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.


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The methylation level at the individual CpG sites was estimated using methylation-  Target amplimers ranged 150 bp to 440 bp in length Control amplimers (resistant to 212 enzymatic digestion) were ten bp larger than the target amplimers (Supplemental Table   213 S3).

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To the amplification products were added formamide (Hi-Di Formamide, Applied 217 Biosystems) and the GeneScan 500 molecular weight standard labeled with the 218 fluorochrome LIZ ™ (orange fluorescence) (Applied Biosystems). The amplification 219 products were separated by high resolution ((1 bp) capillary electrophoresis in the POP4 220 polymer using the ABI Prism ™ 310 Genetic Analyzer platform. The electrophoretic 221 profiles were analyzed using the GeneMapper ID 3.2 programs (Applied Biosystems).

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Computational Cross-Referencing with 5meCpG Levels in Public Methylomes 224 We validated the levels of methylation at each specific CpG site in the target immune  CpG sites with a normal distribution, the groups were compared using the Welch´s t-test 256 (unequal variances t-test). Otherwise, the groups were analyzed using the Wilcoxon 257 Rank Sum Test to calculate p-values and 95% confidence intervals (95%CI) of the 258 difference between the different groups. The range of 95%CI was graphically displayed 259 as a forest plot using ggplot2 graph data analysis.

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To select the target genes, we consider the immune regulatory mechanism of production 263 of anti-FVIII inhibitor antibodies. The exact mechanism of inhibitor production has not 264 yet been clarified, but it is proposed to initiate when antigen-presenting cells (APC) 265 endocytose the exogenous FVIII and degrade it into peptides. In turn, these peptides   Table 2.

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We determined the methylation status of thirteen individual CpG sites in ten target 294 genes (Supplemental Table S3) using restriction enzyme methylation-sensitive PCR 295 triplex assays. For each CpG site, a specific MSRE-PCR assay was developed. We

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Initially, we interrogated all the thirteen specific CpG sites in a subset of samples (up to 303 ten cases and ten controls) and ten non-hemophilia A healthy subjects. At the particular 304 CpG sites, we consistently observed one of three possible methylation statuses:  Table S5). We also noted that all the evaluated CpG sites at the target 328 genes showed concordant levels of methylation reported in several methylome studies 329 from diverse biological samples withdrawn from healthy subjects (Supplemental Table   330 S4 Dataset B).

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Within the IL6 amplimer, there is a two-base pair deletion, referring to the SNP  (Table S6). Cross-referencing with the PhenoScanner GWAS public database, 337 we found that in one study (Astle et al., 2016) the rs35081782 indel variant has been 338 associated at genome-wide scores with white blood cell count traits linked to common 339 complex diseases (Table S7 Dataset A).  We also noted that most of the 5meCpG sites (IL10, IFNG, CST7, IL4, and TNF) 348 exhibited gametic asymmetry in that the oocyte´s DNA was hypomethylated and the 349 sperm´s DNA was hypermethylated (Table S4 Dataset

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We also noted the occurrence of the SNP rs2227282 (NC_000005.9:g.132013179C>G)      (Table S5). Whether the observed differences result in measurable 440 increments in the production of IFN-γ is unclear.

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We note two potential caveats against the results of our study. Firstly, the limited  that tend to be hypermethylated in newborn but intermediately methylated at very 489 advanced age. Since no significant difference was observed in the methylation levels 490 after age stratification, we concluded that the differences in methylation levels at most 491 CpG sites interrogated in the present study are not age-related.

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The authors declare that the research was conducted in the absence of any commercial or 507 financial relationships that could be construed as a potential conflict of interest.                    Table S7. Genome-wide significance of associations between the IL4 rs2227282, IL6 853 rs35081782 and human diseases and traits from cross-referencing with genotype-