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

ROSSETTI LC; MARCHIONE VD; ARIAS M; SUELDO E; RADIC CP; ZIEGLER B; ABELLEYRO MM; WAISMAN K; BAQUES A; DE BRASI CD

Virtual

Congreso; ISTH 2021 Congress; 2021

ISTH

Background:Inapproximately one-third of patients with mild-moderate haemophilia A(HA), there is a discrepancy between the results of FVIII activity(FVIII:C) measured by one-stage-assay (OSA) andchromogenic-substrate-assays (CSA), phenomenon which has beenassociated with particular missense variants.Aims:Characterizethe F8-genotypein persons with HA (PwHA) from Argentina showing discrepancy betweenFVIII:C assays and compare them with those reported in theInternational F8-variantdatabase EAHAD (European Association for Haemophilia and AlliedDisorders, https://f8-db.eahad.org/).Methods:PwHAsamples (N=5 with OSA ranging mild-moderate phenotypes, from 4families) that showed discrepancy in the measurement of FVIII:C wereincluded. OSA and CSA FVIII:C was determined using tworeagent/coagulometer systems, IL/ACL Top 300 and Siemens/SysmexCS-2500.Genotyping: peripheral blood leukocyte-extracted genomic-DNAwas mutational screened by PCR-amplification of all coding andregulatory regions of F8followedby conformation sensitive gel electrophoresis (CSGE) andselected-amplimers were characterized by Sanger sequencing.Pathogenicity ofF8-variantswas classified according the ACMG criteria.Results:TheHA-causative F8-genotypewas identified in all 5 PwHA (Table 1) and classified asmissense-type: one located in FVIII-C1-Domain and 4 inFVIII-A3-Domain. None of them was previously associated with assaydiscrepancy in EAHAD. Two of them were not reported(c.6246C>A/p.(Ser2082Arg); c.5508G>T/p.(Trp1836Cys)), whereasthe reported ones were associated with different(mild-moderate-severe) phenotypes but only based on presumed OSA(Table 1).Conclusions:Thusfar, in EAHAD database, only 28 missense HA-causative variants wereassociated with FVIII:C assay discrepancy, and none of them matchedwith our 5 cases from Argentina, which showed different phenotypeseverities in EAHAD when were reported. Our data may reveal somedegree of misclassificationof phenotype severity in HA, which may be associated with the rareapplication of CSA to measure FVIII:C complementing OSA levels.Consequently, our findings reinforce the convenience to base theinitial diagnosis of non-severe-HA phenotype on the results of both,one-stage and chromogenic assays.p { margin-bottom: 0.1in; direction: ltr; line-height: 115%; text-align: left; orphans: 2; widows: 2; background: transparent }p.western { so-language: en-GB }a:link { color: #000080; so-language: zxx; text-decoration: underline }a:visited { color: #800000; so-language: zxx; text-decoration: underline }