Haemophilia A Genotyping in Patients with Discrepancy in FVIII Activity Levels Measured with One-stage and Chromogenic Assays

ROSSETTI, LC; SUELDO, E; ABELLEYRO, M; DE BRASI, C; MARCHIONE, V; RADIC, P; WAISMAN K; ARIAS, M; ZIEGLER B; BAQUES, A

Congreso; Virtual Meeting of the International Society on Thrombosis and Haemostasis (ISTH); 2021

Background: In approximately one-third of patients withmild-moderate haemophilia A (HA), there is a discrepancy between the results ofFVIII activity (FVIII:C) measured by one-stage-assay (OSA) and chromogenic-substrate-assays(CSA), phenomenon which has been associated with particular missense variants.Aims: Characterizethe F8-genotype in persons with HA (PwHA) from Argentina showingdiscrepancy between FVIII:C assays and compare them with those reported in theInternational F8-variant database EAHAD (European Association forHaemophilia and Allied Disorders, https://f8-db.eahad.org/).Methods: PwHAsamples (N=5 with OSA ranging mild-moderate phenotypes, from 4 families) thatshowed discrepancy in the measurement of FVIII:C were included. OSA and CSAFVIII:C was determined using two reagent/coagulometer systems, IL/ACL Top 300and Siemens/Sysmex CS-2500.Genotyping: peripheral blood leukocyte-extractedgenomic-DNA was mutational screened by PCR-amplification of all coding andregulatory regions of F8 followed by conformation sensitive gel electrophoresis(CSGE) and selected-amplimers were characterized by Sanger sequencing.Pathogenicity ofF8-variants was classified according the ACMG criteria.Results: The HA-causative F8-genotype wasidentified in all 5 PwHA (Table 1) and classified as missense-type: one locatedin FVIII-C1-Domain and 4 in FVIII-A3-Domain. None of them was previouslyassociated with assay discrepancy in EAHAD. Two of them were not reported(c.6246C>A/p.(Ser2082Arg); c.5508G>T/p.(Trp1836Cys)), whereas thereported ones were associated with different (mild-moderate-severe) phenotypes butonly based on presumed OSA (Table 1).Conclusions: Thus far, in EAHAD database, only 28 missenseHA-causative variants were associated with FVIII:C assay discrepancy, and noneof them matched with our 5 cases from Argentina, which showed differentphenotype severities in EAHAD when were reported. Our data may reveal somedegree of misclassification of phenotype severity in HA, whichmay be associated with the rare application of CSA to measure FVIII:Ccomplementing OSA levels. Consequently, our findings reinforce the convenienceto base the initial diagnosis of non-severe-HA phenotype on the results ofboth, one-stage and chromogenic assays.