Peptides from the Exon 22/Exon 23 junction and their affinity for common MHC-II variants. (A) Ribbon diagram of the structure of the C1 domain of FVIII highlighted to emphasize the I22I break point encoded by the 3′- and 5′-ends of exons 22 and 23, respectively. Y2105 and R2150 (red) represent the positions of recurrent mild HA-causing missense mutations that are strongly associated with inhibitor development. Residues 2106 to 2123 and 2126 to 2149 (blue) are 2 segments of C1 on either side of the inversion break point. M2124 and V2125 (orange and white, respectively) are the residues flanking the break point. (B) Forty-six amino acids comprising most of the C-terminal one-third of the C1 domain are shown together with the location of known HA-causing missense mutations; the presence (red asterisk) or absence (black minus sign) of inhibitor development in patients found to have one of these abnormalities with these is also indicated. Note that Y2105C and R2150H have been found in many unrelated alloimmunized HA patients and represent the N- and C-terminal missense mutations, respectively, closest to the exon 22/exon 23 junction, which have been identified in inhibitor patients. Although 18 additional missense mutations (green) have been identified more proximal than Y2105C and R2150H to the I22I break point, none of these patients has yet been reported to have developed inhibitors to date. (C) The immunogenicity potential of wild-type tFVIII-derived peptides encoded by the mRNA sequence spanning the exon 22/exon 23 junction is depicted as promiscuity scores.5,27 The binding affinities of commonly-occurring HLA-II proteins for peptides derived from the C1 domain region corresponding to the exon 22/exon 23 junction were predicted using the NetMHCIIpan-3.0 method (http://www.cbs.dtu.dk/services/NetMHCIIpan/28 ). The method predicts the binding affinity (in nM) for each 15-mer peptide–HLA-II complex. The population-level promiscuity scores for each 15-mer FVIII peptide is then defined as the (normalized) cumulative prevalence of common HLA-II proteins that bind it with high affinity (≤50 nM) in 3 populations: white European (blue), black African (green), and Global (red).
