CURRENT STRATEGY FOR GENETIC-ANALYSIS OF HEMOPHILIA-A FAMILIES

Carrier detection and prenatal diagnosis of haemophilia A, which was b ased in the last decade mainly on linkage polymorphism analysis, has b een greatly facilitated by the recent discovery that two types of inve rsion disrupting the factor VIII, gene are common mutations observed i n 42-48% of severe haemophilia A cases. In this study DNA analysis was performed in 64 unrelated severe haemophilia A patients and 173 women belonging to their families, and in four women from a family with a d eceased haemophilic relative whose DNA was unavailable (a total of 177 females from 65 unrelated families). Factor VIII gene inversions were found in 32 out of the 65 families (49%), 29 involving recombination with the distal A gene and three with the proximal A gene. Definitive information regarding carriership of haemophilia was provided to all 8 1 women belonging to the 32 inversion-positive families, among them on e woman previously uninformative for any of the polymorphisms examined , five women who were informative only for extragenic polymorphisms, a nd four suspected carriers who were relatives of the deceased haemophi liac,. In 33 inversion-negative families, 96 females were examined by analysis of the BclI restriction fragment length polymorphism (RFLP) i n intron 18 and of the multiallelic dinucleotide repeats in introns 13 and 22, followed by analysis of other intragenic polymorphisms. This procedure yielded an informativity rate of almost 100%. Of the 96 fema les examined by linkage polymorphism analysis, 78 belonged to 25 famil ies with more than one haemophiliac and 29 of them were obligate carri ers. In 47 of the 49 suspected carriers linkage polymorphism analysis enabled definition of carriership based on intragenic polymorphisms. 1 8 of the 96 females belonged to eight families with sporadic haemophil ia cases and only eight of the 18 suspected carriers could be diagnose d by exclusion. In nine pregnant women carrying factor VIII gene inver sions, mRNA extracted was analysed gene reverse transcription/polymera se chain (RT/PCR). This procedure enabled rapid prenatal diagnoses in six male fetuses. Taken together, our data indicate that a high rate o f informativity and carrier definition is possible by the strategy of first screening for factor VIII gene inversions, and, if none are foun d, sequential use of highly informative intragenic polymorphisms, foll owed by less informative intragenic and extragenic polymorphisms.