Defects in Sialic Acid Biosynthesis Cause Dysregulation of the Alternative Pathway of Complement

Introduction: Atypical Hemolytic Uremic Syndrome (aHUS) is a disease characterized by microangiopathic hemolytic anemia, thrombocytopenia, and acute kidney injury. aHUS is usually caused by a predisposing germline variant in a complement regulatory gene, and a second hit of a complement amplifying condition such as infection, pregnancy, or inflammation.However, only approximately 50% of aHUS patients have identifiable genetic variants, with variants in factor H (CFH) accounting for 20%-30% of the genetic predisposition, C3 for 7%, membrane cofactor protein (MCP)/CD46 for 8%, factor B (CFB) for 2% and factor I (CFI) for 6%. For the other 50% of the patients, the genetic predisposition remains elusive. CFH binds to α2,3 sialic acid (SA) linked glycans on host cell surfaces and protects against attack by the alternative pathway of complement (APC). We hypothesized that the biosynthesis of SA is essential to complement regulation and SA biosynthesis defects predispose to aHUS. Methods: We performed targeted sequencing on 34 aHUS patients and 43 healthy controls for 4 genes that are responsible for the de novo biosynthesis of sialic acid: GNE, NANS, NANP, and CMAS. Then we used CRISPR-Cas9 and lentivirus systems to manipulate these genes in TF1 or TF1 PIGA-null cells, which lack the glycophosphatidylinositol-linked cell surface complement regulators CD55 and CD59, and allow the APC cascade to proceed once activated. α2,3 SA levels on the cell surface were measured with Maackia Amurensis lectin II (MAL II). Finally, we studied the functional consequences of these genetic changes. Normal human serum (NHS) was used to activate the APC on cells, and factor D (CFD) depleted serum (D-Dpl) was used to specifically block APC activation. C5b-9 deposition and CFH binding capacity on cells were detected by flow cytometry, and complement induced cell killing was detected via a WST-1 cell viability assay (mHam). Results: i) Rare germline variants found in SA biosynthesis genes Two rare germline variants (minor allele frequency < 0.005; data from GnomAD) were identified via targeted sequencing. An NANS M117I variant was found in an aHUS patient, while an NANP A153V variant was found in a control. The aHUS case did not harbor any variants in known complement genes. ii) Loss ofNANS but not NANP decreasesα2,3 SA on TF1 cells NANS knockout TF1 cells showed decreased α2,3 SA, demonstrating that this gene is essential for de novo SA biosynthesis. Conversely, NANP knockout TF1 cells showed no α2,3 SA level change. iii) NANS knockout increases the susceptibility to the APC TF1 PIGA-null cells with NANS knockout (TF1 DKO) had significantly higher C5b-9 deposition when treated with NHS compared to deletion of either gene alone (Figure A), demonstrating the formation of membrane attack complex (MAC). Cell viability assays also showed that TF1 DKO cells had significantly higher complement-induced cell killing when treated with NHS. Both C5b9 deposition and killing were rescued by APC-specific inhibition targeting CFD, demonstrating SA biosynthesis defects will increase the susceptibility to the APC specifically. iv) NANS knockout decreases the binding capacity of CFH To investigate CFH binding to the cell surface, C3b was first evenly loaded onto cells, followed by the addition of recombinant CFH. NANS knockout cells displayed significantly reduced CFH binding capacity, providing a mechanism for APC activation in the NANS knockout. v) NANS M117I decreases de novo biosynthesis of SA To assess the NANS variant identified in a patient with aHUS, TF1 DKO cells were transduced with a lentivirus containing the either wild type or M117I-mutated NANS, and cell-sorted to select individual clones. After treatment with sialidase to remove all SA, cells with NANS M117I had significantly lower SA level recovery compared to NANS wild type cells (Figure B). Conclusion: Targeted sequencing of 34 patients with aHUS identified a germline NANS variant in one case, suggesting an association between aHUS and SA biosynthesis defects. Functional studies showed that NANS knockout and the NANS M117I variant decreased cell surface SA levels. Loss of NANS also caused a decrease in CFH binding capacity and uncontrolled APC activation with increased cell death. Disclosures Chaturvedi: Alexion: Honoraria, Membership on an entity's Board of Directors or advisory committees; Sanofi: Honoraria, Membership on an entity's Board of Directors or advisory committees.