Objective Catastrophic antiphospholipid syndrome (CAPS), characterized by widespread thrombosis and multi-organ failure, is associated with high morbidity and mortality. We previously established complement activation as a pathogenic driver of CAPS and identified rare germline variants in complement-regulatory genes including Complement Receptor 1 (CR1) in 50% of CAPS.

Methods We quantified CR1 expression by flow cytometry across hematopoietic cell types. CRISPR/Cas9 genome editing of TF-1 (erythroleukemia) cells was performed to generate CR1 “knock-out” and “knock-in” lines with patient-specific CR1 variants. Multiomics analysis was performed to investigate the role of methylation in CR1 expression in patients with reduced CR1 expression. Functional impact of low CR1 expression was assessed by complement-mediated cell killing using modified Ham (mHam) assay, cell-bound complement degradation products through flow cytometry and circulatory immune complexes (CIC) in serum samples through ELISA.

Results CR1 expression in erythrocytes was markedly reduced on CAPS erythrocytes (n=9, 21.80%) compared to healthy controls (HC; n=32, 82.40%), with promoter hypermethylation emerging as a plausible epigenetic mechanism for CR1 downregulation. A novel germline variant (CR1-V2125L; rs202148801) mitigated CR1 expression and increased complement-mediated cell death of knock-in cell lines. Erythrocytes from the patient with the CR1-V2125L variant had low CR1 expression. Levels of CIC, which are bound and cleared by CR1 on erythrocytes, were higher in acute CAPS (n=3, 25.55 µg Eq/ml) than healthy controls (n=3, 7.445 µg Eq/ml). Five patients were treated with C5 inhibition which mitigated thrombosis.

Conclusion Genetic or epigenetic-mediated CR1 deficiency is a potential hallmark of CAPS and predicts response to C5 inhibition.

N.R., D.F.G., A.R., K.H.: declares no conflicts of interest. M.A.C.1: served on the advisory board of Alexion Pharmaceuticals and holds individual stock in AstraZeneca, Novo Nordisk, and Omeros Pharmaceuticals. G.F.G.: serves on advisory boards of Apellis Pharmaceutical and Alexion Pharmaceuticals. M.A.C.2: reports consultancy or advisory board fees from Bayer, AstraZeneca, Pfizer, Hemostasis Reference Laboratories, Syneos Health and Eversana. E.M.B.: Incyte Corporation: Current Employment, Current equity holder in publicly traded company. K.R.M.: reports consultancy or advisory board fees from Sanofi, Novartis, and Sobi. S.C.: reports consultancy or advisory board fees from Alexion, Sanofi, Takeda, Sobi and Sanofi. K.R.M. reports consultancy or advisory board fees from Sanofi, Novartis, and Sobi. R.A.B.: Alexion Pharmaceuticals: Consultancy. Under a license agreement between Machaon Diagnostics and Johns Hopkins University, R.A.B. and M.A.C.1 and the University are entitled to royalty distributions related to technology described in the study discussed in this publication. This arrangement has been reviewed and approved by the Johns Hopkins University in accordance with its conflict-of-interest policies.

This work was funded by N.H.L.B.I (R56HL133113) and the Department of Defense (W81XWH2110898). The funders had no role in study design, data collection and analysis, the decision to publish or preparation of the manuscript.

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