Association of factor VIII inhibitors with genetic variants implicated in immune dysregulation Free

Introduction: Congenital hemophilia A (HA) is a severe bleeding disorder that results from the deficiency of coagulation factor VIII (FVIII). Prophylactic FVIII replacement therapy effectively reduces bleeding-related morbidity and mortality but can also trigger the production of anti-FVIII neutralizing alloantibodies, termed inhibitors, in ~30% of people with HA (PwHA) with severe disease (< 1% FVIII activity). While licensed and emerging non-factor therapeutics (NFT) facilitate hemostasis, PwHA still require additional hemostatic treatment (i.e. FVIII) for break-through bleeds. Despite substantial research into the mechanisms underlying FVIII inhibitor development, there is still no definitive way to predict who will form FVIII inhibitors which has hampered the ability to develop approaches to prevent inhibitors. Given that B and T cell responses to FVIII can be detected in ~20% of healthy individuals and that FVIII is the most common coagulation factor targeted by autoantibodies, we hypothesized that single nucleotide variants (SNVs) associated with immune dysregulation disorders may be associated with FVIII inhibitor development.

Aims: This study aims to identify candidate SNVs that associate with FVIII inhibitor status in order to form the basis for investigation of these SNVs as biomarkers of FVIII inhibitor development in a future validation cohort.

Methods: This single-center retrospective study enrolled PwHA with and without inhibitors on an IRB-approved protocol. Demographic and clinical information in the de-identified dataset included age, race, F8 mutation, FVIII inhibitor status, and immune tolerance induction (ITI) status. DNA was extracted from peripheral blood and sequenced using the Illumina Infinium ImmunoArray platform. Following quality control, sequencing data was imputed using GenomeStudio for allele genotype accounting for copy number variation. The odds ratio (OR) for the minor allele frequency (MAF) at each SNV was calculated by comparing allele frequencies between inhibitor positive and negative groups with p < 5x10^-4 considered suggestive of association with FVIII inhibitors for this discovery cohort.

Results: Of 111 PwHA enrolled on the study, 33 (30%) had FVIII inhibitors of whom 24 (73%) had attempted ITI and 13/24 (54%) had achieved FVIII tolerance as of data cutoff (December 2024). PwHA with inhibitors were more likely to be of non-Caucasian race (42% vs 19%, p = 0.02) and have nonsense F8 mutations (64% vs 40%, p = 0.003). In this PwHA study cohort, there were 39 SNVs with significantly different minor allele frequencies between inhibitor and non-inhibitor PwHA. Most of these SNVs were expected to be silent or located in non-coding regions. SNVs of interest with higher MAF in inhibitor PwHA were found in BTN3A2 (OR = 4.4, p = 8.8x10^-5), TNIP1 (OR = 3.3, p = 1.17x10^-4), FAM65B (OR = 3.0, p = 3.18x10^-4), and IL6R (OR 4.2, p = 4.98x10^-4). Three SNPs were identified in TLR7, all with MAF that were lower in inhibitor PwHA (OR 0.1-0.2, p < 4.7x10^-4) suggestive of a protective effect of TLR7 variants in FVIII inhibitor development. The BTN3A2 gene encodes a member of the immunoglobulin superfamily, TNIP1 encodes an A20-binding protein which helps regulate nuclear factor kappa-B activation and plays a role in autoimmunity, FAM65B encodes an atypical inhibitor of RhoA which mediates T cell and neutrophil polarization, IL6R encodes a component of the receptor for IL6 which is a cytokine with pleiotropic effects on inflammation and immunity, and TLR7 is a pathogen recognition protein involved in innate immunity.

Conclusions: This single center retrospective study has identified 5 candidate SNVs in genes involved in the regulation of both the innate and adaptive immune response with FVIII inhibitor development. While these data are intriguing, future work will include validation of these findings in a larger cohort of PwHA including quantitative RNA analysis to determine the impact of these variants on immune function. Further, targeting these genes/proteins in animal HA models will help to determine their ability to prevent or treat FVIII inhibitors.