Genomic profiling and gene-gene interaction in rheumatoid arthritis

Complex disease is characterized by the interplay of multiple genetic and environmental factors. Rheumatoid arthritis (RA) is a complex autoimmune disease with a pronounced genetic component, mainly due to HLA-DRB1 gene, but also a multitude of loci outside the HLA region. In this work we strive to contribute to the understanding of the functional involvement of these susceptibility loci in the pathogenesis of RA.

This study is based on a large material of whole blood samples and peripheral blood mononuclear cells (PBMCs) from RA patients and matched healthy controls from Sweden. The main methods used in this work included probe-based genotyping and gene-expression assays, cell cultures, RNA-sequencing, gene-gene interaction and pathway analysis, as well as a plethora of common molecular genetics and bioinformatics methods.

We investigated the role of expression of known genetic risk factors PTPN22 and PTPN2 in RA, with a special attention to the splicing profile of these genes. Our data indicates significant differences in the expression ratio of splice variants for PTPN22 in whole blood samples from RA patients and healthy controls. For PTPN2 we demonstrate a significant difference in the relative mRNA expression of' transcript TC48 in PBMCs of healthy controls and RA patients. Additionally, we identified new susceptibility SNPs in the PTPN2 locus: rs657555 and rs11080606, by addressing the interaction of PTPN2 variants with HLA-DRB1 shared-epitope (SE) alleles in autoantibody positive RA patients in two independent cohorts.

In this work, we also address the functional genetic role of the members of the MAP signaling pathway upstream of p38 and JNK – crucial enzymes in RA – with a regard to splicing profile and their connection to HLA-DRB1. We found a significant statistical interaction for rs10468473 from MAP2K4 locus with SE alleles in autoantibody-positive RA. Importantly, individuals heterozygous for rs10468473 demonstrated higher expression of total MAP2K4 mRNA in blood, compared to A-allele homozygous. We also describe a novel, putatively translated RNA splice form of MAP2K4, that is differentially expressed in peripheral blood mononuclear cells from 88 RA cases and controls, and is modulated in response to TNF in Jurkat cell line.

Finally, we performed an expression analysis of multiple validated RA risk loci, and pathway analysis to assess functional relationship between RA susceptibility genes and predict new potential study candidates. New candidate molecules suggested by the pathway analysis, genes ERBB2 and HSPB1, as well as HLA-DRB1, were differentially expressed between RA patients and healthy individuals in RNA-seq data. ERBB2 expression profile was similar in whole blood of both treated and untreated patients compared to healthy individuals. A similar expression profile was replicated for ERBB2 in PBMCs in an independent material.

In this work, we approached the task of elucidating the functional aspects of genetic susceptibility of RA, by integrating genetic epidemiology, transcriptomics, proteomics, cellmodels, and bioinformatics. We maintain, that such integrative approach provides the rationale to prioritize genes and genetic events for further functional studies. Our findings also outline the need to consider potential clinical significance of alternative splicing in gene expression studies.

List of scientific papers

I. Ronninger, M., Guo, Y. J., Shchetynsky, K., Hill, A., Khademi, M., Olsson, T., Reddy, P. S., Seddighzadeh, M., Clark, J. D., Lin, L. L., O'Toole, M., & Padyukov, L. (2012). The balance of expression of PTPN22 splice forms is significantly different in rheumatoid arthritis patients compared with controls. Genome Medicine, 4.
https://doi.org/10.1186/gm301

II. Houtman, M., Shchetynsky, K., Catrina, A., Padyukov, L. Strengthening evidence from association studies: PTPN2 genetic variations interact with HLA-DRB1 shared epitope alleles and PTPN2 is differentially expressed in rheumatoid arthritis. [Manuscript]

III. Shchetynsky, K., Protsyuk, D., Ronninger, M., Diaz-Gallo, L. M., Klareskog, L., & Padyukov, L. (2015). Gene-gene interaction and RNA splicing profiles of MAP2K4 gene in rheumatoid arthritis. Clin Immunol, 158, 19-28.
https://doi.org/10.1016/j.clim.2015.02.011

IV. Shchetynsky, K., Diaz-Gallo, L.M., Klareskog, L., Padyukov L. Expression pathway analysis for genes associated with rheumatoid arthritis. [Manuscript]