Association study with Wegener granulomatosis of the human phospholipase Cγ2 gene

BACKGROUND: Wegener Granulomatosis (WG) is a multifactorial disease of yet unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis. Analyses of candidate genes revealed several associations, e.g. with α(1)-antitrypsin, proteinase 3 and with the HLA-DPB1 locus. A mutation in the abnormal limb mutant 5 (ALI5) mouse in the region coding for the hydrophobic ridge loop 3 (HRL3) of the phospholipaseCγ2 (PLCγ-2) gene, corresponding to human PLCγ-2 exon 27, leads to acute and chronic inflammation and granulomatosis. For that reason, we screened exons 11, 12 and 13 coding for the hydrophobic ridge loop 1 and 2 (HRL1 and 2, respectively) and exon 27 of the PLCγ-2 protein by single strand conformation polymorphism (SSCP), sequencing and PCR/ restriction fragment length polymorphism (RFLP) analyses. In addition, we screened indirectly for disease association via 4 microsatellites with pooled DNA in the PLCγ-2 gene. RESULTS: Although a few polymorphisms in these distinct exons were observed, significant differences in allele frequencies were not identified between WG patients and respective controls. In addition, the microsatellite analyses did not reveal a significant difference between our patient and control cohort. CONCLUSION: This report does not reveal any hints for an involvement of the PLCγ-2 gene in the pathogenesis of WG in our case-control study

On the Wegener granulomatosis associated region on chromosome 6p21.3

BACKGROUND: Wegener granulomatosis (WG) belongs to the heterogeneous group of systemic vasculitides. The multifactorial pathophysiology of WG is supposedly caused by yet unknown environmental influence(s) on the basis of genetic predisposition. The presence of anti-neutrophil cytoplasmic antibodies (ANCA) in the plasma of patients and genetic involvement of the human leukocyte antigen system reflect an autoimmune background of the disease. Strong associations were revealed with WG by markers located in the major histocompatibility complex class II (MHC II) region in the vicinity of human leukocyte antigen (HLA)-DPB1 and the retinoid X receptor B (RXRB) loci. In order to define the involvement of the 6p21.3 region in WG in more detail this previous population-based association study was expanded here to the respective 3.6 megabase encompassing this region on chromosome 6. The RXRB gene was analysed as well as a splice-site variation of the butyrophilin-like (BTNL2) gene which is also located within the respective region. The latter polymorphism has been evaluated here as it appears as a HLA independent susceptibility factor in another granulomatous disorder, sarcoidosis. METHODS: 150–180 German WG patients and a corresponding cohort of healthy controls (n = 100–261) were used in a two-step study. A panel of 94 microsatellites was designed for the initial step using a DNA pooling approach. Markers with significantly differing allele frequencies between patient and control pools were individually genotyped. The RXRB gene was analysed for single strand conformation polymorphisms (SSCP) and restriction fragment length polymorphisms (RFLP). The splice-site polymorphism in the BTNL2 gene was also investigated by RFLP analysis. RESULTS: A previously investigated microsatellite (#1.0.3.7, Santa Cruz genome browser (UCSC) May 2004 Freeze localisation: chr6:31257596-34999883), which was used as a positive control, remained associated throughout the whole two-step approach. Yet, no additional evidence for association of other microsatellite markers was found in the entire investigated region. Analysis of the RXRB gene located in the WG associated region revealed associations of two variations (rs10548957 p(allelic )= 0.02 and rs6531 p(allelic )= 5.20 × 10(-5), OR = 1.88). Several alleles of markers located between HLA-DPB1, SNP rs6531 and microsatellite 1.0.3.7 showed linkage disequilibrium with r(2 )values exceeding 0.10. Significant differences were not demonstrable for the sarcoidosis associated splice-site variation (rs2076530 p(allelic )= 0.80) in our WG cohort. CONCLUSION: Since a microsatellite flanking the RXRB gene and two intragenic polymorphisms are associated significantly with WG on chromosome 6p21.3, further investigations should be focussed on extensive fine-mapping in this region by densely mapping with additional markers such as SNPs. This strategy may reveal even deeper insights into the genetic contributions of the respective region for the pathogenesis of WG

Increased neutrophil membrane expression and plasma level of proteinase 3 in systemic vasculitis are not a consequence of the −564 A/G promotor polymorphism

Several findings link proteinase 3 (PR3) to small vessel vasculitis. Besides being a major target of anti-neutrophil cytoplasm antibodies (ANCA), previous findings have shown increased circulating levels of PR3 in vasculitis patients, increased levels of neutrophil membrane-PR3 (mPR3) expression and a skewed distribution of the −564 A/G polymorphism in the promotor region of the PR3 gene. In this study we elucidate how these three findings relate to each other. The plasma concentration of PR3 was measured by enzyme-linked immunosorbent assay (ELISA), mPR3 expression by fluorescence activated cell sorter (FACS) and the gene polymorphism by real-time polymerase chain reaction (PCR). We compared results from 63 patients with ANCA-associated systemic vasculitis (AASV) with 107 healthy blood donors. In accordance with previous reports, AASV patients had increased plasma concentrations of PR3 compared to healthy controls (mean 224 µg/l versus 155 µg/l, P < 0·0001). They also showed an increased number of mPR3-positive neutrophils (60%versus 42%, P < 0·001). However, contrary to a previous report, we found no skewed distribution of the polymorphism in PR3 gene. There was a weak correlation between mPR3 mean fluorescence intensity (MFI) and plasma PR3 among healthy controls and myeloperoxidase–ANCA (MPO–ANCA)-positive patients (r = 0·24, P = 0·015 and r = 0·52, P = 0·011, respectively). In conclusion, increased plasma PR3 and high expression of mPR3 are associated with small vessel vasculitis, but neither of them is a consequence of the −564 A/G polymorphism of the PR3 gene promotor