National Hemophilia Mutation Testing Program Laboratory opened in November 2000 Severe hemophilia A and B Requirement for carrier testing and/or prenatal diagnosis Isolated (sporadic) severe hemophilia History of inhibitors Unusual phenotypes - mild hemophilia with inhibitors - self resolving hemophilia Study patients - ie. prophylaxis VWD patients – selected cases Current Funding - Baxter Bioscience Canada

Genotyping Strategy PCR-Based Heteroduplex Screen Conformation Sensitive Gel Electrophoresis Factor VIII - 37 fragments Factor IX - 9 fragments Identification of Mutant Exon Automated Sequencing of Mutant Exon

Genotyping Turnaround Time Severe Hemophilia - prenatal diagnosis weeks Regular testing - 1 to 4 months Problematic cases (5-10%) - 4+ months

Hemophilia A Genotyping Samples referred from 41 centers 1284 samples referred for testing 1125 reports generated 358 missense mutations (131 not in HAMSTeRS) 56 nonsense mutations (26 not in HAMSTeRS) 87 frameshift mutations (37 not in HAMSTeRS) 32 splice site mutations (24 not in HAMSTeRS) 115 inversion mutations (introns 1 and 22)

Hemophilia B Genotyping Samples referred from 28 centers 282 samples referred for testing 271 reports generated 138 missense mutations (24 not in HMB database) 17 nonsense mutations (3 not in HMB database) 13 frameshift mutations (8 not in HMB database) 9 splice site mutations (1 not in HMB database) 6 Leyden promoter mutations 4 large deletions (most / all of gene)

Hemophilia A Mutation Database - HAMSTeRS Internet Accessible Worldwide Hemophilia Mutation Databases 457 Mutations Reported Novel

Hemophilia B Mutation Database Internet Accessible Worldwide Hemophilia Mutation Databases 154 Mutations Reported - 33 Novel

Test Subject Details Affected Males: FVIII = 791 FIX = 206 Potential Carriers: FVIII = 493 FIX = 76

von Willebrand Disease Testing Rationale As a complement to standard hemostasis laboratory testing for VWD (hemostasis tests difficult to interpret or variable)

von Willebrand Disease Testing Testing Performed for : Type 2 A Type 2B Type 2M Type 2N PT-VWD

Localization of Type 2 VWD Mutations D’ D3D4 A2A3A1 B1-3 2N NH2 COOH 2B 2M FVIII GPIb

von Willebrand Disease Testing Type 2A 6 patients tested Sequencing of VWF exon 28 Type 2A mutations identified in 4 cases

Type 2B 26 patients tested Sequencing of VWF exon 28 Type 2B mutations identified in 16 cases Platelet-type VWD mutations identified in the GPIbα gene in 2 cases von Willebrand Disease Testing

Type 2M 35 patients tested Sequencing of VWF exon 28 Type 2M mutations identified in 15 cases

von Willebrand Disease Testing Type 2N 61 patients tested Sequencing of VWF exons 18,19,20,21,24,25,26 and 27 Type 2N mutations identified in 15 cases Mild hemophilia A mutations identified in 10 cases

von Willebrand Disease Testing Type 3 2 families tested Sequencing of VWF Exons 1-52 Mutations identified in exons 18 and 31 Prenatal testing performed in conjunction with the Kingston DNA Diagnostic Laboratory.

Total Referred Cases HMAHMBType 2N VWD

Acknowledgements Baxter Bioscience Canada Jayne Leggo Shawn Tinlin