Molecular algorithm for genetic diagnosis of women with hemophilia A in Argentina

ZIEGLER B, ROSSETTI L, ABELLEYRO M, MARCHIONE V, NEME D, MEDINA-ACOSTA E, DE BRASI C, RADIC P

Londres

Congreso; XXX Congress of the International Society on Thrombosis and Haemostasis ISTH 2022.; 2022

International Society on Thrombosis and Haemostasis ISTH

Background: Hemophilia A (HA) is an X-linked recessive coagulopathy caused by pathogenic variants in FVIII gene (F8), typically expressed in males but rarely in females. Recently a new nomenclature defines hemophilia carrier women: women/girls with severe (FVIII < 0.01IU/mL), moderate (0.01-0.05IU/mL) or mild HA (0.05-0.40IU/mL), symptomatic and asymptomatic HA carriers (HAC) (FVIII≥0.40IU/mL) with and without a bleeding phenotype, respectively. HA expression in females associates with different causes: in most cases, heterozygous-F8 variant and skewed X-chromosome inactivation (XCI) silencing the normal F8, and rarely: combining two F8-variants (in homozygous or compound heterozygous cases) or F8-hemizygosity associated with female phenotype.Aims: Analyze the molecular basis of HA expression in women through a simple algorithm.Methods: 150 women suspected of being carriers of HA were studied. In cases with a known HA-causative familiar variant: point-mutations and indels were carrier diagnosed by Sanger-sequencing, gross-deletions by breakpoint-specific-PCR, or F8-inversions (Inv22/Inv1) by inverse-shifting-PCR.In cases with no history of hemophilia or an unknown familial variant, the complete F8-analysis algorithm was applied: -inverse-shifting-PCR for Inv22/Inv1, small-variant screening by CSGE (conformation-sensitive-gel-electrophoresis) on all relevant F8 amplicons (38 PCR-products), and Sanger-sequencing. Large deletions were investigated in suspected cases by MLPA or qPCR dosage. Variant pathogenicity was evaluated following ACMG criteria (American College of Medical Genetics).The XCI status was investigated in peripheral blood by XCI-sensitive markers at AR (Xq) and RP2 (Xp).Results: 105 women resulted HACs, including severe, moderate, mild and non-symptomatic carriers. 107 F8-variants were found. 102 HACs resulted heterozygous, one hemizygous (X0), one homozygous and a compound heterozygous (Figure 1).Conclusion(s): Causes of bleeding phenotype were stated in eight HACs. Full F8 genotyping of HA-causative variants and XCI patterns allow understanding the molecular basis of FVIII:C levels and clinical expression in HAC. The cost-effectiveness of our approach makes it suitable also for developing countries