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The allelic-specific fluorescence was measured using an ABI PRISM 7900 Sequence Detector System (Applied Biosystems). Water control and previously genotyped samples were included in each plate to ensure accuracy of genotyping. When possible, the BTNL2 genotype was verified by sequencing using the following primers adapted from (16): BTNL2-e5-F, 5��-GGTTTCTAAACTCCAATGGGACTGTT-3�� and BTNL2-e5-R, 5��-CAAATGTCGAGAAAATTGTCCGAGA-3��. PCR amplification was performed in 50??l containing 0.5??M primers, approximately 50?ng genomic DNA, 1.0?mM MgCl, 0.2?mM dNTP, and 2.0?U AccuPOL polymerase (Ampliqon, Bie & Berntsen, Copenhagen, Denmark). Thermocycling was performed on an Eppendorf Mastercycler (Eppendorf Nordic Aps, H?rsholm, Denmark) with initial denaturing MG-132 in vivo at 95��C for 5?min. This was followed by 35 cycles each consisting of 45?s denaturation at 95��C, 45?s annealing at 60��C, and 90?s extension ATPase at 72��C. The process was concluded with a final extension of 10?min at 72��C. The PCR products were purified using Nucleofast 96 PCR plates (Macherey-Nagel GmbH & Co. KG, Dueren, Germany) and sequenced using the BigDye Terminator v1.1 Cycle Sequencing kit (Applied Biosystems) and analysed on an ABI3730 DNA analyser (Applied Biosystems). Sequencing results were assembled and analysed using the SeqScape v2.5 software program (Applied Biosystems). We performed the statistical analyses using SPSS 15.0 for Windows [Release 15.0.1.1 (July 3, 2007), SPSS Inc., Chicago, IL, USA]. BTNL2 variation frequencies in the sarcoidosis group and the control group were compared using the ��2-test and odds ratios (OR) were calculated for disease risk. Variables concerning clinical parameters and lung function parameters were compared using the Mann�CWhitney test and ��2-test as appropriate. The risk of developing disease when carrying a specific genotype was assessed by odds ratio. The population attributable fraction (PAF) was calculated using the formula by Browner and Newman (21). PAF indicates the proportion of Epacadostat sarcoidosis cases that could be avoided or prevented if the gene A allele was not present as a risk factor The significance level was set at P?=?0.05. The distribution of the BTNL2 genotypes was in Hardy-Weinberg equilibrium in both patients (P?=?0.7) and controls (P?=?0.2). The genotype distribution is shown in Table?2. The A allele frequency was significantly increased in sarcoidosis patients (A?=?73.9%, G?=?26.1%) compared with controls (A?=?55.8%, G?=?44.2%; P?
