E067 Four SNPs associated with monocyte/macrophage cell lineage uniquely associated with CRPS-1 in discovery and replication cohorts and suggest predisposition to regional osteopaenia and digit misperception

Abstract

Abstract Background/Aims Do patients with CRPS1 have overexpressed SNPs in their exome sequencing? Do these SNP alleles correlate with phenotype? Methods We performed exome sequencing in a discovery cohort of well-characterised patients with CRPS using the Agilent 51M kit, sequencing to an average of 50-fold coverage. We used the vcf, bam and bam.bai files for genome wide SNP allele frequency assessment using the fSNPd programme. Deviations from expected frequency against 3 normative data sources were assessed for significance using a Chi-squared test with two tails and Yates correction. The resulting P values were subjected to a false discovery rate (FDR) correction as approximately 100,000 SNPs were assessed. Significant P-value SNPs were then filtered using bioinformatics, seeking SNPs where the alternative allele was predicted to have deleterious effects on the translated protein. We used the Human Genome Browser, NCBI BLASTP for protein sequence comparisons, Conserved Domains (CD search) for detecting if the amino acid change occurred within a known protein domain, and the SIFT and REVEL programs to predict if mis-sense mutations were deleterious. SNPs identified in the Discovery cohort were individually Sanger sequenced using genomic DNA in a Replication cohort. Patients with fibromyalgia were recruited as controls. Samples were collected from all the cohorts between 2014-19 after NRES approval (12/EE/40486; Cambridge Central). A subgroup of Cambridge patients were assessed for pain phenotype, including novel clinical signs, and radiographic osteopaenia (2 independent, blinded assessors (MSK Radiologist and Rheumatologist Consultants)). Results In total 5 SNPs were found to be overexpressed in the Discovery cohort (n = 37). All 5 SNPs were to be found to be functionally active and expressed in cells from the macrophage-monocyte lineage. 4 of these 5 SNPs were then found to be overrepresented in the Replication cohort (n = 48). The Fibromyalgia cohort did not overexpress these SNPs.24/84 (28.6%) patients expressed these SNPs. More males (8/15 (53.3%)) than females (16/69 (23.2%)) expressed SNPs. 7/27 patients from the phenotyped Cambridge cohort expressed these SNPs. 2/15 had osteopaenia as judged by 2 independent blinded assessors., both having the same SNP. 2/13 without osteopaenia expressed a SNP. There was a 1 in 16 chance of such clustering. Patients with SNPs had more depression and anxiety; less activity; and more likely to have digit misperception. There were no differences in functioning, pain severity, or examination findings. Conclusion Patients with CRPS-1 overexpress the rare allele of 4 SNPs. Remarkably, all 4 SNPs are expressed and functionally active in the same pathway within Monocyte-Macrophage cell lineages. Patients who carry these SNPs are more likely to be male and demonstrate radiographic osteopaenia, anxiety, depression, less activity and digit misperception. These new targets direct further basic and clinical research into pathophysiological mechanisms underpinning CRPS-type 1 and possibly other diseases. Disclosure N. Shenker: Honoraria; Lilly, Roche, Pfizer.