Disentangling Effects of the DR and DQ Isomers Encoded by the HLA class II Haplotype DRB1*15:01/DQB1*06:02 to Help Establish the True Risk Allele for FVIII Inhibitor Development in Hemophilia A

Vincent P Diego ^1* Bernadette W. Luu ² Marcio Almeida ¹ Rajalingam Raja ³ Marco Hofmann ⁴ Jacob A Galan ¹ Eron Grant Manusov ¹ Jerry S Powell ^2,5 Long V Dinh ² Henry Mead ⁶ Huy Huynh ⁷ Anne M Verhagen ⁷ Juan Peralta ¹ Paul V Lehmann ^8,9 Satish Kumar ¹ Eli J Fine ¹⁰ Joanne E Curran ¹ Harald Goring ¹ Miguel A Escobar ^11,12 Sarah Williams-Blangero ¹ Eugene Maraskovsky ⁷ John Blangero ¹ Tom E. Howard ^1,13,2

• ¹ Department of Human Genetics, School of Medicine, University of Texas Rio Grande Valley, Brownsville, TX, United States
• ² Haplogenics Corporation, Brownsville, TX, United States
• ³ Immunogenetics and Transplantation Laboratory, Department of Surgery, School of Medicine, University of California at San Francisco, San Francisco, CA, United States
• ⁴ CSL Innovation GmbH, Marburg, Germany
• ⁵ Division of Hematology and Oncology, Department of Internal Medicine, School of Medicine, University of California Davis, Davis, CA, United States
• ⁶ Global Medical Affairs, BioMarin, Novato, CA, United States
• ⁷ CSL Limited Research, Bio21 Institute, Melbourne, Australia
• ⁸ Cellular Technology Limited (CTL), Shaker Heights, OH, United States
• ⁹ Departments of Pathology and Neurology, School of Medicine, Case Western Reserve University, Cleveland, OH, United States
• ¹⁰ Consultant, Atlanta, GA, United States
• ¹¹ Gulf States Hemophilia and Thrombophilia Center, Houston, TX, United States
• ¹² Division of Hematology and Oncology, Department of Medicine, University of Texas Health Science Center, Houston, TX, United States
• ¹³ VA Texas Valley Coastal Bend Health Care System, Veterans Health Administration, United States Department of Veterans Affairs, Harlingen, Texas, United States

Hemophilia-A (HA) patients (HAPs) with the human-leukocyte-antigen (HLA)-class-II (HLAII) haplotype DRB1*15:01/DQB1*06:02, and thus antigen-presenting-cells which express HLAII β-polypeptide-chains that form heterodimers of DR15- and DQ6-serotypes, respectively, have an increased risk of developing "factor (F)VIII inhibitors (FEIs)", i.e., neutralizing-antibodies against the therapeutic-FVIII-proteins (tFVIIIs) infused to prevent/arrest bleeding. As DRB1*15:01 and DQB1*06:02 exist in strong linkage-disequilibrium, association-analysis cannot disentangle which is the actual risk allele. To establish the true risk-allele of this haplotype, we analyzed the tFVIII-derived-peptides (tFVIII-dPs) bound to either the DR- or DQ-molecules comprising the individual-HLAII-repertoires expressed by monocyte-derived dendritic-cells obtained from 25 normal-blood-donors and six HAPs, four without and two with FEIs. We performed log-linear-mixed model analyses, where the dependent-variable is the log of the measured peptide count. Under Model 1, we analyzed an HLAII allele predictor consisting of 10 levels (four DRB1 and six DQB1 alleles) in the fixed effects, and variables in the random effects to account for non-independence. Model 2---where the HLAII-allele variable consisted of only DRB1*15:01 and DQB1*06:02---compares the HLAII alleles. Relative to the Model 1 reference, DRB1*15:01 and DQB1*06:02 significantly increased tFVIII-derived-peptide counts, and DRB1*15:01 contributed significantly more than DQB1*06:02. Reported as risk-ratios (RRs) and their 95%-confidence interval (CI) lower-bound (LB) and upper-bound (UB), we found a RR (95%-CI-LB, -UB) of 14.16 (10.38, 19.33) and 1.76 (1.24, 2.50) for DRB1*15:01 and DQB1*06:02, respectively. Under Model 2, we found a RR for DRB1*15:01 against DQB1*06:02 of 7.00 (5.80, 8.44). Our results suggest that DRB1*15:01 is the offending-HLAII-allele and DR15-allotypes underlie the increased FEI-risk in HAPs.