The - 564 A/G polymorphism in the promoter region of the proteinase 3 gene associated with Wegener's granulomatosis does not increase the promoter activity.
Proteinase 3 is the major autoantigen in patients with Wegener's granulomatosis. Earlier studies have shown that circulating leucocytes from patients with Wegener's granulomatosis show elevated proteinase 3 surface expression and mRNA levels. Wegener's granulomatosis patients also have increased levels of proteinase 3 in plasma. A single nucleotide polymorphism (SNP) (-564 A/G SNP) in the promoter region has been associated with disease. This SNP introduces a new potential Sp1 transcription factor binding site that may be responsible for the observed up-regulated expression of proteinase 3. To investigate this a 740 base pair long region of the promoter was cloned from genomic DNA. The disease-associated -564 A/G, as well as a control -621 A/G exchange, were introduced by polymerase chain reaction mutagenesis and cloned into a luciferase reporter vector. Endogenous expression levels of proteinase 3 mRNA and promoter activity of the cloned constructs were measured in three myeloid cell lines, HL-60, U937 and NB-4, and in epithelial HeLa cells. The results demonstrate a good correlation between the endogenous proteinase 3 mRNA expression and the promoter activity, as judged by luciferase activity. However, no significant differences in activity between the wild-type, polymorphic and the mutated control variant were found. In conclusion, the -564 A/G polymorphism is not responsible for the increased expression levels seen in myeloid cells from patients with Wegener's granulomatosis.
Pieters
K
K
Department of Haematology, Lund University, Lund, Sweden. Katrien.Pieters@hematologi.lu.se
Pettersson
A
A
Gullberg
U
U
Hellmark
T
T
D019268:Antibodies, Antineutrophil Cytoplasmic / Q000235:genetics / Q000276:immunology; D045744:Cell Line, Tumor; D014890:Granulomatosis with Polyangiitis / Q000235:genetics* / Q000276:immunology; D018922:HL-60 Cells; D006367:HeLa Cells; D006801:Humans; D053146:Myeloblastin; D020641:Polymorphism, Single Nucleotide / Q000235:genetics* / Q000276:immunology; D011401:Promoter Regions, Genetic / Q000235:genetics* / Q000276:immunology; D012333:RNA, Messenger / Q000032:analysis; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D012697:Serine Endopeptidases / Q000235:genetics* / Q000276:immunology; D014158:Transcription, Genetic; D014162:Transfection / Q000379:methods; D020298:U937 Cells
D019268:Antibodies, Antineutrophil Cytoplasmic; D012333:RNA, Messenger; D012697:Serine Endopeptidases; D053146:Myeloblastin