shown).
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Discussion
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In ANCA-associated vasculitis, reactive oxygen species play an important role in tissue injury and the inflammatory reaction. Therefore, A allele carriage of G-463A MPO gene polymorphism, leading to reduced MPO activity with the presumable consequences of reduced production of radicals, might be protective against the development of an ANCA-associated vasculitis. However, our results demonstrate that allelic as well as genotype frequencies did not differ between MPOANCA- and PR3ANCA-associated vasculitis patients. Furthermore, the frequencies of ANCA-associated vasculitis patients observed were comparable with those previously published in various Caucasian reference populations [7,8,13].
In contrast to the findings of Reynolds et al. [13], no differences were observed when the ANCA-associated vasculitis patients were stratified for gender or organ manifestation. As the data of Reynolds et al. were not available at the time when our study was planned, no study design concerning the results of Reynolds et al. was made. Using the allele frequencies of ethnically matched controls of Reynolds et al. and a significance of <0.05, the number of patients included in our population could be sufficient to find at least a tendency in female patients. However, since the number of patients studied is very small, the power to find a significant statistical difference in our patient group is only 30%.
In the study of Reynolds et al., the frequency of G/G genotype was increased in female, but not in male, MPOANCA-associated vasculitis patients. Therefore, the presence of the A allele appeared to prevent formation of MPOANCA-associated vasculitis in females. In the same study, however, patients with the A allele had in an earlier onset of MPOANCA-associated vasculitis. Furthermore, the presence of the A allele was associated with a higher frequency of nasal, sinus and/or ear involvement and a shorter relapse-free period.
The contrasting results of the present study and the investigation by Reynolds et al., might be explained by the fact that reliable data are difficult to obtain in small patient groups, particularly when polymorphism is used for multiple comparisons. In the study of Reynolds et al., P values were not corrected for multiple comparison. The subgroups were very small (n = 16 for patients with nose/sinus involvement, n = 6 for ear involvement). Moreover, it seems unlikely that, on the one hand, the presence of A allele mediates protection against the disease and, on the other hand, implies the higher susceptibility to special organ involvement and the development of relapses.
In conclusion, our data suggest that G-463A MPO polymorphism contributes neither to MPOANCA- nor PR3ANCA-associated vasculitis. However, a marginal impact of genetic polymorphism of the MPO gene regarding a trend towards decreased frequency of the A allele in MPOANCA positive compared with PR3ANCA positive vasculitis disease would have to be assessed in a large study group.
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Acknowledgments
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The authors thank Martina Flechsig, Hannelore Gros and Heide Regelsberger for excellent technical assistance and Dr Andreas Busjahn for helping with the statistical analysis.
Conflict of interest statement. None declared.
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References
|
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- Kallenberg CG, Rarok A, Stegeman CA. Genetics of ANCA-associated vasculitides. Cleve Clin J Med 2002; 69 [Suppl 2]: SII6163[Medline]
- Gencik M, Meller S, Borgmann S, Fricke H. Proteinase 3 gene polymorphisms and Wegener's granulomatosis. Kidney Int 2000; 58: 24732477[CrossRef][ISI][Medline]
- Schreiber A, Busjahn A, Luft CF, Kettritz R. Membrane expression of proteinase 3 is genetically controlled. J Am Soc Nephrol 2002; 13: 172A
- Rarok AA, Stegeman CA, Limburg PC, Kallenberg GM. Neutrophil membrane expression of proteinase 3 is related to relapse in PR3-ANCA-associated vasculitis. J Am Soc Nephrol 2002; 13: 22322238[Abstract/Free Full Text]
- Schonermarck U, Lamprecht P, Csernok E, Gross WL. Prevalence and spectrum of rheumatic diseases associated with proteinase 3-antineutrophil cytoplasmic antibodies (ANCA) and myeloperoxidase-ANCA. Rheumatology 2001; 40: 178184[Abstract/Free Full Text]
- Hagen EC, Daha MR, Hermans J et al. Diagnostic value of standardized assays for anti-neutrophil cytoplasmic antibodies in idiopathic systemic vasculitis. EC/BCR Project for ANCA Assay Standardization. Kidney Int 1998; 53: 743753[CrossRef][ISI][Medline]
- Cascorbi I, Henning S, Brockmoller J et al. Substantially reduced risk of cancer of the aerodigestive tract in subjects with variant 463A of the myeloperoxidase gene. Cancer Res 2000; 60: 644649[Abstract/Free Full Text]
- London SJ, Lehman TA, Taylor JA. Myeloperoxidase genetic polymorphism and lung cancer risk. Cancer Res 1997; 57: 50015003[Abstract]
- Charles LA, Caldas ML, Flak RJ, Terrell RS, Jennette JC. Antibodies against granule proteins activate neutrophils in vitro. J Leukoc Biol 1991; 50: 539546[Abstract]
- Kallenberg CG. Autoantibodies to myeloperoxidase: clinical and pathophysiological significance. J Mol Med 1998; 76: 682687[CrossRef][ISI][Medline]
- Reumaux D, De Boer M, Duthilleul P, Roos D. Neutrophils from a completely myeloperoxidase-deficient donor are not activated by anti-MPO monoclonal antibodies. Clin Exp Immunol 2000; 120 [Suppl]: 40
- Sham PC, Curtis D, MacLean CJ. Likelihood ratio tests for linkeage and linkeage disequilibrium:asymptotic distribution and power. Am J hum Genet 1996; 58: 10931096[ISI][Medline]
- Reynolds WF, Stegeman CA, Cohen Tervaert JW. 463 G/A myeloperoxidase promoter polymorphism is associated with clinical manifestations and the course of disease in MPO-ANCA-associated vasculitis. Clin Immunol 2002; 103: 154160[CrossRef][ISI][Medline]
Received for publication: 28. 4.03
Accepted in revised form: 5.11.03