Factor VIII and Factor IX Mutation Analysis in 600 U.S. Hemophilia Patients: Correlation of Mutation Type with History of Inhibitor
Authors: Miller Connie H. , Hooper Craig , Abshire Thomas C. , Bockenstedt Paula L. , Brettler Doreen B. , Di Paola Jorge , Massey Gita , Neff Anne T. , Shapiro Amy D. , Tarantino Michael , Wicklund Brian M. , Manco-Johnson Marilyn J. , Dunn Amy L. , Knoll Christine , Creary Melissa , Benson Jane , Ellingsen Dorothy , Driggers Jennifer , Soucie J. Michael 
Published: April 2020

Abstract
More than 600 U.S. hemophilia patients have been genotyped as part of the pilot study for a prospective surveillance system for factor inhibitors conducted at 12 U.S. Hemophilia Treatment Centers. 80% of enrolled subjects had hemophilia A, 58% with severe disease, 24% moderate, and 18% mild. Age ranged from &lt;1 to 84 years. 83% were white, 8% black, and 4% Hispanic. In hemophilia A patients, all exons, all intron-exon junction regions, and the 3′ untranslated region of the factor VIII (F8) gene were resequenced in both directions by automated sequencer. The VariantSEQr™ protocol was used for resequencing on a 3730 DNA Analyzer from Applied Biosystems. The PCR primers and M13 sequencing primers are described at http://www.ncbi.nlm.nih.gov/sites/entrez?db=probe with a few modifications to the PCR primers to enhance throughput and reproducibility. Data were analyzed with SeqScape®. Inversions of intron 22 and intron 1 in the F8 gene were examined by PCR. Among 477 hemophilia A patients, missense mutations were found in 196 (41%), intron 22 inversions in 139 (29%), frameshifts in 50 (10%), nonsense mutations in 41 (9%), large deletions in 18 (4%), intron 1 inversions in 9 (2%), splice site changes in 5 (0.6%), and insertion in 1 (0.2%). Two mutations were identified in 4 (0.8%). No mutation was identified in 18 (4%). 124/139 of int22 inversions were reported to result in severe hemophilia, as well as 18/18 large deletions, 44/50 frameshifts, 37/41 nonsense mutations, and 8/9 int1 inversions. Of 196 missense mutations, 56 resulted in severe disease, 55 in moderate, and 80 in mild. History of inhibitor was reported in 79 patients, 22.4% of those with severe, 11.8% of those with moderate, and 2.4% of those with mild disease. Inhibitors occurred in 61% of those with large deletions, 26% of intron 22 inversions, 22% of nonsense mutations, 14% of frameshifts, 11% of intron 1 inversions, 6% of missense mutations, 20% of splice site changes, and 11% of those with no mutation identified. 173 distinct mutations were observed, 81 of which have not been reported previously in the Hemophilia A Mutation Database (HAMSTeRS). Among the patients enrolled in the study, black patients with hemophilia A were more likely to have a history of inhibitor than white patients (p=0.02). In hemophilia B patients, the promoter, coding regions, and intron-exon junctions of the factor IX (F9) gene were resequenced as above. Among 123 hemophilia B patients, 90 (73%) had missense mutations, 9 (7%) had frameshift mutations, 8 (7%) had nonsense mutations, and 3 (2%) had deletions. Two enrolled patients had history of FIX inhibitor, one with a large deletion and one a missense mutation. Centralized testing with high-throughput systems allows genotype to be used as a variable in ongoing studies of inhibitor risk.
This project is supported by the CDC Foundation through a grant from Wyeth Pharmaceuticals, which had no role in data analysis or abstract preparation.