Functional genomics of ankylosing spondylitis
Keywords: fine-mapping
functional genomics
NPEPPS
ankylosing spondylitis

Ankylosing spondylitis (AS) is a chronic inflammatory arthritis primarily affecting the joints of the spine and pelvis. Its prevalence is highly correlated with the HLA-B*27 allele but not all of the genetic risk for this complex disease is explained by this association. At least 113 other susceptibility loci have been identified, with the NPEPPS locus implicated by multiple high impact studies.
This thesis aimed to fine-map the AS disease-associated signal that overlaps the NPEPPS region on chromosome 17q21. Using statistical methods and epigenomic annotation it attempts to identify and prioritise the causal variant(s) and/or gene at this region. It then aims to validate the mechanistic role these prioritised variants play in AS.
Illumina CoreExome genotyping data from 8,244 AS cases and 14,542 healthy controls of European ancestry was used to fine-map the NPEPPS region (total cohort=22,786). Frequentist and Bayesian analysis were performed to identify sets of casual SNPs. The NPEPPS region was then annotated using epigenomic data from publicly available datasets and in-house disease-specific data to identify prioritised causal variants that overlapped with functional elements in the genomic landscape of AS. RT-qPCR was performed to compare the gene expression levels of NPEPPS and TBKBP1 between AS cases and controls in PBMCs. Luciferase assays were performed to assess the function of the prioritised putative regulatory elements and the impact of specific genotypes.
Frequentist analysis in PLINK identified rs4794057 (risk allele=T, OR=1.12, p=2.4x10-11) as the lead SNP at the NPEPPS region. 96 SNPs were identified at genome-wide significance (p<5x10-8), across 307,006bp. Bayesian analysis improved on this approach to identify a 99% credible set containing 62 SNPs across 211,521bp overlapping three genes (NPEPPS, KPNB1, TBKBP1). Annotation of the NPEPPS region with public epigenomic datasets and in-house AS-specific data identified three regulatory regions with a putative role in disease: a CD4-specific putative enhancer region upstream of NPEPPS, a CD8-specific putative enhancer region between NPEPPS and KPNB1, and a putative promoter for TBKBP1. Following RT-qPCR experiments, lower expression of NPEPPS and TBKBP1 was observed in AS cases compared to controls with a significant expression fold change of <0.64 and 0.02, respectively. In luciferase reporter assays the NPEPPS-KPNB1 putative enhancer was shown to significantly increase relative luciferase activity (>2.5-fold).
This study has successfully improved on previous fine-mapping results of the NPEPPS region. Functional experiments identified lower expression of NPEPPS and TBKBP1 in AS cases compared to healthy controls and an enhancer region was identified in the NPEPPS-KPNB1 intergenic region tagged by the AS-associated SNP rs4375701. Further work is required to identify the regulatory region responsible for the differential expression observed and explain its mechanistic role in disease. This fine-mapping strategy should be implemented to improve the current understanding of known AS-associated loci which could potentially lead to the discovery of new disease pathways and new therapies for this debilitating disease.