Genetic or Epigenetic CR1 Deficiency Defines Catastrophic Antiphospholipid Syndrome (CAPS) and Response to Complement Inhibition

Introduction: CAPS is a life-threatening manifestation of the antiphospholipid syndrome (APS) characterized by widespread thrombosis and multi-organ failure. Despite standard-of-care therapies, CAPS is associated with >30% mortality. Complement is implicated in the pathophysiology of thrombosis in CAPS, and complement inhibition shows efficacy as salvage therapy. We previously used the modified HAM (mHam) assay to demonstrate complement activation in >85% of CAPS sera (Chaturvedi, Blood, 2020). Further, 48% (9/19) of CAPS patients had rare germline variants in complement regulatory genes, a frequency similar to atypical hemolytic uremic syndrome. Of these, 33% (3/9) harbored rare variants in Complement Receptor 1 (CR1), which have not been reported in aHUS. CR1 is expressed predominantly on erythrocytes, and essential for the clearance of immune complexes and complement regulation. We investigated the functional significance of the CR1 variants and molecular mechanisms regulating associated CR1 expression in CAPS. Methods: We performed CRISPR/Cas9 genome editing of TF-1 (erythroleukemia) cells to generate CR1 “knock-out (KO)” (positive control) and “knock-in (KI)” lines with patient specific CR1 variants. CR1 mRNA and protein expression were quantified by real-time quantitative PCR (RT-qPCR), immunoblots, and flow cytometry. To assess complement activity directed against these cell lines, we measured complement-mediated cell killing using the mHam assay. Briefly, cells were incubated with 20% normal human serum and cell killing was measured using a WST-1 dye. Next whole blood was collected from patients with history of CAPS or anticoagulant refractory thrombotic APS. We quantified surface CR1 expression on erythrocytes by flow cytometry. To assess the role of methylation in CR1 expression, we performed reduced-representation bisulfite sequencing (RRBS) of the CR1 promoter region of healthy controls and CAPS cohort. Results: We previously identified a germline CR1 V2125L variant (rs202148801) in a woman with triple positive APS and pregnancy-associated CAPS. Pedigree analysis confirmed that this variant was inherited from her mother. We generated a TF-1V2125L cell line and found that the variant leads to significantly reduced expression of CR1 at the transcriptional and translational levels. Another CR1 variant G2109S, identified in a woman with preeclampsia but no thrombosis did not lead to diminished CR1 expression and therefore, served as a control. Functionally, the mHam showed that V2125L led to increased complement-mediated cell death induced by NHS (23% cell kill in TF-1CR1-/- vs 21% in TF-1V2125Lvs 7% in TF-1WT cells). Lastly, to confirm this variant leads to reduced CR1 expression in the patient, we performed flow cytometry of the patient's RBCs and found reduced surface CR1 expression. This observation led us to analyze erythrocyte CR1 expression by flow cytometry in 3 additional triple-positive APS patients with a history of CAPS or recurrent thrombosis refractory to anticoagulation. While these patients did not possess germline variants in CR1, we found a significant reduction in surface CR1 expression (n=4, 13.22%±3.77) as compared to healthy controls (n=22; 78.89%±2.98) and APS patients (n=6; 82.43%±6.34). To investigate the molecular mechanisms behind reduced CR1 expression, we performed RRBS of the CR1 promoter region, which demonstrated hypermethylation in these patients but not in the patient with the CR1 V2125L variant. This explains CR1 downregulation in CAPS patients without germline mutations. Lastly, these 3 patients were initiated on C5 inhibition (C5i) at the discretion of the treating clinician (duration of therapy 2 mo-9 y). Thrombosis rates reduced from 54.3 events per 100-patient-years to 10.2 events/100 patient years after starting C5i. Notably the one thrombotic event on C5i occurred in the postoperative period in the setting of delayed C5i administration (1 week late). Conclusion: CR1 deficiency, due to genetic or epigenetic down-regulation identifies a subset of CAPS that is particularly responsive to C5i.

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