Multiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of haemophilia A.

Use of molecular genetic studies for carrier detection and prenatal diagnosis of haemophilia A will be informative in approximately 75% of kindreds with a prior history of the disorder if previously reported bi-allelic intragenic polymorphisms are used. In this study we report the use of two multi-allelic, microsatellite repeat polymorphisms within the factor VIII gene in genetic testing for this disease. These two, dinucleotide repeat, polymorphisms have been analysed using a multiplex polymerase chain reaction (PCR) protocol, and results can be available within 3 d of receipt of samples. At the intron 13 polymorphic locus we have confirmed the original observation of eight alleles, whilst at the intron 22 locus, in contrast to a preliminary report of this polymorphism, we have seen five alleles. We have analysed 32 families (174 subjects) with the two microsatellite repeat markers and have found that using these two polymorphisms alone, 81% of families are informative for linkage analysis. The intron 13 repeat was informative in 22/31 families tested (71%) and the intron 22 polymorphism was informative in 12/17 families (71%). We have also found that in 11/32 families tested (34%) the microsatellite repeats were the only informative intragenic markers. In view of these observations, we believe that the most effective strategy for initiating haemophilia A genetic testing is to combine the multiplex PCR analysis of the intron 13 and 22 dinucleotide repeats with the PCR analysis of the intron 18 BclI marker. In our population this protocol provided informative results in approximately 88% of families and results can be available within 3 d of receiving the samples for testing.