F8 genotype characterisation of the first Argentine series of patients with mild haemophilia A: notable prevalence of recurrent mutations

Abstract

HaemophiliaA (HA) is the commonest X-linked coagulopathy caused by deleterious mutationsin F8. Mild-HA associates with minor reduction in the clotting activityof factor VIII (FVIII:C) to 5-40 UI/dL. Perhaps due to their mild phenotypeexpression, mild-HA patients are rarely genotyped although they represent35-40% of HA cases worldwide. The scarce published data indicate that mild-HAshows notable differences with severe-HA (FVIII:C<1 IU/dL) in its populationgenetics and mutational pool turnover.Objective:to characterise the F8-genotype in a large series of Argentine patientswith mild-HA and to discuss its mutational dynamics.Population:64 apparently unrelated families affected with mild-HA countrywide, 97individuals including index-cases and relatives. Our F8 analysisalgorithm includes: -genomic DNA extraction from peripheral blood leukocytes,-a mutational screening by PCR-amplification of all coding and regulatoryregions of F8 over all 26 exons (38 amplimers) and conformationsensitive gel electrophoresis (CSGE), -mutational characterisation by Sangersequencing of CSGE anomalous amplimers. Duplication of exon 13 (Dup13) wasdetected by tail-to-head PCR-analysis.Themild-HA-causative mutation (established by genotype/phenotype assignmentcriteria) was identified in 61 families (detection efficiency 95%). Thirty-fourfamilies (56%) showed 14 recurrent mutations (repeated 2-5 times), whereas theremnant 27 families, non-recurrent F8-defects. Among the recurrentmutations, we found 11 missense in 26 families highlighting p.Arg612Cys* (n=5)and p.Arg550Cys (n=3) among others (n=2), a splicing defect on c.601+5G>A(n=3), the Dup13* (n=3) and a synonym change (n=2) (*reported with Italianorigin). Non-recurrent mutations included 24 missense, an ins-del and twosplicing defects.Ourfindings demonstrate that our practical approach is adequate to characterisethe mild-HA-causative F8-genotype in patients and relatives, highlightthe prevalence of missense defects in mild-HA (50/61, 82%) and indicate thatthe higher frequency assessed for recurrent mutations in mild-HA respect tosevere-HA may reflect the higher mutational turnover of the severe-HA pool.