Background/Purpose:

SNPs in ERAP2 are strongly associated with ankylosing spondylitis (AS). One associated SNP is rs2248374 which changes the strength of the exon 10 donor splice site, with the G allele resulting in a truncated protein which is degraded by nonsense-mediated decay, resulting in a complete absence of ERAP2. This allele has a frequency of approximately 0.5 in the European descendent population, therefore 25% of the population (those who are GG homozygous) are natural ERAP2 knockouts. We investigated the effect of this associated variant on HLA class I allele presentation, HLA class I heavy chain presentation, ER stress markers and pro-inflammatory cytokine gene transcription in AS.

Methods:

AS patients and healthy controls with either AA or GG homozygous status for rs2248374 were recruited. Ficoll gradient separated PBMCs were analysed with flow cytometry. Anti-CD14-Pacific Blue, anti-CD19-ECD, anti-HLA-A,B,C-PECy7, anti-V alpha7.2-PE, anti-CD161-PerCy5.5, anti-HC10 and anti-HLA-B27 were used to analyse the PBMCs. Cells were analysed as monocytes, B cells and mucosal associated invariant T (MAIT) cells.  Expression levels of endoplasmic reticulum (ER) stress markers (GRP78 and CHOP) and pro-inflammatory genes (TNF, IL-6, IL-17 and IL-22) were assessed by RT-PCR in unsorted PBMCs, normalised to RPL32 housekeeping gene-expression.

Results:

7 AS cases and 8 controls with GG genotype, and 8 AS cases and 8 controls with the AA genotype (all HLA-B27 positive) were analysed. rtPCR patient numbers were: B27+ERAP2 AA = 8 ; B27+ ERAP2 GG = 12; B27-ERAP2 AA = 16; B27-ERAP2 GG = 15. Comparing rs2248374 AA and GG genotype carriers, there were no significant differences in HLA Class I expression or free HLA Class I heavy chain levels either intracellularly or extracellularly, ER stress as measured by expression of markers GRP78 and CHOP, or pro-inflammatory gene expression.  Also, no difference in ER stress markers or free HLA Class I heavy chain levels was noted between AS cases and healthy controls in any cell type studied.

Conclusion:

The study demonstrates that there are not large differences in surface expression of class I antigens or heavy chains, ER stress or proinflammatory cytokine gene expression between the two genotypes in AS cases. This suggests that ERAP2 loss of function variants which are protective against AS do not operate by effects on ER stress or HLA Class I misfolding.  Further, the absence of a difference between cases and controls in measures of either free HLA Class I heavy chain levels or ER stress markers is not consistent with HLA-B27 operating to cause AS through either misfolding or ER stress induction, at least in the cell types studied.

ACR Meeting Abstracts - https://acrabstracts.org/abstract/erap2-functional-knockout-in-humans-does-not-alter-er-stress-or-evidence-of-hla-b27-misfolding-in-ankylosing-spondylitis/