ClinGen-Based Reassessment of F8 and F9 Variants Improves Diagnostic Certainty in Hemophilia

Introduction Accurate interpretation of variants in F8 and F9 , the genes responsible for hemophilia A and B, respectively, is essential for diagnosis and counseling. Variants of uncertain significance (VUS) remain a major clinical challenge. Following the recent release of the ClinGen Coagulation Factor Deficiency Expert Panel specifications to the ACMG/AMP Variant Interpretation Guidelines, we systematically re-evaluated all F8 and F9 variants identified in our laboratory since 2018, with a particular focus on the reclassification of ACMG class 3 variants. Fig. 1 ClinGen-based reclassification of F8 variants. Reclassification of F8 variants after application of ClinGen gene-specific ACMG/AMP specifications. Flows illustrate changes between original (left) and updated (right) ACMG classes (LP/P=likely pathogenic/pathogenic, VUS=variant of uncertain significance, LB/B=likely benign/benign) Method All F8 (n=51) and F9 (n=22) variants identified in our diagnostic database between 01/2018 and 09/2025 were re-assessed using the ClinGen gene-specific ACMG/AMP specifications [ 3 ] [ 4 ]. Variant evidence was curated based on population frequency data, in silico predictions, functional assays, segregation information, clinical data, and information from F8 and F9 variant databases, as well as hemophilia-specific criteria. Each variant was independently reviewed by two experts (a physician and molecular geneticist) and classified according to the five-tier ACMG system (pathogenic, likely pathogenic, VUS, likely benign, benign) [ 1 ] [ 2 ]. Results For F9 , two VUS were successfully reclassified as either likely benign/benign (ACMG class 1 or 2) or likely pathogenic/pathogenic (ACMG class 4 or 5). One variant previously considered likely pathogenic (class 4) was downgraded to VUS (class 3), and another to likely benign (class 2) after applying the ClinGen specifications. No changes were observed for variants originally classified as class 1 or 2. Fig. 2 ClinGen-based reclassification of F9 variants. Reclassification of F9 variants after application of ClinGen gene-specific ACMG/AMP specifications. Flows illustrate changes between original (left) and updated (right) ACMG classes (LP/P=likely pathogenic/pathogenic, VUS=variant of uncertain significance, LB/B=likely benign/benign). For F8 , more unexpected changes were observed. Of seven VUS, three could be confidently reassigned to ACMG classes 1, 2, 4, or 5. Among 39 variants originally classified as class 4 or 5, one was downgraded to class 3 and six to classes 1 or 2. No changes were observed for variants originally categorized as class 1 or 2. Overall, the application of ClinGen specifications resulted in clinically relevant reclassifications for both genes, with a notable reduction in the number of VUS. Conclusion Systematic application of ClinGen gene-specific ACMG/AMP specifications for F8 and F9 significantly refines variant interpretation in hemophilia diagnostics. It enables clarification of previously uncertain variants and correction of previous overclassifications, thereby improving diagnostic accuracy and clinical management. The integration of detailed clinical data—including a thorough patient (family) history and relevant laboratory findings—is equally crucial for accurate variant interpretation. Further refinement of these specifications—such as defining additional mutational hotspots and well-established functional domains—will increase classification precision. Moreover, continued data sharing across diagnostic laboratories and variant databases is essential, as criteria such as PS1, PS4, and PM5 rely on sufficiently large and diverse datasets to be applied with confidence. Publication History Article published online: 17 February 2026 © 2026. Thieme. All rights reserved. Georg Thieme Verlag KG Oswald-Hesse-Straße 50, 70469 Stuttgart, Germany