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Human proteome and hemophilia A inhibitor risk
coverages of >70%, >90% and >80%, respectively,22 using UniProt23 FVIII sequence P00451.
For the full set of 25 HLA class II alleles considered for this research, over four million peptide-HLA isoforms combinations were evaluated in order to identify the combinations that are pre- dicted to form a novel pMHC surface – plus a similar order of additional evaluations necessary to identify potential cross-match- es to the human proteome. A detailed breakdown of the calcula- tions performed is given in Online Supplementary Table S1.
Scanning the human proteome for cross-matches
Our preliminary risk assessment focused exclusively on the location of the disease-causing F8 missense mutation – that is, we assessed whether one or more peptides would be perceived as for- eign, given the binding properties and side-chain orientations of both tFVIII and endogenous FVIII peptides spanning the location of the missense mutation (Figure 1A). The innovative hypothesis explored here is that some pMHC surfaces that were identified as risk-associated by this preliminary approach may not be novel within the broader context of the whole human proteome.
For this research, we compared pMHC surfaces formed by tFVIII peptides spanning the location of the F8 missense mutation with those formed by peptides from the human proteome. Following research showing that the “maximal representation of the ‘immunological self’”17 is made available for tolerance induc- tion in the thymus, we used the complete human proteome from Ensembl24 containing over 100,000 proteins, including alternative isoforms that have an associated protein product.
A
As previously, we confined our analysis to 15-mers, this being the most common peptide length chosen for MHC class II binding experiments. The canonical proteome was sub-divided into all possible 9-mers. The resultant dataset consists of nearly 38 million 9-mers of which more than 11 million are non-identical.
A summary of the computational pipeline used to identify novel pMHC surfaces is shown in Figure 2.
Statistical analysis
We evaluated the accuracy of our method using F8 mutation data downloaded from the largest source of F8 mutation data in the public domain (The European Association for Haemophilia and Allied Disorders. The Factor VIII Gene (F8) Variant Database. http://www.factorviii-db.org. Accessed November 26, 2016). The dataset contained 956 distinct F8 missense mutations at 605 differ- ent loci from 3,243 individuals. Ninety of the missense mutations were associated with at least one reported case of inhibitor forma- tion, with a total of 160 individuals (prevalence 4.9%) listed as having inhibitors.
We tested the null hypothesis that our predicted rate and the database reported rate of inhibitor formation are independent. In the absence of HLA-typing information for the patients, we pre- dicted whether a patient with a given missense mutation has a risk of inhibitor formation based on the combined predictions for our chosen set of 25 common HLA class II isoforms. We evaluated dif- ferent IC50 binding thresholds for tFVIII peptides, on the grounds that binding strength is likely to be an important factor in inhibitor risk.25 However, when considering peptide-MHC binding in the
B
Figure 1. Schematic diagram showing how side-chain differ- ences may, or may not, lead to novel peptide-MHC surfaces. (A) If a tFVIII peptide spanning the location of the F8 missense mutation is a non-binder, it poses no risk of forming a novel pMHC surface capable of inducing an immune response. Otherwise, one needs to consider the position of the F8 mis- sense mutation within the MHC groove of the tFVIII peptide and the corresponding endogenous peptide. For most HLA class II isoforms, positions 1,4 6 and 9 are MHC-facing, and positions 2, 3, 5, 7 and 8 are TCR-facing. Where the F8 mis- sense mutation is at a downward, MHC-facing position (top row of A, denoted by a diamond), there are two scenarios: both tFVIII and endogenous peptides are binders, implying no risk; or the tFVIII peptide is a binder and the endogenous pep- tide is a non-binder, implying a potential risk. Where the F8 missense mutation is at an upward, TCR-facing position (bot- tom row of A, denoted by a diamond) and both peptides are binders, there is a potential risk. (B) Where a tFVIII peptide is associated with a potential risk according to the preceding assessment, a peptide from elsewhere in the human pro- teome that (i) has the same TCR-facing residues and (ii) is a binder, will militate against this risk, as no novel pMHC sur- face will be formed. FVIII: factor VIII; tFVIII: therapeutic factor VIII; TCR: T-cell receptor; MHC: major histocompatibility com- plex; pMHC: peptide-major histocompatibility complex.
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