Novel Approach to and Results of Genetic Analysis of 3000 Hemophilia Patients Enrolled in the MyLifeOurFuture Initiative
Authors: Johnsen Jill , Fletcher Shelley N , Huston Haley , Roberge Sarah , Martin Beth K , Kircher Martin , Josephson Neil C , Shendure Jay , Ruuska Sarah , Koerper Marion A , Meltzer Leslie , Pierce Glenn F , Aschman Diane , Konkle Barbara 
Published: October 2019

Abstract
Background Hemophilia A and B are rare X-linked bleeding disorders affecting ~1:5000 male births. Hemophilia genotype is important to inform reproductive planning, pregnancy, and neonatal management, risk of inhibitor formation and bleeding severity, and basic understanding of mechanisms of disease. In 2012, two separate surveys found only ~20% of patients with hemophilia had a genotype determined. MyLifeOurFuture (MLOF) was formed as a multi-sector collaboration between the American Thrombosis and Hemostasis Network (ATHN), the National Hemophilia Foundation (NHF), BloodworksNW (BWNW), and Biogen to provide hemophilia genotype analysis for patients in the U.S. and to create a Research Repository for future scientific discovery.
Methods Participating hemophilia treatment centers (HTCs) contract through ATHN, enroll patients, obtain samples, and provide clinical results to patients. ATHN offers HTC provider education, a secure infrastructure for clinical data collection, and access for research proposals. NHF educates the bleeding disorders community about the initiative and supports recruitment. Biogen provides scientific collaboration and financial support. BWNW serves as the central sample processing and genotyping laboratory and houses the research sample repository. Genotyping was performed custom molecular inversion probes (MIPs) targeting the F8 and F9 genes and F8 inversions for simultaneous next generation sequencing (NGS) followed by confirmation of variants using standard genotyping methods.. Clinical results were returned to providers, and new variants were submitted to public databases.
Results 69 HTCs enrolled the first 3000 patients in under 3 years. Clinically reportable DNA variants were detected in 98.1% (2357/2401) of hemophilia A and 99.3% (595/599) of hemophilia B patients. 924 unique variants were found; 285 were novel. Predicted gene disrupting variants were common in severe disease, while missense variants predominated in mild-moderate disease. The custom MIP-based NGS inversion screening method successfully detected F8 gene proximal and distal intron 22 inversion and intron 1 inversion variants. Unexpectedly, the NGS approach detected more than one reportable variants in 40 patients (10 females), a finding with potential clinical implications. NGS also detected 108 unique incidental variants unlikely to cause disease; 11 variants were previously reported associated with hemophilia. Interrogation of the ExAC database, which has data from >66,000 individuals without hemophilia, reports DNA variants distributed across the coding regions of both genes.
Conclusions MLOF is the largest hemophilia genetics project performed to date, with plans to genotype over 6000 U.S. hemophilia patients. In the first 3000 patients, clinically reportable DNA variants were identifiedin nearly all patients. Our hemophilia NGS approach accurately identified F8 and F9 gene variants and is, to our knowledge, the first NGS method which can detect F8 inversions. The incidence of discovery of novel variation was high (30%) and novel variants were discovered continuously (per patient) over the course of the study, indicating that additional genetic variation in hemophilia likely remains undiscovered. Although both the F8 and F9 genes are thought to be conserved, we identified incidental variation in both genes, supporting caution in the interpretation of new variants. In summary, MLOF is a successful nationwide collaboration to genotype two rare bleeding disorders at scale which has contributed significantly towards DNA variant identification in the F8 and F9 genes in hemophilia. Through a consented research repository, MLOF data and samples, including phenotypic data from the ATHNdataset, will be accessible to providers and research communities for advancing our understanding of hemophilia and other disorders.

Disclosures
Johnsen: Octapharma: Consultancy; CSL Behring: Consultancy. Meltzer:Biogen: Employment.