Multiple sclerosis risk variant HLA-DRB1*1501 associates with high expression of DRB1 gene in different human populations.

The human leukocyte antigen (HLA) DRB1*1501 has been consistently associated with multiple sclerosis (MS) in nearly all populations tested. This points to a specific antigen presentation as the pathogenic mechanism though this does not fully explain the disease association. The identification of expression quantitative trait loci (eQTL) for genes in the HLA locus poses the question of the role of gene expression in MS susceptibility. We analyzed the eQTLs in the HLA region with respect to MS-associated HLA-variants obtained from genome-wide association studies (GWAS). We found that the Tag of DRB1*1501, rs3135388 A allele, correlated with high expression of DRB1, DRB5 and DQB1 genes in a Caucasian population. In quantitative terms, the MS-risk AA genotype carriers of rs3135388 were associated with 15.7-, 5.2- and 8.3-fold higher expression of DQB1, DRB5 and DRB1, respectively, than the non-risk GG carriers. The haplotype analysis of expression-associated variants in a Spanish MS cohort revealed that high expression of DRB1 and DQB1 alone did not contribute to the disease. However, in Caucasian, Asian and African American populations, the DRB1*1501 allele was always highly expressed. In other immune related diseases such as type 1 diabetes, inflammatory bowel disease, ulcerative colitis, asthma and IgA deficiency, the best GWAS-associated HLA SNPs were also eQTLs for different HLA Class II genes. Our data suggest that the DR/DQ expression levels, together with specific structural properties of alleles, seem to be the causal effect in MS and in other immunopathologies rather than specific antigen presentation alone.

DRB1*03:01 haplotypes: differential contribution to multiple sclerosis risk and specific association with the presence of intrathecal IgM bands.

BACKGROUND Multiple sclerosis (MS) is a multifactorial disease with a genetic basis. The strongest associations with the disease lie in the Human Leukocyte Antigen (HLA) region. However, except for the DRB1*15:01 allele, the main risk factor associated to MS so far, no consistent effect has been described for any other variant. One example is HLA-DRB1*03:01, with a heterogeneous effect across populations and studies. We postulate that those discrepancies could be due to differences in the diverse haplotypes bearing that allele. Thus, we aimed at studying the association of DRB1*03:01 with MS susceptibility considering this allele globally and stratified by haplotypes. We also evaluated the association with the presence of oligoclonal IgM bands against myelin lipids (OCMB) in cerebrospinal fluid. METHODS Genotyping of HLA-B, -DRB1 and -DQA1 was performed in 1068 MS patients and 624 ethnically matched healthy controls. One hundred and thirty-nine MS patients were classified according to the presence (M+, 58 patients)/absence (M-, 81 patients) of OCMB. Comparisons between groups (MS patients vs. controls and M+ vs. M-) were performed with the chi-square test or the Fisher exact test. RESULTS Association of DRB1*03:01 with MS susceptibility was observed but with different haplotypic contribution, being the ancestral haplotype (AH) 18.2 the one causing the highest risk. Comparisons between M+, M- and controls showed that the AH 18.2 was affecting only M+ individuals, conferring a risk similar to that caused by DRB1*15:01. CONCLUSIONS The diverse DRB1*03:01-containing haplotypes contribute with different risk to MS susceptibility. The AH 18.2 causes the highest risk and affects only to individuals showing OCMB.

HLA and non-HLA genes in Behçet's disease: a multicentric study in the Spanish population.

INTRODUCTION According to genome wide association (GWA) studies as well as candidate gene approaches, Behçet's disease (BD) is associated with human leukocyte antigen (HLA)-A and HLA-B gene regions. The HLA-B51 has been consistently associated with the disease, but the role of other HLA class I molecules remains controversial. Recently, variants in non-HLA genes have also been associated with BD. The aims of this study were to further investigate the influence of the HLA region in BD and to explore the relationship with non-HLA genes recently described to be associated in other populations. METHODS This study included 304 BD patients and 313 ethnically matched controls. HLA-A and HLA-B low resolution typing was carried out by PCR-SSOP Luminex. Eleven tag single nucleotide polymorphisms (SNPs) located outside of the HLA-region, previously described associated with the disease in GWA studies and having a minor allele frequency in Caucasians greater than 0.15 were genotyped using TaqMan assays. Phenotypic and genotypic frequencies were estimated by direct counting and distributions were compared using the χ(2) test. RESULTS In addition to HLA-B*51, HLA-B*57 was found as a risk factor in BD, whereas, B*35 was found to be protective. Other HLA-A and B specificities were suggestive of association with the disease as risk (A*02 and A*24) or protective factors (A*03 and B*58). Regarding the non-HLA genes, the three SNPs located in IL23R and one of the SNPs in IL10 were found to be significantly associated with susceptibility to BD in our population. CONCLUSION Different HLA specificities are associated with Behçet's disease in addition to B*51. Other non-HLA genes, such as IL23R and IL-10, play a role in the susceptibility to the disease.

Natalizumab-related anaphylactoid reactions in MS patients are associated with HLA class II alleles.

OBJECTIVES We aimed to investigate potential associations between human leukocyte antigen (HLA) class I and class II alleles and the development of anaphylactic/anaphylactoid reactions in patients with multiple sclerosis (MS) treated with natalizumab. METHODS HLA class I and II genotyping was performed in patients with MS who experienced anaphylactic/anaphylactoid reactions and in patients who did not develop infusion-related allergic reactions following natalizumab administration. RESULTS A total of 119 patients with MS from 3 different cohorts were included in the study: 54 with natalizumab-related anaphylactic/anaphylactoid reactions and 65 without allergic reactions. HLA-DRB1*13 and HLA-DRB1*14 alleles were significantly increased in patients who developed anaphylactic/anaphylactoid reactions (p M-H = 3 × 10(-7); odds ratio [OR]M-H = 8.96, 95% confidence interval [CI] = 3.40-23.64), with a positive predictive value (PPV) of 82%. In contrast, the HLA-DRB1*15 allele was significantly more represented in patients who did not develop anaphylactic/anaphylactoid reactions to natalizumab (p M-H = 6 × 10(-4); ORM-H = 0.2, 95% CI = 0.08-0.50), with a PPV of 81%. CONCLUSIONS HLA-DRB1 genotyping before natalizumab treatment may help neurologists to identify patients with MS at risk for developing serious systemic hypersensitivity reactions associated with natalizumab administration.

The systemic lupus erythematosus IRF5 risk haplotype is associated with systemic sclerosis

Systemic sclerosis (SSc) is a fibrotic autoimmune disease in which the genetic component plays an important role. One of the strongest SSc association signals outside the human leukocyte antigen (HLA) region corresponds to interferon (IFN) regulatory factor 5 (IRF5), a major regulator of the type I IFN pathway. In this study we aimed to evaluate whether three different haplotypic blocks within this locus, which have been shown to alter the protein function influencing systemic lupus erythematosus (SLE) susceptibility, are involved in SSc susceptibility and clinical phenotypes. For that purpose, we genotyped one representative single-nucleotide polymorphism (SNP) of each block (rs10488631, rs2004640, and rs4728142) in a total of 3,361 SSc patients and 4,012 unaffected controls of Caucasian origin from Spain, Germany, The Netherlands, Italy and United Kingdom. A meta-analysis of the allele frequencies was performed to analyse the overall effect of these IRF5 genetic variants on SSc. Allelic combination and dependency tests were also carried out. The three SNPs showed strong associations with the global disease (rs4728142: P  = 1.34×10(-8), OR  = 1.22, CI 95%  = 1.14-1.30; rs2004640: P  = 4.60×10(-7), OR  = 0.84, CI 95%  = 0.78-0.90; rs10488631: P  = 7.53×10(-20), OR  = 1.63, CI 95%  = 1.47-1.81). However, the association of rs2004640 with SSc was not independent of rs4728142 (conditioned P  = 0.598). The haplotype containing the risk alleles (rs4728142*A-rs2004640*T-rs10488631*C: P  = 9.04×10(-22), OR  = 1.75, CI 95%  = 1.56-1.97) better explained the observed association (likelihood P-value  = 1.48×10(-4)), suggesting an additive effect of the three haplotypic blocks. No statistical significance was observed in the comparisons amongst SSc patients with and without the main clinical characteristics. Our data clearly indicate that the SLE risk haplotype also influences SSc predisposition, and that this association is not sub-phenotype-specific.

Distinct mechanisms of loss of IFN-gamma mediated HLA class I inducibility in two melanoma cell lines

BACKGROUND The inability of cancer cells to present antigen on the cell surface via MHC class I molecules is one of the mechanisms by which tumor cells evade anti-tumor immunity. Alterations of Jak-STAT components of interferon (IFN)-mediated signaling can contribute to the mechanism of cell resistance to IFN, leading to lack of MHC class I inducibility. Hence, the identification of IFN-gamma-resistant tumors may have prognostic and/or therapeutic relevance. In the present study, we investigated a mechanism of MHC class I inducibility in response to IFN-gamma treatment in human melanoma cell lines. METHODS Basal and IFN-induced expression of HLA class I antigens was analyzed by means of indirect immunofluorescence flow cytometry, Western Blot, RT-PCR, and quantitative real-time RT-PCR (TaqMan(R) Gene Expression Assays). In demethylation studies cells were cultured with 5-aza-2'-deoxycytidine. Electrophoretic Mobility Shift Assay (EMSA) was used to assay whether IRF-1 promoter binding activity is induced in IFN-gamma-treated cells. RESULTS Altered IFN-gamma mediated HLA-class I induction was observed in two melanoma cells lines (ESTDAB-004 and ESTDAB-159) out of 57 studied, while treatment of these two cell lines with IFN-alpha led to normal induction of HLA class I antigen expression. Examination of STAT-1 in ESTDAB-004 after IFN-gamma treatment demonstrated that the STAT-1 protein was expressed but not phosphorylated. Interestingly, IFN-alpha treatment induced normal STAT-1 phosphorylation and HLA class I expression. In contrast, the absence of response to IFN-gamma in ESTDAB-159 was found to be associated with alterations in downstream components of the IFN-gamma signaling pathway. CONCLUSION We observed two distinct mechanisms of loss of IFN-gamma inducibility of HLA class I antigens in two melanoma cell lines. Our findings suggest that loss of HLA class I induction in ESTDAB-004 cells results from a defect in the earliest steps of the IFN-gamma signaling pathway due to absence of STAT-1 tyrosine-phosphorylation, while absence of IFN-gamma-mediated HLA class I expression in ESTDAB-159 cells is due to epigenetic blocking of IFN-regulatory factor 1 (IRF-1) transactivation.

Role of gene methylation in antitumor immune response: Implication for tumor progression

Cancer immunosurveillance theory has emphasized the role of escape mechanisms in tumor growth. In this respect, a very important factor is the molecular characterization of the mechanisms by which tumor cells evade immune recognition and destruction. Among the many escape mechanisms identified, alterations in classical and non-classical HLA (Human Leucocyte Antigens) class I and class II expression by tumor cells are of particular interest. In addition to the importance of HLA molecules, tumor-associated antigens and accessory/co-stimulatory molecules are also involved in immune recognition. The loss of HLA class I antigen expression and of co-stimulatory molecules can occur at genetic, transcriptional and post-transcriptional levels. Epigenetic defects are involved in at least some mechanisms that preclude mounting a successful host-antitumor response involving the HLA system, tumor-associated antigens, and accessory/co-stimulatory molecules. This review summarizes our current understanding of the role of methylation in the regulation of molecules involved in the tumor immune response.

Epigenetic regulation of human cancer/testis antigen gene, HAGE, in chronic myeloid leukemia.

BACKGROUND AND OBJECTIVES Cancer testis antigens (CTA) provide attractive targets for cancer-specific immunotherapy. Although CTA genes are expressed in some normal tissues, such as the testis, this immunologically protected site lacks MHC I expression and as such, does not present self antigens to T cells. To date, CTA genes have been shown to be expressed in a range of solid tumors via demethylation of their promoter CpG islands, but rarely in chronic myeloid leukemia (CML) or other hematologic malignancies. DESIGN AND METHODS In this study, the methylation status of the HAGE CTA gene promoter was analyzed by quantitative methylation-specific polymerase chain reaction (MSP) and sequencing in four Philadelphia-positive cell lines (TCC-S, K562, KU812 and KYO-1) and in CML samples taken from patients in chronic phase (CP n=215) or blast crisis (BC n=47). HAGE expression was assessed by quantitative reverse transcriptase-polymerase chain reaction. RESULTS The TCC-S cell line showed demethylation of HAGE that was associated with over-expression of this gene. HAGE hypomethylation was significantly more frequent in BC (46%) than in CP (22%) (p=0.01) and was correlated with high expression levels of HAGE transcripts (p<0.0001). Of note, in CP-CML, extensive HAGE hypomethylation was associated with poorer prognosis in terms of cytogenetic response to interferon (p=0.01) or imatinib (p=0.01), molecular response to imatinib (p=0.003) and progression-free survival (p=0.05). INTERPRETATIONS AND CONCLUSION: The methylation status of the HAGE promoter directly correlates with its expression in both CML cell lines and patients and is associated with advanced disease and poor outcome.

Influence of the LILRA3 Deletion on Multiple Sclerosis Risk: Original Data and Meta-Analysis.

BACKGROUND Multiple sclerosis (MS) is a neurodegenerative, autoimmune disease of the central nervous system. Genome-wide association studies (GWAS) have identified over hundred polymorphisms with modest individual effects in MS susceptibility and they have confirmed the main individual effect of the Major Histocompatibility Complex. Additional risk loci with immunologically relevant genes were found significantly overrepresented. Nonetheless, it is accepted that most of the genetic architecture underlying susceptibility to the disease remains to be defined. Candidate association studies of the leukocyte immunoglobulin-like receptor LILRA3 gene in MS have been repeatedly reported with inconsistent results. OBJECTIVES In an attempt to shed some light on these controversial findings, a combined analysis was performed including the previously published datasets and three newly genotyped cohorts. Both wild-type and deleted LILRA3 alleles were discriminated in a single-tube PCR amplification and the resulting products were visualized by their different electrophoretic mobilities. RESULTS AND CONCLUSION Overall, this meta-analysis involved 3200 MS patients and 3069 matched healthy controls and it did not evidence significant association of the LILRA3 deletion [carriers of LILRA3 deletion: p = 0.25, OR (95% CI) = 1.07 (0.95-1.19)], even after stratification by gender and the HLA-DRB1*15:01 risk allele.