MOLECULAR DIAGNOSIS OF HAEMOPHILIA A IN ARGENTINA: OPTIMISED ALGORITHM FOR FACTOR VIII GENE ANALYSIS.

DE BRASI CD, ROSSETTI LC, CANDELA, M, LARRIPA IB, PEREZ BIANCO R, DE TEZANOS PINTO M.

Río de Janeiro, Brasil

Congreso; XVIII Congreso Internacional de Hemostasis y Trombosis del Grupo Cooperativo Latinoamericano de Hemostasis y Trombosis (CLAHT).; 2003

CLAHT

Introduction: Haemophilia A (HA) is a sex-linked inherited, bleeding disorder due to deficiency in coagulation factor VIII (FVIII). Except for large DNA inversions that involves either the intron 22 (Inv22) or the intron 1 (Inv1) that account for about one half of severe cases, HA mutation diagnosis is challenging by the heterogeneous types of changes and the size and complexity of the gene. Besides, certain cases require indirect analysis using intragenic DNA polymorphisms. Herein, we present the first HA molecular series from Argentina, describe a rapid method to detect the Inv22, the XbaI A (X) and the MspI A (M) polymorphisms, and propose an algorithm for FVIII gene analysis. Materials and Methods: The new method for Inv22 detection involves the splitting of a previously described single-tube long distance PCR (LD-PCR) into two separated LD-PCRs: one specific for the wild type (wt) and the other for the mutated allele. The X analysis can be directly achieved on the wt-amplimer, and M, by nested PCR. The Inv22 was classically studied by Southern blot. Large deletions were detected by FVIII gene exon-specific PCR, and further analysed by LD-PCR. The screening of small mutations was performed by conformation sensitive gel electrophoresis, and its final characterisation by DNA sequencing, among other techniques.  Results: We studied 60 severe HA families and found the Inv22 in 42% (32% distal and 10% proximal type), the Inv1 in one case (2%), and large deletions in 6 cases (10%). We also found 10 small mutations including: 3 small deletions, 2 small insertions (only one involving an A-run), and 6 nucleotide changes [4 nonsense and 2 missense mutations (one related to moderate HA). The inhibitor formation of all these severe HA cases roughly coincides with those reported in the HA mutation database. In addition, the proposed method for streamline genotyping of Inv22+X+M was validated and the informativity of the DNA polymorphisms, estimated in our population. Conclusions: An algorithm for FVIII gene analysis for virtually all cases of HA, was presented. This includes the new method for Inv22+X+M genotyping, which is particularly valuable when there is not time to perform the analysis of the entire FVIII gene. The determination of the causative mutation will benefit both the genetic counselling (carrier and prenatal diagnosis) and the provision of important information for the treatment design.