Rapid hemophilia A molecular diagnosis by a simple DNA sequencing procedure: identification of 14 novel mutations.

We here describe a simple, efficient DNA sequencing procedure for hemophilia A molecular diagnosis. In severe patients we first test for the presence of factor VIII gene intron 22 inversion using a recently described single-tube PCR method. In moderate, mild, or inversion-negative severe patients we systematically sequence the promoter, all exons and splice junctions of factor VIII gene. Specially designed primers allow amplification of 23 PCR products under the same salt conditions and thermocycling parameters. The whole sequencing procedure, from blood extraction to mutation identification, can be readily done within 42 h when using regular instruments or in just 14 h when using a high-throughput sequencer. Thus, this is a versatile and cost-effective strategy with little hands-on time requirements. Since its implementation we have identified mutations in 45/46 hemophilia A patients, 14 of which are novel. Once the genetic defect has been identified, accurate genetic counseling is then easily performed.

Factor IX gene sequencing by a simple and sensitive 15-hour procedure for haemophilia B diagnosis: identification of two novel mutations.

We have developed a simple, sensitive and cost-effective direct DNA sequencing procedure for the molecular diagnosis of haemophilia B. All factor IX gene essential regions were amplified under identical thermocycling parameters allowing mutation identification in less than 15 h from blood sample collection. Identical results in terms of accuracy and speed were obtained when using a single hair as the source of DNA. Using this approach, we have found mutations in 10 out of 10 haemophilia B patients, two of which are novel (P287T and S123C). Very suitable for individual studies, this procedure can be easily scaled up for higher throughput.