Variants in Complement Regulatory Genes in Patients with Thrombotic Storm

Background: Thrombotic storm (TS) is a rare condition characterized by rapid-onset of thromboembolic events affecting multiple vascular beds. Catastrophic antiphospholipid syndrome (APS) is a type of thrombotic storm that occurs in patients with antiphospholipid antibodies and frequently includes microvascular manifestations. Many patients with TS do not have a history of APS or have detectable aPL, however, suggesting that aPL is not a requirement for the development of TS. Recent research has identified a higher occurrence of rare functional variations in complement genes among CAPS patients compared to non-CAPS APS cases and healthy controls.The relationship of functional variations in complement genes to patients with non-aPL-related catastrophic thrombotic syndromes is unknown.

Aim: We hypothesized that the presence of complement gene variants would be (1) more frequent in TS patients than in controls, and (2) more frequent in aPL+ TS patients than in aPL- TS patients.

Methods: We included all patients who met diagnostic criteria for TS from our prior report. Testing for aPL was performed on blood samples collected at enrollment. Controls were from previous studies on autism and Alzheimer’s disease with available sequencing and genotyping data. Whole exome sequencing (WES) was conducted on all TS patients and controls. The entire dataset was screened for variants in genes known to play roles in the complement system, including genes involved in both activation or regulation. TS patients were genotyped using the Illumina HumanExome-12v1_A Beadchip. Copy number variations (CNVs) in CHFR1, 3 and 4 were identified and/or confirmed using Illumina’s CNV partition program for GenomeStudio and TaqMan CNV assays. All variants were also scored using the Combined Annotation Dependent Depletion algorithm (CADD).

Results: The cohort included 26 patients with TS and up to 262 controls. Fourteen of the TS patients tested positive for aPL, including 8 with a diagnosis of CAPS at enrollment; the remaining 12 were aPL negative. None of the controls were known to have APS. We found 154 rare variants (MAF<1%) in genes regulating the complement system and 110 rare variants (MAF<1%) in genes involved in complement activation, across both cases and controls. Regarding genes regulating complement, there was no significant difference in variant carriers between TS patients overall, aPL+ or aPL- cases, and controls when considering variants based solely on their frequency (<1%) or frequency (<0.5%) and presence in specific gene sets previously reported. When variants were filtered based on frequency (<1%) and additional evidence of functional potential (conservation and CADD>15), TS patients were significantly more likely to carry two or more of these variants compared to controls (5/26 vs 10/262; p=0.006). This difference was primarily driven by aPL+ TS cases (3/14, p=0.02), with a similar trend observed in aPL- cases (2/12, p=0.09). For genes involved in complement activation, a nominal significant difference (p=0.046) was observed in carriers of multiple rare variants (MAF<1%) between TS patients and controls, driven mainly by aPL- patients (4/12 vs 25/262, p=0.03). No additional differences were found between TS patients, aPL+ or aPL- cases, and controls in further filtering scenarios for activation genes. We did not find a difference in CNVs (frequency >1%) between the aPL+ and aPL- TS patients, nor between the TS patients and the control population.

Conclusions: An increased frequency of variants predicted to have a functional impact in complement regulatory genes, and in multiple rare variants in complement activation genes, was identified between patients with TS and controls, but no significant differences were observed between aPL- and aPL+ TS patients. These findings suggest that patients with catastrophic thrombotic phenotypes have a similar frequency of alterations in complement regulatory and activation genes regardless of aPL status.