The binding site for the SP1 transcription factor at 463G/A in the G allele is a plausible factor for the observed higher expression of MPO [5]

The binding site for the SP1 transcription factor at 463G/A in the G allele is a plausible factor for the observed higher expression of MPO [5]. Our study examined the hypothesis that if MPO was involved in the pathogenesis of vascular injury then individuals with AASV who had the MPO 463G allele, which was associated with higher MPO levels, would have more severe disease than patients with the A allele. (2 = 0904, = 06362) or in presenting serum creatinine concentration based on MPO genotype (2 = 0389, = 08232). There was no significant difference in genotype frequencies between controls (13AA, 102GG, 35GA) and patients (14AA, 97GG, 23GA: 2 = 175, = 0417), patients with Wegener’s granulomatosis (5AA, 53GG, 11GA: 2 = 1864, = 03938) or patients with microscopic polyangiitis (9AA, 44GG, 12GA: 2 = 1682, = 04317). A meta-analysis of our study and two previous studies showed that there was no association between the myeloperoxidase G-463/A polymorphism and the risk of developing ANCA-associated vasculitis; GG GA plus AA (odds ratio 114; 95% confidence interval 086C150). The MPO G-463/A polymorphism is not a risk factor for the development or severity of AASV. polymerase. After denaturation at 95C for 5 min, the cycling conditions were as follows: 35 cycles at 94C for 1 min, 56C for 1 min and 72C for 1 min with a final extension at 72C for 7 min. The PCR products were then digested with 5 units of AciI overnight at 37C and separated on a 2% agarose gel. The G to A substitution at position 463 leads to the loss of a AciI restriction site which yields a 61-base pairs (bp) fragment that serves as an internal control (Fig. 1). The PCR products were then analysed through a 1% agarose gel in TBE buffer [90 mM borate acid, 25 mM ethylenediamine tetraacetic acid (EDTA)] containing 1 g/ml of ethidium bromide and visualized by UV transillumination. Open in a separate window Fig. 1 Agarose gel electrophoresis of amplified DNA by restriction fragment length polymorphism polymerase chain reaction using allele specific primers for myeloperoxidase. Neutrophil isolation Neutrophils were isolated using a method adapted from Toothill using isotonic Percoll. Neutrophils were 99% viable by trypan blue exclusion and were 98C99% pure when stained with haematoxylin. Myeloperoxidase assay MPO activity was analysed in neutrophils from individuals of known MPO allotypes (3A/A, 3G/A and 3G/G). MPO activity was assayed spectrophotometrically by determining the decomposition of hydrogen peroxide using o-dianisidine as the hydrogen donor [12]. Thus, 5 106 neutrophils were lysed with ice-cold 05% hexadecyltrimethylCammonium bromide in 50 mmol/l phosphate buffer (pH 60) (50 l) for 30 min at room temperature. The cell lysate was then centrifuged for 15 min at 12 FGF2 Pafuramidine 500 GA and AA, GG and GA AA, and GG AA in patients with AASV compared with controls. A further analysis was carried out comparing GG GA and AA in patients with anti-MPO antibodies or with anti-PR3 antibodies, with controls. We analysed the data using the fixed effects model and generated odds ratios with 95% confidence intervals (CI) using Review Manager 42. We estimated between study heterogeneity using the 2-based Cochrane Q statistic. Results Patient demographics Of the 134 patients with AASV, 69 had Pafuramidine Wegener’s granulomatosis and 65 had microscopic polyangiitis. Ninety-one patients had PR3CANCA and 43 had MPOCANCA. All patients and controls were genotyped successfully. KaplainCMeier survival analysis There was no difference in survival to renal failure or death in patients with the different MPO alleles (2 = 0904, = 06362) (Fig. 2). Open in a separate window Fig. 2 KaplainCMeier survival estimates Pafuramidine by myeloperoxidase 463G/A polymorphism. MPO genotype and renal function There was no significant difference in serum creatinine concentration at presentation in patients with AASV based on MPO genotype (2 = 0389, = 08232) (Fig. 3). Open in a Pafuramidine separate window Fig. 3 Serum creatinine (Creat) analysis by myeloperoxidase 463G/A polymorphism. Overall distribution of MPO genotypes There was no difference in the overall distribution of MPO genotypes.