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

Abstract

Introduction: 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)-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 determine which is the actual risk allele.

Methods: 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 that comprise 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 ten 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.

Results: 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- (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 an RR for DRB1*15:01 against DQB1*06:02 of 7.00 (5.80, 8.44).

Discussion/conclusion: Our results suggest that DRB1*15:01 is the offending HLAII allele and that DR15 allotypes underlie the increased FEI risk in HAPs.

Keywords: FVIII inhibitors; Hemophilia A; MHC-associated peptide proteomics; dendritic cell protein processing and presentation assays; immunogenic potential; linkage disequilibrium and HLAII haplotype DRB1*15:01/DQB1*06:02; therapeutic FVIII derived peptides; therapeutic FVIII proteins.

FIGURE 1

Comparison of the DQ- and DR-derived peptide counts. (A) Peptide counts of the DQB1 (blue) and DRB1 (red) fractions at each amino acid position along the FVIII protein, where the A1, A2, B, A3, C1, and C2 domains are indicated at the top of the figure and where the vertical dashed lines mark contiguous domains. The asterisks at the top indicate the midpoint of ranges from the N-termini to the C-termini of the tFVIII-derived IPSs (relative to the amino acid sequence of the FL-rFVIII parent protein) reported in other studies of potentially immunogenic peptides as well as the corresponding locations from the current study. The purple asterisks indicate peptides that were reported in van Haren et al. with residue midpoints—after rounding to the nearest integer—of 89 and 466 (van Haren et al., 2011; van Haren et al., 2013). The green asterisk located at residue 376 represents a peptide in van Haren et al. (2011), van Haren et al. (2013), Sorvillo et al. (2016), and Hu et al. (2004). The study by Hu et al. (2004) is of interest because they demonstrated in CD4 T-cell stimulation assays that their reported peptide, which spanned residues 371 to 400, consistently had the highest immunogenicity index in NDs as well as in HAPs both those with (FEI+) and without (FEI−) FEIs. The orange asterisk located at midpoint residue 1775 represents a peptide reported in van Haren et al. (2011), van Haren et al. (2013), Sorvillo et al. (2016), and Reding et al. (2004).

FIGURE 2

Risk ratios for (A) Model 1. DRB1 alleles (red) and DQB1 alleles (blue) with DQB1*02:01 being the baseline (i.e., reference) allele; (B) Model 2. Head-to-head comparison of DRB1*15:01 (red) to DQB1*06:02 with DQB1*06:02 being the baseline/reference.

FIGURE 3

Tetrachoric correlations between DRB1*15:01 (presence versus absence) with (A) hemophilia-A (HA) status (affected versus unaffected) and (B) FVIII inhibitor (FEI) status (yes versus no). Liabilities to HA status and FEI status are on the horizontal axes in (A) and (B), espectively, whereas liability to DRB1*15:01 is on the vertical axis in (A) and (B). The ellipses are the 95% confidence intervals (CIs) for the bivariate normal of the two latent, normally distributed liabilities in (A) and (B).