Optimization of methods to study associations between genetic variations, clinical outcomes, and response to biologic treatments in patients with juvenile idiopathic arthritis

Abstract

Juvenile idiopathic arthritis (JIA) is an autoimmune disease seen in children younger than 16 years of age. The immunological background of JIA is driven by dysregulated activation of innate and adaptive immune pathways, where pro-inflammatory cytokines such as interleukin-6 (IL-6) and tumour necrosis factor alpha (TNF-α) play a central role. Single nucleotide polymorphisms (SNPs) in cytokine encoding genes can contribute to its pathogenesis. The first line of biologic treatment for JIA usually involves TNF-α and IL-6 inhibitors. However, about 20-40% JIA patients do not respond to the prescribed biologic treatment. Ineffectiveness of biologic treatment has been linked to certain SNPs related to cytokine genes among other contributing factors. To associate cytokine levels and genotype, identification of these SNPs and quantification of cytokines from stimulated peripheral blood mononuclear cells (PBMCs) of patients are important. In this project, six and five relevant SNPs of TNF-α promoter region and IL-6 receptor (IL-6R) gene were targeted based on their reported associations with the expression of the encoded proteins and response to biologic medication. PCR primers were designed using NCBI primer blast. PCR conditions were optimized to amplify segments of DNA containing one or multiple of the target SNPs. The optimization scheme for the PBMC stimulation assay was made targeting density of cells, stimulating agents and their concentration, and incubation time before measuring cytokines from cell culture supernatants. All TNF-α SNPs and four of the IL-6R SNPs were successfully amplified by PCR and sequenced by Sanger sequencing. DNA samples of 108 JIA patients were analysed and compared to 99 healthy Finns from the 1000 Genome Project. In IL-6R, SNPs rs4329505 (T/C) (p = 0.007) and rs11265618 (C/T) (p = 0.006), the presence of minor alleles was associated with more active disease based on the number of biological medications needed. Moreover, minor alleles in rs12083537 (A/G) and rs4329505 (T/C) are associated with higher likelihood of polyarthritis (p = 0.039) and extended oligoarthritis (p = 0.024) respectively. No statistically significant correlations were found in the TNF-α SNPs. However, TNF-α SNPs, rs1799964(T>C) and rs1800630(C>A) showed significantly stronger linkage disequilibrium (LD) in the JIA cohort compared to healthy controls. Moreover, it is possible to measure TNF-α concentrations if ≥2×105 PBMCs per well are stimulated with 1 µg/mL LPS for 6 hours. More in-depth analysis of genetic information can reveal details of these associations and may aid in treatment decisions. The PBMC stimulation assay needs further optimization.