Functional characterization of 105 Factor H variants associ-ated with atypical HUS: lessons for variant classification

1 p.Background: Atypical hemolytic uremic syndrome (aHUS) isa life-threatening thrombotic microangiopathy that can progress,when untreated, to end-stage renal disease. Most frequently, aHUSis caused by complement dysregulation due to pathogenic vari-ants in genes that encode complement components and regulators.Amongst these genes, the Factor H (FH) gene, CFH, presents withthe highest frequency (15-20%) of variants and is associated withthe poorest prognosis. Correct classification of CFH variants aspathogenic or benign is essential to clinical care but remains chal-lenging owing to the dearth of functional studies. As a result,significant numbers of variants are reported as variants of uncertainsignificance.Methods: To address this knowledge gap, we expressed andfunctionally characterized 105 aHUS-associated FH variants witha battery of hemolytic and plate-based assays to determine theirimpact on its regulatory and binding properties. We used 26 pre-viously characterized FH variants to validate the robustness of thefunctional assays used.Results: All FH variants were categorized as pathogenic orbenign, and for each, we fully documented the nature of thepathogenicity. Among the 79 uncharacterized variants, only 29(36.7%) alter FH in vitro expression or function and are thereforeproposed to be pathogenic. We show that rarity in control databasesis not informative for variant classification, and we identify impor-tant limitations in applying prediction algorithms to FH variants.Based on structural and functional data, we suggest ways to circum-vent these difficulties and thereby improve variant classification.Conclusions: Our work reveals limitations of routinely usedvariant classification methods. Rarity in control databases can bemisleading and prediction algorithms fails classifying up to 17%of the variants. This highlights the need for functional assays tointerpret FH variants accurately if clinical care of patients withaHUS is to be individualized and optimized.Peer reviewe

Functional characterization of 105 Factor H variants associated with HUS:lessons for variant classification

36 p.-3 fig.-3 tab.Atypical hemolytic uremic syndrome (aHUS) is alife-threatening thrombotic microangiopathy that can progress, when untreated, to end-stage renal disease. Most frequently, aHUS is caused by complement dysregulationdue topathogenic variants in genes that encode complement components and regulators. Amongst these genes, the Factor H (FH) gene, CFH, presents with the highest frequency (15-20%) of variantsand is associated with the poorest prognosis. Correct classification of CFH variants as pathogenic or benign is essential to clinical care but remainschallenging owing to the dearth of functional studies. As a result, significant numbers of variants are reported as variants of uncertain significance. Toaddress this knowledge gap, we expressed and functionally characterized105 aHUS-associated FH variants. All FH variants were categorized as pathogenic or benign, and for each,we fully documented the nature of the pathogenicity. Twenty-sixpreviously characterizedFH variantswere used as controls to validate and confirm the robustness of the functional assays used. Of the remaining 79 uncharacterized variants, only 29 (36.7%)alter FH in vitroexpression or function and are therefore proposed to be pathogenic. We show that rarityin control databases isnot informative for variant classification,andwe identifyimportant limitations in applying prediction algorithms toFH variants. Based on structural and functional data, we suggest ways to circumvent these difficultiesand thereby improve variant classification.Our work highlights the need for functional assays to interpret FH variants accuratelyif clinical care of patients with aHUS is to be individualized and optimized.SRdC was supported by grants from the Spanish Ministerio de Economía y Competitividad-FEDER (PID2019-104912RB-I00) and Autonomous Region of Madrid (S2017/BMD-3673). RJHS was supported, in part, by a grant from the National Institute of Diabetes and Digestive and Kidney Diseases (R01 110023). Conception, design, data collection and analysis, as well as writing of the manuscript, was performed with support from Alexion Pharmaceuticals, Inc. Editorial support, funded by Alexion Pharmaceuticals Inc., Boston, MA, was provided by Oxford PharmaGenesis, Oxford, UK.Peer reviewe