Clinical and genetic factors associated with radiographic damage in patients with ankylosing spondylitis

Abstract

Objectives: To identify clinical and genetic factors associated with severe radiographic damage in patients with ankylosing spondylitis (AS).

Methods: We newly generated genome-wide single nucleotide polymorphism data (833K) for 444 patients with AS. The severity of radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). To identify clinical and genetic factors associated with severe radiographic damage, multiple linear regression analyses were performed. Human AS-osteoprogenitor and control-osteoprogenitor cells were used for functional validation.

Results: The significant clinical factors of final mSASSS were baseline mSASSS (β=0.796, p=3.22×10^-75), peripheral joint arthritis (β=-0.246, p=6.85×10^-6), uveitis (β=0.157, p=1.95×10^-3), and smoking (β=0.130, p=2.72×10^-2) after adjusting for sex, age and disease duration. After adjusting significant clinical factors, the Ryanodine receptor 3 (RYR3) gene was associated with severe radiographic damage (p=1.00×10^-6). For pathway analysis, the PI3K-Akt signalling pathway was associated with severe radiographic damage in AS (p=2.21×10^-4, false discovery rate=0.040). Treatment with rhodamine B, a ligand of RYR3, dose-dependently induced matrix mineralisation of AS osteoprogenitors. However, the rhodamine B-induced accelerated matrix mineralisation was not definitive in control osteoprogenitors. Knockdown of RYR3 inhibited matrix mineralisation in SaOS2 cell lines.

Conclusions: This study identified clinical and genetic factors that contributed to better understanding of the pathogenesis and biology associated with radiographic damage in AS.

Keywords: Ankylosing Spondylitis; Autoimmune Diseases; Polymorphism, Genetic.

Figure 1

Rhodamine B, a ligand of RYR3, promotes the matrix mineralisation in AS osteoprogenitors. Control (CON) and AS osteoprogenitors were differentiated into osteoblasts and continually stimulated by vehicle or rhodamine B with indicated dose during osteoblast differentiation. Osteogenic differentiation activity was assessed by (A) alkaline phosphates (ALP) and collagen (COL) staining, (B) ALP activity of (A), (C) Alizarin red staining (ARS) and ARS quantification, (D) von Kossa staining (VON) and mineralisation area (%), (E) hydroxyapatite (HA) staining and HA quantification.

Figure 2

RYR3 knockdown inhibits matrix mineralisation in SaOS2 cells. SaOS2 cells were transfected with siRNA against human RYR3 and control (CON), incubated for 48 hours, and differentiated for day 7. The stimulated cells were subjected to (A) RT-PCR, (B) immunoblotting, and (C) immunofluorescence. (D) Osteogenic differentiation activity was assessed by alkaline phosphates (ALP), collagen (COL), alizarin red staining (ARS), von Kossa staining (VON), and hydroxyapatite (HA). (E) Quantification data of figure D (n=4). Representative images are shown.