S JIANG1,2,3, V ATHANASOPOULOS1,2, J ELLYARD1,2, A CHUAH4, J CAPPELLO1,2, S PRABHU1, J CARDENAS5, J GU5, J ROCO1,2, I PAPA1,2, M YABAS1, G WALTERS1,2,3, G BURGIO1, K MCKEON1,2, A ENDERS1,2, P CANETE1,2, S ALEXANDER2,6, A KITCHING2,7, N SHEN8, J BABON9, M FIELD2, T ANDREWS2, V PASCUAL5, M COOK1,2, C VINUESA1,2
1Department of Immunology and Infectious Disease, ANU, Australia, 2Centre for Personalised Immunology, ANU, Australia, 3Department of Renal Medicine, The Canberra Hospital, Canberra, Australia, 4Genome Informatics Laboratory, ANU, Australia, 5Baylor Medical Institute, , Baylor, USA, 6Westmead Children’s Hospital, Sydney, Australia, 7Centre for Inflammatory Diseases, Department of Medicine, Monash University , Melbourne, Australia, 8China Australia Centre for Personalised Immunology, Renji Hospital, JiaoTong University, Shanghai , China, 9Walter and Eliza Hall Institute,, Melbourne, Australia
Aim: To assess the contribution of rare genetic variants in BLK to SLE
Background: SLE is a relapsing autoimmune disease of which excessive Type 1 Interferon (T1IFN) production is central to SLE pathogenesis. Genetics represent one of the most potent risk factors for SLE, yet no GWAS-associated variant has a proven contribution to pathophysiology. Although BLK is reproducibly associated with SLE by GWAS, its mechanism remains unelucidated.
Methods: SLE patients and controls underwent whole exome sequencing. BLK, BLK variants and interacting partners were purchased or generated with site directed mutagenesis. IRF5 interaction and phosphorylation was tested by western blot. IFNb activity was measured using a dual luciferase assay in HEK293T and CRISPR-Cas9 engineered Ramos cells. Patient lymphocyte RNA profiling was performed by microarray with QuSAGE algorithm analysis.
Results: Seven unique rare BLK SNPs were found in 22.9% (n=20/87) of SLE patients compared with 6.2% (n=6/97, p<0.0001) of healthy controls or 2.8% (n=3/102, p<0.002) of an immunodeficiency cohort. Computational modelling predicted these SNPs disrupted residues critical to BLK function. Overexpression confirmed significant impairment of kinase ability of BLK SNPs. Similar to the related Src-kinase LYN, wildtype BLK was capable of interacting with and repressing IRF5-mediated IFNb production, although BLK SNPs impaired repression of IFNb expression. CRISPR-Cas9 engineered BLK mutation into Ramos cells resulted in enhanced IFNb expression in response to R848 stimulation. Microarray confirmed increased T1 IFN responsive gene expression in SLE patients with BLK SNPs.
Conclusions: Rare variants in BLK are observed at high frequency in SLE patients. These variants are deleterious, altering protein function enhancing T1IFN expression directly linking genetic variation in BLK to SLE pathogenesis.