Polymorphisms within the RANK and RANKL Encoding Genes in Patients with Rheumatoid Arthritis: Association with Disease P
- PDF / 614,710 Bytes
- 9 Pages / 595.276 x 790.866 pts Page_size
- 18 Downloads / 158 Views
(2020) 68:24
ORIGINAL ARTICLE
Polymorphisms within the RANK and RANKL Encoding Genes in Patients with Rheumatoid Arthritis: Association with Disease Progression and Effectiveness of the Biological Treatment Joanna Wielińska1 · Katarzyna Kolossa2 · Jerzy Świerkot3 · Marta Dratwa1 · Milena Iwaszko1 · Bartosz Bugaj3 · Barbara Wysoczańska1 · Monika Chaszczewska‑Markowska1 · Sławomir Jeka2,4 · Katarzyna Bogunia‑Kubik1 Received: 10 December 2019 / Accepted: 17 June 2020 © The Author(s) 2020
Abstract Inconsistency of the results regarding the genetic variability within genes coding for receptor activator of nuclear factor κB (RANK) and its ligand (RANKL) in rheumatoid arthritis (RA) prompted us to study the RANK and RANKL polymorphisms as potential biomarkers associated with disease predisposition and response to anti-TNF treatment in a group of Polish patients with RA. This study enrolled 318 RA patients and 163 controls. RANK (rs8086340, C > G; rs1805034, C > T) and RANKL (rs7325635, G > A; rs7988338 G > A) alleles were determined by real-time PCR with melting curve analysis and related with clinical parameters. In addition, RANKL serum levels were measured by ELISA. The RANK rs8086340-G allele was overrepresented among patients as compared to controls (OD = 1.777, p = 0.038). C-reactive protein (CRP) levels were significantly (p 1.2 and DAS28 at endpoint > 3.2 or 0.6 G) is placed within intron 1 and rs1805034 (C > T) missense substitution in exon 6 leads to an amino acid change from alanine to valine. The RANKL gene is located on chromosome 13 and both rs7325635 (G > A) and rs7988338 (G > A) are situated in intron 2 in possible transcription factor binding sites. The minor allele frequencies (MAF) of all the selected SNPs were higher than 0.15.
24
Genomic DNA was isolated from peripheral blood of RA patients and controls using QIAamp DNA Blood Midi Kit (Qiagen, Hilden, Germany) according to recommendations of the manufacturer. RANK (rs8086340, rs1805034) and RANKL (rs7325635, rs7988338) alleles were determined by real-time polymerase chain reaction (PCR) amplification and melting-curve analysis using LightSNiP assay (TIB MOLBIOL, Berlin, Germany) on the LightCycler 480 Real-Time PCR system (Roche Diagnostics, Rotkreuz, Switzerland).
RANKL Serum Level Analysis Serum concentration of RANKL was measured by commercial ELISA kits (DY626, R&D Systems, Minneapolis, MN, USA) according to protocols provided by the manufacturer. The analyses were performed for a subgroup of RA patients consisting of 109 individuals before anti-TNF treatment and 99 controls. The absorbance was measured in a Tecan Sunrise absorbance reader and Magellan software (Tecan Trading AG, Switzerland). The optical density of each well run in duplicate was determined by microplate reader set to 450 nm with wavelength correction set to 570 nm. Peptide concentration in the samples was measured by comparing the optical density of the sample with a computer-generated four parameters logistic curve-fit standard curve.
Statistical Analysis The Hardy–Wei
Data Loading...
