ALBERT

Description: 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.

Description: Background Hemophilia A (HA) and hemophilia B (HB) are rare X-linked bleeding disorders. Hemophilia genotype is important to inform reproductive planning, pregnancy, neonatal management, inhibitor risk, disease severity, and understanding of disease mechanisms. MyLifeOurFuture (MLOF) was formed as a collaboration between the American Thrombosis and Hemostasis Network (ATHN), the National Hemophilia Foundation (NHF), Bloodworks (BW), and Biogen. Together with participating hemophilia treatment centers (HTCs), MLOF provided hemophilia genotype analysis for patients in the U.S. and created a Research Repository. Methods HTCs contracted through ATHN, enrolled patients, obtained samples, and provided clinical results to patients. ATHN offered provider education, secure infrastructure for clinical data collection, and access for research proposals. NHF educated the bleeding disorders community about the initiative and supported recruitment. Biogen provided scientific collaboration and financial support. BW served as the central genotyping laboratory and houses the research sample repository. Genotyping was performed using a custom next generation sequencing screen followed by confirmation with a second method. Clinical results were returned to providers. Results 107 HTCs enrolled 11,341 patients for testing: 8976 for HA (6180 males, 2796 females), 2358 for HB (1616 males, 742 females), 3 for HA/HB, 4 for hemophilia NOS. Clinically reportable variants were detected in 98.2%% of male HA and 98.1% of male HB patients. 1919 unique variants were found, 1486 in F8 and 433 in F9. Of these, 744 were novel (F8 n=610, F9 n=134). Two variants were detected in 95 patients, including 36 females. In severe HA (n=3419), the most common variants were F8 inversions (44.7%), missense (16.8%), frameshift (16.1%), stop-gain (11.3%), large structural variants (SV) (5.7%), and splice (3.4%). In severe HB (n=564), the most common variants were missense (46.8%), stop-gain (24.1%), frameshift (9.4%), SV (8.3%), and splice (4.1%). Missense variants were the most common variants found in non-severe HA (81.1%) and HB (88.2%). Inhibitor information is reported for 6986 MLOF patients (HA n=5583; HB n=1403) in the ATHNdataset. Inhibitors were more common in severe disease than non-severe disease in HA [29.8% (n=950/3193) vs. 9.0% (n=216/2390)] and HB [12.4% (n=63/508) vs. 1.9% (n=17/895)]. In severe HA, inhibitors were reported in ~50% of patients with large deletions (n=77/80), complex intron 22 inversions (n=9/17), or no variant found (n=7/14). Other gene-disrupting genotypes had intermediate inhibitor rates (25-36%): intron 1 inversions, intron 22 type 1 and type 2 inversions, stop-gain, splice, and frameshift. Lower rates (~7-14%) were reported with in-frame insertion-deletions, missense, and large duplications. In severe hemophilia B, inhibitors were most common with large deletions (57%, n=24/42). Conclusions We here report our analysis of the complete dataset of MLOF, the largest hemophilia genetics project performed to date. Clinically reportable DNA variants were identified in nearly all patients. Our findings support the need for comprehensive gene sequencing and SV detection. Deletions, complex inversions, and "No variant found" were the highest risk genotypes for inhibitors in severe hemophilia. The incidence of discovery of novel variation was high (38%) and continued throughout the study, indicating additional variation remains undiscovered. In summary, MLOF has been a successful nationwide collaboration to genotype two rare bleeding disorders at a scale not previously done. This effort has contributed significantly towards identifying and better understanding DNA variation in the F8 and F9 genes in hemophilia. Disclosures Johnsen: Octapharma: Research Funding. Pierce:Voyager Therapeutics: Membership on an entity's Board of Directors or advisory committees; CRISPR Therapeutics: Consultancy; Decibel Therapeutics: Consultancy; Third Rock Ventures: Consultancy; Takeda: Consultancy; Geneception: Consultancy; Generation Bio: Consultancy; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; BioMarin: Consultancy; Ambys Medicines: Consultancy; World Federation of Hemophilia.: Membership on an entity's Board of Directors or advisory committees. Recht:Genentech: Consultancy, Other: personal fees, Research Funding; CSL Behring: Consultancy, Other: personal fees; uniQure: Consultancy, Other: personal fees, Research Funding; Novo Nordisk: Consultancy, Other: personal fees, Research Funding; Spark: Research Funding; Takeda: Consultancy, Other: personal fees, Research Funding; BioMarin: Research Funding; Pfizer: Consultancy, Other: personal fees. Konkle:BioMarin: Consultancy; Sanofi: Consultancy, Research Funding; Sigilon: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Baxalta: Research Funding; Spark: Consultancy, Research Funding; Takeda: Research Funding; Uniquire: Research Funding; CSL Behring: Consultancy; Roche: Consultancy.