Rapid Hemophilia A Molecular Diagnosis by a Simple DNA Sequencing Procedure: Identification of 14 Novel Mutations

Abstract

SummaryWe 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.

Prevalence of small rearrangements in the factor VIII gene F8C among patients with severe hemophilia A

“…Although this finding could be biased by the relatively small number of patients, it supports the high frequency of this type molecular defects in the B domain of the factor VIII gene. Some of the mutations in this exon identified in our study have already been described in several other patients, giving evidence for mutation hot-spots, like the A-stretch at position c.3629-3638 (codons 1191-1194) -mutations in 25 unrelated patients described [Becker et al, 1996;Lin et al, 1993;Economou 1992;Pieneman et al, 1995;Becher et al, 1996;Freson et al, 1998;Vidal et al, 2001;Goodeve et al, 2000;Akkarapatumwong et al, 2000, present study]; c.4372-4379, codons 1439-1441 (13 mutations described) [http://europium.csc.mrc.ac.uk]; c.4820-4825, codons 1588-1590 (4 mutations described) [Lin et al, 1993;Freson et al, 1998;Goodeve et al, 2000;present study). Furthermore c.3702-3705delTACA, codon 1215-1216 identified in one of our patients has been described in two other patients [Tavassoli et al, 1998b;Ivaskevicius et al, 2001], showing that this repetitive sequence could be also prone to mutations.…”

Influence of the type of factor VIII concentrate on the incidence of factor VIII inhibitors in previously untreated patients with severe hemophilia A

“…Regarding the F8 genotype, the mutations profile in our patients was typical of what would be expected in a group of severe hemophilia A patients (63.5% had severe molecular defects: 50% inversions, 13.5% nonsense). [36][37][38] It is well established that the F8 genotype is a critical risk factor for the development of inhibitors. 4,27,28 With the exception of large insertion/deletion of multiple exons (a very rare mutation) that are associated with the highest risk of inhibitor (68%-88%), the other high-risk mutations (inversions, nonsense light chain) are at 30% to 40% risk of inhibitor-about twice more than other mutations such as small insertion/deletion non-A run or nonsense heavy chain, and large deletion/insertion single exon, which are at 15% to 20% risk.…”

Severe and moderate hemophilia A: identification of 38 new genetic alterations

“…Negative patients for either mutations were included in the study to search for mutations by sequencing methodology. 6 The promoter region and the 26 exons and their boundaries from the F8 gene were amplified with the Ex Takara Taq TM polymerase under the same PCR conditions. These and primer localization can be provided upon request.…”