Association study with Wegener granulomatosis of the human phospholipase Cγ2 gene
- Peter Jagiello†1Email author,
- Stefan Wieczorek†1,
- Philipp Yu2,
- Elena Csernok3,
- Wolfgang L Gross3 and
- Joerg T Epplen1
© Jagiello et al; licensee BioMed Central Ltd. 2005
Received: 07 October 2004
Accepted: 09 February 2005
Published: 09 February 2005
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.
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.
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.
Wegener granulomatosis (WG) is a systemic inflammatory disease of unknown aetiology characterized by granulomata of the respiratory tract and systemic necrotizing vasculitis . There is a strong and specific association with presence of anti-neutrophil cytoplasmatic antibodies to a defined target antigen, proteinase 3 (PR3-ANCA), which is present within primary azurophil granules of neutrophils (PMN) and lysozymes of monocytes . Upon cytokine priming of PMNs, this enzyme translocates to the cell surface, where PR3-ANCAs can interact with their antigens and activate PMNs . It has been shown that PMNs from patients with active WG expressing PR3 on their surfaces produce respiratory burst and release proteolytic enzymes after activation with PR3-ANCA . The consequence is a self-sustaining chronifying inflammatory process. WG appears as a multifactorial disease, and environmental influences still remain elusive. Several factors, such as bacterial infections, have been proposed as probable initiators of the disease . It has been reported that chronic carrier status of Staphylococcus aureus is a risk factor for disease exacerbation in WG . Recently a strong association of WG with distinct HLA-DPB1 alleles or rather an extended haplotype, respectively, in the MHC class II region has been reported . In addition, analyses of candidate genes revealed several associations, e.g. with α(1)-antitrypsin, and proteinase 3 [ and ].
A primary candidate gene, PLCγ-2, was proposed on the basis of a novel animal model system. A mutation in the abnormal limb mutant 5 (ALI5) mouse  causes a phenotype comparable to human autoimmunity disease like WG: inflammations, granulomatosis, affected organs (lung, kidney with glomerulonephritis, eye and skin) and ANCAs were detected in the ALI5 mouse initially. Yet, this result has to be confirmed in further studies. The ALI5 mutation is located in the genomic region coding for the hydrophobic ridge loop 3. In mouse PLCγ-2 mutation leads to acute and chronic inflammations with a phenotype comparable to WG. Therefore, human PLCγ-2 is a good candidate gene for seeking predisposing genetic factors for WG. The human gene is a member of the PLC family comprising 12 closely related molecules involved in signal transduction from numerous receptors . The protein is activated by cytoplasmatic tyrosine kinases (Lyn, Syk, and Btk) which are induced by engagement of B-cell receptors. In turn, the PLCγ-2 activation leads to the generation of diacylglycerol (DAG) and inositol 1,4,5-trisphosphat (IP3). While DAG activates protein kinase C (PKC, ), IP3 mediates Ca2+ mobilization, which is required for activation of B-cells . PLCγ-2 itself is expressed mainly in B-cell, hematopoetic cells, macrophages, granulocytes, testis, sperm, skin and brain . The protein consists of two catalytic domains, which are separated by two SH2 and one SH3 domains . It was established that PLCγ-2 mediates the coupling of G-protein-coupled receptors (GPCRs) and Ca2+ entry in cell lines . The human PLCγ-2 gene is localized on chromosome 16 (16q24.1) spanning 179 kb of genomic DNA. The 4.2 kb mRNA consists of 33 Exon coding for a M r 140,000 protein with 1265 amino acids . PLCγ-2 is highly polymorphic .
Here, we report on an indirect association screen by microsatellite analysis and pooling of DNA in a case-control study design. Furthermore, we screened exons 11, 12 and 13, partly coding for the hydrophobic ridge loop 1 and 2 of PLCγ-2 protein, by single stranded conformation polymorphism (SSCP), sequencing and the PCR/RFLP method. As the ALI5 mutation is located in the region corresponding to the human exon 27, this exon was also screened by SSCP.
Analyses of microsatellites
Primer sequences and information about microsatellites used in study
SSCP, sequencing and PCR/RFLP analyses
Summary of found variations in the human PLCγ-2 gene in exons 11, 12, 13 and 27
frequency of alleles
As recently reported a mutation in the PLCγ-2 gene in the ALI5 mouse leads to acute and chronic inflammation and granulomatosis. In addition, the ALI5 mice show similar symptoms as WG patients. For this reason screening of PLCγ-2 appears as a logical consequence in seeking candidate genes for WG. In the present study PLCγ-2 was analysed by an indirect microsatellite approach using pooled DNA from WG patients with a defined PR3-ANCA+ status and a matched control cohort as reported before . Furthermore, exons partly representing the catalytic domains of the PLCγ-2 protein, HRL1, 2 and 3, were analysed by SSCP, sequencing and by the PCR/RFLP method. We did not find any hints of an involvement of the PLCγ-2 gene in the pathophysiology of WG. Hence, we exclude at this time that the PLCγ-2 gene predisposes for WG in our cohort.
Our study revealed 4 single base substitutions, 2 of which were reported before . A further silent and low frequent SNP was detected in exon 12 which did not differ significantly between patient and control cohorts after PCR/RFLP analyses. As this SNP is the first basepair (bp) after the 3'-splice site one might hypothesise an influence on splicing but to present knowledge this SNP does not change a consensus sequence required for splicing. The ALI5 mutation is located in the HRL3 domain partly corresponding to the human exon 27 of PLCγ-2. Our analysis revealed a SNP (G>A) at position 3030 in one WG patient.
The indirect microsatellite based approach did not reveal any association of PLCγ-2 with WG. Altogether 4 microsatellites spread in the PLCγ-2 gene were analysed. The approach using pooled DNA and ad hoc designed markers intragenic or in the immediate vicinity of a distinct gene has proven to be a reliable and efficient method to detected predisposing loci in WG before . Here, none of the markers did show a significant allele distribution between the patient and control group.
In conclusion, our analysis of the human PLCγ-2 gene did not reveal an association of PLCγ-2 with WG. In contrast to ALI5 mice, where a single mutation leads to distinct symptoms of inflammatory autoimmunity, human WG depends on a more complex genetic background. Further analysis of all exons of PLCγ-2 might yield an association with WG but our microsatellite analysis strongly suggests that predisposition for WG is not due to variations in the PLCγ-2 gene.
Material and Methods
Patients and controls
175 well-characterized patients of German origin with a clinical diagnosis of WG and a defined PR3-ANCA+ status were included in present study. Diagnosis of WG was established according international standards [ and ]. All patients were biopsy-proven. Biopsies were seen in German reference centre for vasculitis (Department of Pathology, University of Schleswig-Holstein Campus Luebeck, Germany) by 2 different observers. A group of 165 healthy individuals of German origin were used as controls. All persons gave informed consent.
Pooling of DNA was performed as reported before . Patient (n = 150) and healthy control (n = 100) individuals from the abovementioned groups were divided into 3 and 2 sub-pools, respectively, containing 50 persons each.
In this study 3 intragenic microsatellites as well as one in the immediate vicinity of the gene were included (table 1; see also UCSC Database, June 2002 Freeze; URL). Oligonucleotides were designed by Primer Express 2.0 software (ABI) adjusted to an annealing temperature of 55°C.
For PCR we used the 'tailed primer PCR' as described before . For amplification three oligonucleotides were used: 1. tailed sense primer (tailed F), 2. anti-sense primer and 3. labeled primer (labeled F) corresponding to the 5'-tail sequence of tailed F. PCR conditions were as follows: 1 × PCR buffer (Qiagen), 1.5 pmol labeled F, 0.2 mM each dNTP, 3 mM MgCl2, 0.2 pmol tailed F, 1.5 pmol reverse primer, 0.25 U Qiagen Hot Start Taq (Qiagen) and 50 ng DNA.
Electrophoreses were run using ABI standard protocols. Raw data were analyzed by the Genotyper software (ABI) producing a marker-specific allele image profile (AIP, see  and ). AIP consists of series of peaks with different heights reflecting the allele frequency within each analyzed DNA pool.
Statistics for comparisons of allele frequencies of patients and controls was performed as described before [ and ]. Case and control distributions were compared statistically by means of contingency tables.
SSCP, sequencing and PCR/RFLP analyses
Exons 11, 12, 13 and 27 were analysed by the SSCP method. PCR was performed using oligonucleotides reported before (, exon 27 corresponding to exon 26). Heat-denaturated fragments were then separated by polyacrylamide gel electrophoresis under non-denaturating conditions yielding specific band patterns for each of the alleles. Results were visualized by autoradiography. Alleles of representative probes were determined by direct DNA sequence analysis on a 377 ABI automatic sequencer (ABI). Afterwards, variations were genotyped individually by PCR/RFLP method with restriction enzymes specific for the respective change (for details see table 2). The variation in exon 27 was individually genotyped by the SSCP method.
- Lamprecht P, Gross WL: Wegener's granulomatosis. Herz. 2004, 29: 47-56. 10.1007/s00059-004-2525-0.View ArticlePubMedGoogle Scholar
- Csernok E, Muller A, Gross WL: Immunopathology of ANCA-associated vasculitis. Intern Med. 1999, 38: 759-765.View ArticlePubMedGoogle Scholar
- Csernok E, Ernst M, Schmitt W, Bainton DF, Gross WL: Activated neutrophils express proteinase 3 on their plasma membrane in vitro and in vivo. Clin Exp Immunol. 1994, 95: 244-250.View ArticlePubMedPubMed CentralGoogle Scholar
- van der Geld YM, Limburg PC, Kallenberg CG: Proteinase 3, Wegener's autoantigen: from gene to antigen. J Leukoc Biol. 2001, 69: 177-190.PubMedGoogle Scholar
- Brons RH, Bakker HI, Van Wijk RT, Van Dijk NW, Muller Kobold AC, Limburg PC, Manson WL, Kallenberg CG, Tervaert JW: Staphylococcal acid phosphatase binds to endothelial cells via charge interaction; a pathogenic role in Wegener's granulomatosis?. Clin Exp Immunol. 2000, 119: 566-573. 10.1046/j.1365-2249.2000.01172.x.View ArticlePubMedPubMed CentralGoogle Scholar
- Popa ER, Tervaert JW: The relation between Staphylococcus aureus and Wegener's granulomatosis: current knowledge and future directions. Intern Med. 2003, 42: 771-780.View ArticlePubMedGoogle Scholar
- 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. 2004, 114: 468-477. 10.1007/s00439-004-1092-z.View ArticlePubMedGoogle Scholar
- Esnault VL, Testa A, Audrain M, Roge C, Hamidou M, Barrier JH, Sesboue R, Martin JP, Lesavre P: Alpha 1-antitrypsin genetic polymorphism in ANCA-positive systemic vasculitis. Kidney Int. 1993, 43: 1329-1332.View ArticlePubMedGoogle Scholar
- Gencik M, Meller S, Borgmann S, Fricke H: Proteinase 3 gene polymorphisms and Wegener's granulomatosis. Kidney Int. 2000, 58: 2473-2477. 10.1046/j.1523-1755.2000.00430.x.View ArticlePubMedGoogle Scholar
- Yu P, Constien R, Dear N, Katan M, Hanke P, Bunney TD, Kunder S, Quintanilla-Martinez L, Huffstadt U, Schroeder A, Jones NP, Peters T, Fuchs H, Hrabe de Angelis M, Nehls M, Grosse J, Wabnitz P, Meyer TPH, Yasuda K, Schiemann M, Schneider-Fresenius C, Jagla W, Russ A, Popp A, Josephs M, Marquardt A, Laufs J, Schmittwolf C, Wagner H, Pfeffer K, Mudde GC: Autoimmunity and inflammation due to a gain-of-function mutation in phospholipase Cγ2 that specifically increases external Ca2+ entry. Immunity.
- Rhee SG: Regulation of phosphoinositide-specific phospholipase C. Annu Rev Biochem. 2001, 70: 281-312. 10.1146/annurev.biochem.70.1.281.View ArticlePubMedPubMed CentralGoogle Scholar
- Rhee SG, Bae YS: Regulation of phosphoinositide-specific phospholipase C isozymes. J Biol Chem. 1997, 272: 15045-15048. 10.1074/jbc.272.24.15045.View ArticlePubMedGoogle Scholar
- Kurosaki T, Maeda A, Ishiai M, Hashimoto A, Inabe K, Takata M: Regulation of the phospholipase C-gamma2 pathway in B cells. Immunol Rev. 2000, 176: 19-29. 10.1034/j.1600-065X.2000.00605.x.View ArticlePubMedGoogle Scholar
- Marshall AJ, Niiro H, Yun TJ, Clark EA: Regulation of B-cell activation and differentiation by the phosphatidylinositol 3-kinase and phospholipase Cgamma pathway. Immunol Rev. 2000, 176: 30-46. 10.1034/j.1600-065X.2000.00611.x.View ArticlePubMedGoogle Scholar
- Manning CM, Mathews WR, Fico LP, Thackeray JR: Phospholipase C-gamma contains introns shared by src homology 2 domains in many unrelated proteins. Genetics. 2003, 164: 433-442.PubMedPubMed CentralGoogle Scholar
- Patterson RL, van Rossum DB, Ford DL, Hurt KJ, Bae SS, Suh PG, Kurosaki T, Snyder SH, Gill DL: Phospholipase C-gamma is required for agonist-induced Ca2+ entry. Cell. 2002, 111: 529-541. 10.1016/S0092-8674(02)01045-0.View ArticlePubMedGoogle Scholar
- Emori Y, Homma Y, Sorimachi H, Kawasaki H, Nakanishi O, Suzuki K, Takenawa T: A second type of rat phosphoinositide-specific phospholipase C containing a src-related sequence not essential for phosphoinositide-hydrolyzing activity. J Biol Chem. 1989, 264: 21885-21890.PubMedGoogle Scholar
- Wang D, Boylin EC, Minegishi Y, Wen R, Smith CI, Ihle JN, Conley ME: Variations in the human phospholipase Cgamma2 gene in patients with B-cell defects of unknown etiology. Immunogenetics. 2001, 53: 550-556. 10.1007/s002510100356.View ArticlePubMedGoogle Scholar
- Leavitt RY, Fauci AS, Bloch DA, Michel BA, Hunder GG, Arend WP, Calabrese LH, Fries JF, Lie JT, Lightfoot RW: The American College of Rheumatology 1990 criteria for the classification of Wegener's granulomatosis. Arthritis Rheum. 1990, 33: 1101-1107.View ArticlePubMedGoogle Scholar
- Jennette JC, Falk RJ, Andrassy K, Bacon PA, Churg J, Gross WL, Hagen EC, Hoffman GS, Hunder GG, Kallenberg CG: Nomenclature of systemic vasculitides: Proposal of an international consensus conference. Arthritis Rheum. 1994, 37: 187-192.View ArticlePubMedGoogle Scholar
- Goedde R, Sawcer S, Boehringer S, Miterski B, Sindern E, Haupts M, Schimrigk S, Compston A, Epplen JT: A genome screen for linkage disequilibrium in HLA-DRB1*15-positive Germans with multiple sclerosis based on 4666 microsatellite markers. Hum Genet. 2002, 111: 270-277. 10.1007/s00439-002-0801-8.View ArticlePubMedGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.