A Differentially Methylated CpG Site in the IL4 Gene Associated with Anti-FVIII Inhibitor Antibody Development in Hemophilia A

Hemophilia A is the most common clotting disorder in humans. It affects one in five thousand live-born children. Mutations in the X-chromosome linked F8 gene lead to the deficiency of circulating factor VIII (FVIII). The defect is characterized by severe bleeding. The standard therapy is to replace the deficient factor intravenously. The main adverse event of the therapy is the development of anti-FVIII inhibitor antibodies that impair coagulation and result in increased complications and risk of death. Several risk factors have been described for the development of inhibitor antibodies, among them age, type of FVIII administered, ethnicity, and variant alleles in immune response genes. Epigenetic risks factors have not yet been explored. This work aimed to evaluate the methylation statuses at thirteen CpG sites (5meCpG) in regulatory regions of the IL1B, IL2, IL4, IL6, IL10, TNF, IFNG, CTLA4, CD28, and CST7 immune regulation genes in hemophilia A affected males on replacement therapy who develop or do not develop inhibitor antibodies. At each of the thirteen specific CpG sites, we observed one of three possible statuses: hypermethylated, hypomethylated or intermediate methylated. We found a statistically significant (p = 0.04) decrease in the methylation level at one CpG site in the IL4 intron 1 (CpG-3) in the affected group of patients presenting with anti-FVIII inhibitors as compared with the group of patients without inhibitors. The differential 5meCpG-3 maps within a predicted enhancer region in IL4 intron 1 that overlaps DNase I hypersensitive chromatin region of the Th2 IL5, IL13, and IL4 cytokine gene cluster and, therefore, permissive for gene expression. Six-bp upstream of the differentially 5meCpG-3 is the rs2227282 cis expression quantitative trait locus that influences the transcript levels of the PDLIM4, SLC22A4, SLC22A5, RAD50, IL4, KIF3A, SEPT8 genes. We consider the IL4 (CpG-3) site a promising lead epigenetic mark, the potential value of which must be appraised in a larger group of patients. The methodology employed also allowed to evaluate the distribution of the IL6 rs35081782 insertion/deletion variant, associated with white blood cell count traits in genome-wide association studies, and which showed no difference in distribution between the groups of patients.