Rapid genotyping of XbaI and MspI DNA polymorphisms of the human factor VIII gene: estimation of their combined heterozygosity in the Argentinean population.

DE BRASI CD, ROSSETTI LC, LARRIPA IB.

HAEMATOLOGICA

Ferrata Storti Foundation

Lugar: Pavia, Italia; Año: 2003 vol. 88 p. 232 - 234

0390-6078

Background and Objective: Haemophilia A (HA) is a X-linked disorder due to deficiency in the coagulation factor VIII  (FVIII). Except for intron 22 inversions (Inv22) (42% of severe cases), HA mutation diagnosis is time-consuming by features of the gene. This paper presents an alternative route to investigate the XbaI A (X) and MspI A (M) DNA polymorphisms within intron 22 (FVIII gene) and estimates their utility for linkage analysis in HA. Design and Methods: X and M genotyping relies upon the use of a long-distance PCR (LD-PCR) amplicon from a previously described multiplex-LD-PCR to analyse the Inv22. Primers P and Q amplify 12 kb from intron 22 (FVIII gene) that can be subjected to XbaI digestion and to nested PCR followed by MspI digestion. For heterozygosity estimations, DNA samples from Argentinean healthy males representing 37 haplotypes were analysed.  Results: PQ-primed amplimers (12 kb), specific for the intron 22 (FVIII gene) were obtained from samples previously X and M genotyped by validated methods. Their restriction analysis coincides with the predicted polymorphic status showing the X and M polymorphic sites and non-polymorphic sites to control the digestion. X, M and BclI (intron 18) (B) showed single heterozygosities of 49%, 50%, and 47%, respectively, a combined X+M of 63%, and X+M+B of 65%. Interpretation: PQ LD-PCR amplimer is an intragenic-specific substrate for reliable X and M genotyping and would provide informative markers for linkage analysis in most cases of HA. Conclusions: This procedure can be performed in all laboratories that analyses the Inv22 by LD-PCR because of easy accessibility of primers and enzymes. This will improve the provision of rapid information for genetic counselling in Inv22-uninformative HA-families.