The Functional Characterisation of Risk Loci Associated to Musculoskeletal Diseases

  • Antonios Frantzeskos

Student thesis: Phd

Abstract

Background: Autoimmune disorders such as Rheumatoid Arthritis (RA) and Juvenile Idiopathic Arthritis (JIA) are influenced by genetic and environmental factors. Genome-wide association studies (GWAS) have identified numerous single nucleotide polymorphisms (SNPs) associated with these diseases, yet their functional implications remain elusive. A significant fraction of these SNPs are located in non-coding regions and are enriched in regulatory elements, underscoring their potential functional relevance. Aims: The primary aim of this study was to functionally characterise risk-associated SNPs in RA and JIA through an integrative approach that combines genomic and epigenomic data, followed by validation using CRISPR-Cas9 approaches. Methods: With a focus on CD4+ T-cells, epigenomic data was utilised, including chromatin marks, open chromatin and chromatin interactions as well as expression quantitative trait loci (eQTLs) for annotation of GWAS loci in RA and JIA to prioritise putative causal SNPs. For functional validation, a suite of CRISPR-based methodologies was employed. CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) were used to modulate the expression levels of target genes implicated by the prioritised SNPs. Additionally, CRISPR knockout (KO) and base editing (BE) techniques were utilised to create more targeted alterations in the genomic regions of interest. Results: In RA, fine-mapped SNPs rs3087243, rs117701653, and rs55686954 within the CTLA4 locus were examined. Despite their location in predicted enhancer regions and functioning as eQTLs, CRISPRa/i targeting did not reveal a link to any surrounding genes. However, a modest yet statistically significant upregulation of ICA1L was observed when targeting rs3087243. This was not observed in KO of the same SNP in primary CD4+ T-cells. In JIA, four loci—CCR3, HIPK1, CCR4, and AHI1—were identified for functional validation. Notably, rs80054040 in the CCR3 locus demonstrated a clear regulatory role in the upregulation of CCR2 and CCR5. At the AHI1 locus, rs7750586 and rs13197384 were prioritised, with the latter leading to a substantial increase in AHI1 expression upon CRISPR activation. In contrast, SNPs in the HIPK1 and CCR4 loci did not exhibit a discernible regulatory role. Conclusion: This study contributes to the elucidation of the complex genetic architecture of RA and JIA by integrating genomic and epigenomic data for the functional validation of risk SNPs. While some SNPs did not exhibit a clear regulatory role, others like rs80054040 in the CCR3 locus and rs13197384 in the AHI1 locus were functionally validated, offering novel insights into the mechanistic pathways modulated by these SNPs. These findings underscore the importance of empirical validation in understanding the functional consequences of risk SNPs, thereby advancing our comprehension of the pathogenesis of RA and JIA.
Date of Award1 Aug 2024
Original languageEnglish
Awarding Institution
  • The University of Manchester
SupervisorStephen Eyre (Supervisor) & Gisela Orozco (Supervisor)

Keywords

  • Functional genomics
  • CRISPRa/i
  • rheumatoid arthritis
  • juvenile idiopathic arthritis
  • complex disease genetics

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