Page 98 - Recognizing axial spondyloarthritis - Janneke de Winter

CHAPTER SIX
for HLA-B (Hs00818803_g1), HLA-C (Hs00740298_g1 and Hs03044135_m1), beta-2 microglobulin (Hs00187842_m1) and GAPDH (4310884E) according to the manufacturer’s protocol (ThermoFisher, MA, USA). mRNA data were missing for n=2 healthy donors because of unreliable results.
Data analyses
Categorical data are presented as numbers (%), continuous data are presented as the mean (SD) or as median (interquartile range, IQR) as appropriate. We used a χ2 test for categorical data and an unpaired T test or Mann-Whitney U for continuous data when data were or were not normally distributed, respectively. Statistical tests were 2-sided, and p-values less than 0.05 were considered significant.
We investigated the association between HLA-C*07 status and disease characteristics in axSpA patients by logistic regression. Variables were first selected by backward elimination using a p-value of <.20 for inclusion and the model was secondly tested by forward selection and included sex, HLA-B*27 status, the presence of peripheral arthritis, enthesitis, dactylitis, inflammatory bowel disease (IBD), psoriasis and sacroiliitis on MRI or X-ray. Since the SPACE cohort includes both axSpA patients and CBP controls, the SPACE cohort enabled us to investigate the association of HLA-C*07 and HLA-B*27 with a diagnosis of axSpA in a multivariate regression analysis. For that purpose, we used the same logistic regression approach as described earlier: firstly, using backwards elimination and secondly testing the model by forward selection. As a sensitivity model we included all axSpA patients in the analysis, irrespective of fulfilling the ASAS criteria.
RESULTS
mRNA expression of HLA-C*07 is decreased in PBMCs of AS patients versus healthy controls
Analysis of mRNA of MHC class 1 molecules by qPCR demonstrated that the expression levels of HLA-B and Beta-2 microglobulin was not different in peripheral blood of patients with AS and healthy controls (Figure 1, panel A and B). On the contrary, expression of HLA-C was absent in 35 out of 51 AS patients, compared to 14 out of 40 healthy controls (68.7% versus 35.0%, p=0.004) (Figure 1C). Further examination revealed that this absence of expression is explained by the binding of the primers and probe of Hs00740298_g1 Taqman assay within the region transcribed from HLA-C serotype group Cw7 (HLA-C*07). qPCR analysis of the same cohort with Hs03044135_m1 HLA-C Taqman assay revealed similar expression levels of HLA-C between AS patients and healthy controls (Figure 1D).
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