Interleukin-10 Promoter Microsatellite Polymorphisms Influence the Immune Response to Heparin and the risk of Heparin-Induced Thrombocytopenia.

Heparin-induced thrombocytopenia (HIT) results from an atypical immune response to platelet factor 4/heparin complexes (PF4/H), with rapid synthesis of platelet-activating IgG antibodies (Abs). However, the reasons explaining why only a subset of patients treated with heparin develop IgG to PF4/H and why most of immunized patients do not develop HIT have yet to be fully defined. The immune response in HIT involves both B and T cells, and we hypothesized that polymorphisms located in the promoter of the IL10 gene that encodes IL10, a major regulatory cytokine, have an influence on the risk of HIT.

To investigate whether IL10 promoter single nucleotide polymorphisms (SNPs) and variations in IL10R and IL10G microsatellites are associated with the synthesis of Abs to PF4 and HIT.

A cohort of 82 patients with definite HIT (both PF4-specific ELISA and serotonin release assay were positive) and two control groups were studied. The first control group (Ab^neg) consisted of 85 patients who had undergone cardiopulmonary bypass (CBP) with high doses of heparin and who did not develop Abs to PF4 post-operatively. The second control group (Ab^pos) consisted of 84 patients who had also undergone cardiac surgery with CPB and had developed significant levels of PF4-specific antibodies but not HIT.

The SNPs rs1800896 (-1082A>G), rs1800871 (-819C>T) and rs180072 (592A>C), and the polymorphic CA repeat microsatellites IL10 R [5325CA(11_15)] and IL10 G [8134CA(14_29)] (all previously identified in IL10 promoter) were screened. Genotyping of SNPs was achieved by allelic discrimination using a real-time PCR method (ICycler Bio-Rad). Twenty nanograms of purified gDNA were used for each experiment with TaqMan^® Universal PCR Master Mix, using the two primers and two fluorescent probes of the TaqMan^® SNP Genotyping Assays (Applied Biosystems). Genotyping of IL10 microsatellites was determined as previously described (Cochery-Nouvellon et al. Biol Reprod 80, 1115–20. 2009). Linkage disequilibrium analysis was assessed using HAPLOVIEW 4.1, and haplotype reconstruction was carried-out with PHASE 2.1. Statistical analyses were performed with StatView 5.0 and Abacus: logistic regression was used to calculate genetic associations, and ANOVA to determine genotype-phenotype correlations. The Chi-square test was used to compare genotype distributions between the different groups.

The genotypes and allele frequencies of the 3 SNPs studied were similar in HIT patients and controls, with very high linkage disequilibrium between the -592 A>C and -819 C>T polymorphisms. Fourteen different alleles in the IL10G microsatellite (A16 to A29) and 3 alleles in IL10R (A13 to A15) could be defined depending on the number of CA repeat elements identified in the promoter sequence. The short A20 allele of IL10G was more frequent in Ab^neg patients (8.2%) than in Ab^pos (2.3%) and HIT patients (3%), thereby appearing highly protective from the risk of developing Abs to PF4 (OR 0.29; 95% CI [0.12-0.70], p < 0.006). Moreover, the short IL10R A13 allele was also less frequent in HIT patients (70.1%) than in controls (75.9%)(OR 0.37; 95% CI [0.15-0.89], p=0.026). Combinations of the 3 SNPs with IL10G and IL10R variations (short or long alleles) defined nine different representative haplotypes (H1 to H9). Interestingly, the combined H1/H8 haplotype comprising the short alleles for both IL10G and IL10R (A13) was less represented in HIT patients (4.9% vs. 13.6% in controls)(OR 0.33; 95% CI [0.11-0.97], p=0.036). In addition, levels of Abs to PF4 measured by ELISA in Ab^pos patients were also lower in H1/H8 subjects (median OD 0.67 vs. 1.21 in non-H1/H8 individuals, p=0.019).

IL10 promoter microsatellite polymorphisms appear to have a significant influence on the immune response to heparin. The haplotypes combining short IL10G and IL10R alleles are associated with lower levels of antibodies to PF4 and a reduced risk of HIT.

No relevant conflicts of interest to declare.