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D.P. Hart et al.
context of self-tolerance prediction (e.g. the binding potential of proteome cross-matches), we maintained a binding threshold of 1000 nM. For a given tFVIII-peptide binding threshold, a patient’s predicted risk of inhibitor formation was deemed to fall within one of the following three categories: “predicted low/negligible risk”, “predicted at risk”, or “unknown risk”. A patient was deemed to be at low/negligible risk if no novel pMHC surfaces were predicted to be formed with any of the 25 HLA isoforms. A patient was deemed to be “at risk” if novel pMHC surfaces were predicted to be formed by all, or all but one, of the HLA isoforms encoded by at least one HLA gene complex – HLA-DRB1, HLA- DRB3/4/5, HLA-DP, and/or HLA-DQ isoforms. (Here the “all, or all but one” condition was designed to rule out the possibility that
the patient was a heterozygous individual having a risk-free com- bination of common HLA isoforms, i.e. two or more low/negligi- ble risk isoforms per gene complex.) The inhibitor risk for patients in neither of the preceding categories was considered “unknown”. Patients with unknown inhibitor risk were omitted from the sta- tistical test.
The statistical test was undertaken using the two-tailed Fisher exact test implemented in the R statistical programming language. The Fisher exact test is necessary because the sample size and background inhibitor rate are both relatively low; in such circum- stances, the calculation of exact P values is important. Following standard convention, a P value of <0.05 was used to define statis- tical significance.
Figure 2. Flowchart for the assessment of human proteome cross-matching and missense muta- tion hemophilia A inhibitor risk. The flowchart shows the process by which the presence, or oth- erwise, of a novel peptide-MHC surface is determined given a specific combination of endogenous F8 missense mutation and human leukocyte antigen isoform. tFVIII: therapeutic factor VIII; FVIII: factor VIII; TCR: T-cell receptor.
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haematologica | 2019; 104(3)