Analysis of Immunogenetic Factors in Paroxysmal Nocturnal Hemoglobinuria Reveal Increased Frequency of IL-10 Low Secretor Genotype and Correlates with Induced IL-10 Production.

Abstract

Aplastic anemia (AA) and paroxysmal nocturnal hemoglobinuria (PNH) have a common pathogenesis related to an immune attack on hematopoietic progenitor and stem cells. While the inciting events are not known, it is possible that complex immunogenetic predisposition factors exist which, in the context of exogenous influences, determine risk for these diseases. They include HLA background, KIR genotype, cytokine and immunomodulatory gene polymorphisms as well as gene variants involved in peptide processing and presentation. We have empirically selected a number of important polymorphisms that were described in the context of various immunologic diseases and studied their frequency in a large cohort (N=57) of PNH patients. As binding of KIR to the appropriate HLA ligand (KIR-L) can modulate activation of Nk-cells and cytotoxic T lymphocytes, we examined the combined impact of KIR/KIR-L genotypes on the risk of PNH and PNH/AA syndrome. PNH showed a decreased frequency of inhibitory KIR-2DL1 and KIR-2DL3 genes (79% vs. 95%, p=.0054; 67% vs. 89%, p=.0032). Analysis of the KIR genotype in correlation with the corresponding KIR-L profile, deduced from HLA typing, revealed an increased frequency of unopposed 2DS2 (2DS2/C1 type, 37% vs.10%, p=.012) and 2DL2 (2DL2/C1 mismatch 37% vs 11%, p=.031) but these mismatches have a potentially opposing functional effect. Using sequence-specific PCR amplification and/or direct sequencing, we have genotyped DNA samples derived from our cohort who presented with different subtypes of PNH and studied single nucleotide polymorphisms (SNPs) in cytokine genes such as TNF-a (−308 G/A), TGF-b 1 (C/T codon 10, C/G codon 25), IL-10 (−1082 G/A), and IFN-g (+874 A/T) and immunomodulatory receptor genes like CTLA-4 exon 6 (+49 G/A), FcγRIIIa (158 F/V) and CD45-exons 6 (+138 A/G), and 4 (+54 A/G, +77 C/G). These SNPs are responsible for intrinsic differences in cytokine production and receptor function and can thereby influence immune physiologic and pathologic responses. PNH patients showed a significantly higher frequency of A/A polymorphisms at intron −1082 of the IL-10 gene consistent with a “low secretor phenotype” (36% PNH (N=50) vs 13.8% controls (N=363), p=0.0001). In contrast to a few smaller studies, no association with any other SNP tested was found. To establish whether this finding translates into functional consequences, we examined basal and PMA-induced IL-10 secretion in PNH patients andcontrols. Upon induction, IL-10 production increased in controls (N=5, 14.11± 9.8), while in general, stimulation resulted in a much weaker IL-10 response in PNH patients (N=6, 4.51± 2.16). However, when compared genotypic IL-10 “low secretors” (−1082 A/A) among PNH patients showed significantly lower induction levels of IL-10 (N=2, 3.1± 1.58) as compared to PNH patients with “normal/high secretor” genotypes (−1082 A/G,G/G) ((N=4, 5.22 +/−3.1). The low secretor IL-10 genotype may correspond to the exaggerated TH1 response in PNH and in general supports the notion that complex inherited traits may exist that genetically determine propensity to PNH evolution.