The P2X7R is an ATP-gated cation channel expressed in hemopoietic cells that participates in both cell proliferation and apoptosis. Expression and function of the P2X7R have been associated with the clinical course of patients affected by chronic lymphocytic leukemia (CLL). Functional variants causing loss-of-function of the P2X7R have been identified, namely, polymorphisms 1513A>C (E496A), 1729T>A (I568N), and 946G>A (R307Q). Here we investigated other nonsynonymous polymorphisms located either in the extracellular portion of the receptor, such as the 489C>T (H155Y) variant, or in the long cytoplasmic tail of the receptor, such as the 1068G>A (A348T), 1096C>G (T357S), and 1405A>G (Q460R) variants. P2X7R function was monitored by measuring ATP-induced Ca2+ influx in PBL of patients affected by CLL and in recombinant human embryonic kidney (HEK) 293 cells stably transfected with each single P2X7 allelic variant. Ca2+ influx was markedly reduced in association with the 1513C allele, whereas variants located in the same intracellular domain, such as the 1068A, 1096G, or 1405G variants, were associated with a minor functional decrease. Significant Ca2+ flux increase was observed in lymphocytes from CLL patients bearing the 489C/T and 489T/T genotypes in association with the 1513A/A genotype. Functional analysis in recombinant HEK293 cells expressing P2X7R confirmed an increased ATP-dependent activation of the P2X7 489T mutant with respect to the wild type receptor, as assessed by both by [Ca2+]i influx and ethidium uptake experiments. These data identify the 489C>T as a gain-of-function polymorphism of the P2X7R.
Human leukocyte antigen (HLA)-G is an MHC class Ib molecule that is expressed at the feto-maternal interface during pregnancy. However, recent results have also shown that it may have important functions as an immuno-modulatory factor in adult life. Differences in the pattern of alternative splicing and in the stability of HLA-G mRNA transcripts have been associated with HLA-G polymorphisms, especially a 14 bp deletion/insertion polymorphism in the 3' untranslated region of the HLA-G gene. We have investigated the secretion of HLA-G5/soluble HLA-G1 and interleukin-10 (IL-10) in lipopolysaccharide (LPS)-activated peripheral blood mononuclear lymphocytes (PBMCs) in relation to the HLA-G 14 bp genotype. No HLA-G5/sHLA-G1 could be detected in the non-activated control PBMC culture media, and there were no significant differences among the three HLA-G 14 bp genotypes regarding IL-10 concentrations. In LPS-activated PBMC cultures, no significant differences among the three HLA-G 14 bp genotypes regarding HLA-G5/sHLA-G1 concentrations were observed. However, this was in contrast to the IL-10 levels (P=0.0004, Kruskal-Wallis test). The +14/+14 bp PBMC samples expressed higher levels of IL-10 when compared to the -14/+14 bp genotype and the -14/-14 bp genotype. Interestingly, the IL-10 G/G polymorphism at position -1082 was more frequent in the +14/+14 bp genotype (P=0.024, chi2 test). These results support an autocrine loop between HLA-G5/sHLA-G1 and IL-10 expression in activated PBMCs, which may result in higher IL-10 levels in +14/+14 bp HLA-G genotypes.
The results of this study indicate that HLA-regulated immune mechanisms are involved in TDI-induced asthma and that, in exposed subjects, specific factors may increase or decrease the risk of developing disease.
Toluene diisocyanate (TDI) is the most common cause of occupational asthma in western countries. The aim of this study was to investigate whether genetic factors are involved in toluene diisocyanate-induced asthma.We studied the frequency of human leucocyte antigen (HLA) class II genetic markers in three groups of subjects: 1) subjects with TDI-induced asthma (n=30); 2) exposed subjects with no history of TDI-induced asthma (n=12); and 3) normal subjects not exposed to TDI (n=126). Venous blood samples were collected from the three groups and the polymorphic second exon of DQA and DQB genes was amplified by the polymerase chain reaction (PCR) method.Evaluation of HLA class II gene products in TDI-induced asthma cases showed a positive association with HLA-DQB1*0503 and a negative association with HLA-DQB1*0501 alleles, which differed at residue 57 for a single amino acid, i.e. aspartic acid in DQB1*0503 and valine in DQB1*0501. No significant difference was found in the distribution of DQA1 alleles between asthmatics and controls.Our results confirm the hypothesis that HLA-DQB1*0503 has a role in conferring susceptibility to TDI-induced asthma and that residue 57 of HLA-DQB1 is a potentially critical location.
BackgroundMultiple sclerosis (MS) is a complex disorder thought to result from an interaction between environmental and genetic predisposing factors which have not yet been characterised, although it is known to be associated with the HLA region on 6p21.32. Recently, a picture of chronic cerebrospinal venous insufficiency (CCSVI), consequent to stenosing venous malformation of the main extra-cranial outflow routes (VM), has been described in patients affected with MS, introducing an additional phenotype with possible pathogenic significance.MethodsIn order to explore the presence of copy number variations (CNVs) within the HLA locus, a custom CGH array was designed to cover 7 Mb of the HLA locus region (6,899,999 bp; chr6:29,900,001-36,800,000). Genomic DNA of the 15 patients with CCSVI/VM and MS was hybridised in duplicate.ResultsIn total, 322 CNVs, of which 225 were extragenic and 97 intragenic, were identified in 15 patients. 234 known polymorphic CNVs were detected, the majority of these being situated in non-coding or extragenic regions. The overall number of CNVs (both extra- and intragenic) showed a robust and significant correlation with the number of stenosing VMs (Spearman: r = 0.6590, p = 0.0104; linear regression analysis r = 0.6577, p = 0.0106).The region we analysed contains 211 known genes. By using pathway analysis focused on angiogenesis and venous development, MS, and immunity, we tentatively highlight several genes as possible susceptibility factor candidates involved in this peculiar phenotype.ConclusionsThe CNVs contained in the HLA locus region in patients with the novel phenotype of CCSVI/VM and MS were mapped in detail, demonstrating a significant correlation between the number of known CNVs found in the HLA region and the number of CCSVI-VMs identified in patients. Pathway analysis revealed common routes of interaction of several of the genes involved in angiogenesis and immunity contained within this region. Despite the small sample size in this pilot study, it does suggest that the number of multiple polymorphic CNVs in the HLA locus deserves further study, owing to their possible involvement in susceptibility to this novel MS/VM plus phenotype, and perhaps even other types of the disease.
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