Host immune genetic variations influence the risk of developing acute myeloid leukaemia: results from the NuCLEAR consortium.

The purpose of this study was to conduct a two-stage case control association study including 654 acute myeloid leukaemia (AML) patients and 3477 controls ascertained through the NuCLEAR consortium to evaluate the effect of 27 immune-related single nucleotide polymorphisms (SNPs) on AML risk. In a pooled analysis of cohort studies, we found that carriers of the IL13rs1295686A/A genotype had an increased risk of AML (PCorr = 0.0144) whereas carriers of the VEGFArs25648T allele had a decreased risk of developing the disease (PCorr = 0.00086). In addition, we found an association of the IL8rs2227307 SNP with a decreased risk of developing AML that remained marginally significant after multiple testing (PCorr = 0.072). Functional experiments suggested that the effect of the IL13rs1295686 SNP on AML risk might be explained by its role in regulating IL1Ra secretion that modulates AML blast proliferation. Likewise, the protective effect of the IL8rs2227307 SNP might be mediated by TLR2-mediated immune responses that affect AML blast viability, proliferation and chemorresistance. Despite the potential interest of these results, additional functional studies are still warranted to unravel the mechanisms by which these variants modulate the risk of AML. These findings suggested that IL13, VEGFA and IL8 SNPs play a role in modulating AML risk.

MICA*A4 protects against ulcerative colitis, whereas MICA*A5.1 is associated with abscess formation and age of onset.

Ulcerative colitis (UC) is one of the two major forms of inflammatory bowel disease, the aetiology of which remains unknown. Several studies have demonstrated the genetic basis of disease, identifying more than 130 susceptibility loci. The major histocompatibility complex class I chain-related gene A (MICA) is a useful candidate to be involved in UC pathogenesis, because it could be important in recognizing the integrity of the epithelial cell and its response to stress. The aim of this study was to analyse the relationship between polymorphisms in the transmembrane domain of MICA and susceptibility to develop UC. A total of 340 patients with UC and 636 healthy controls were genotyped for MICA transmembrane polymorphism using a polymerase chain reaction (PCR) combined with fluorescent technology. Different MICA alleles were determined depending on the PCR product size. The allele MICA*A4 was less frequent in patients than in controls (P = 0·003; OR = 0·643), and this protective role is higher when it forms haplotype with B*27 (P = 0·002; OR = 0·294). The haplotype HLA-B*52/MICA*A6 was also associated with UC [P = 0·001; odds ratio (OR) = 2·914]. No other alleles, genotypes or haplotypes were related with UC risk. Moreover, MICA*A5.1 is associated independently with abscesses (P = 0·002; OR = 3·096) and its frequency is lower in patients diagnosed between ages 17 and 40 years (P = 0·007; OR = 0·633), meaning an extreme age on onset. No association with location, extra-intestinal manifestations or need for surgery was found.

Polymorphisms in Host Immunity-Modulating Genes and Risk of Invasive Aspergillosis: Results from the AspBIOmics Consortium.

Recent studies suggest that immune-modulating single-nucleotide polymorphisms (SNPs) influence the risk of developing cancer-related infections. Here, we evaluated whether 36 SNPs within 14 immune-related genes are associated with the risk of invasive aspergillosis (IA) and whether genotyping of these variants might improve disease risk prediction. We conducted a case-control association study of 781 immunocompromised patients, 149 of whom were diagnosed with IA. Association analysis showed that the IL4Rrs2107356 and IL8rs2227307 SNPs (using dbSNP numbering) were associated with an increased risk of IA (IL4Rrs2107356 odds ratio [OR], 1.92; 95% confidence interval [CI], 1.20 to 3.09; IL8rs2227307 OR, 1.73; 95% CI, 1.06 to 2.81), whereas the IL12Brs3212227 and IFNγrs2069705 variants were significantly associated with a decreased risk of developing the infection (IL12Brs3212227 OR, 0.60; 95% CI, 0.38 to 0.96; IFNγrs2069705 OR, 0.63; 95% CI, 0.41 to 0.97). An allogeneic hematopoietic stem cell transplantation (allo-HSCT)-stratified analysis revealed that the effect observed for the IL4Rrs2107356 and IFNγrs2069705 SNPs was stronger in allo-HSCT (IL4Rrs2107356 OR, 5.63; 95% CI, 1.20 to 3.09; IFNγrs2069705 OR, 0.24; 95% CI, 0.10 to 0.59) than in non-HSCT patients, suggesting that the presence of these SNPs renders patients more vulnerable to infection, especially under severe and prolonged immunosuppressive conditions. Importantly, in vitro studies revealed that carriers of the IFNγrs2069705C allele showed a significantly increased macrophage-mediated neutralization of fungal conidia (P = 0.0003) and, under stimulation conditions, produced higher levels of gamma interferon (IFNγ) mRNA (P = 0.049) and IFNγ and tumor necrosis factor alpha (TNF-α) cytokines (P value for 96 h of treatment with lipopolysaccharide [PLPS-96 h], 0.057; P value for 96 h of treatment with phytohemagglutinin [PPHA-96 h], 0.036; PLPS+PHA-96 h = 0.030; PPHA-72 h = 0.045; PLPS+PHA-72 h = 0.018; PLPS-96 h = 0.058; PLPS+PHA-96 h = 0.0058). Finally, we also observed that the addition of SNPs significantly associated with IA to a model including clinical variables led to a substantial improvement in the discriminatory ability to predict disease (area under the concentration-time curve [AUC] of 0.659 versus AUC of 0.564; P-2 log likehood ratio test = 5.2 · 10(-4) and P50.000 permutation test = 9.34 · 10(-5)). These findings suggest that the IFNγrs2069705 SNP influences the risk of IA and that predictive models built with IFNγ, IL8, IL12p70, and VEGFA variants can used to predict disease risk and to implement risk-adapted prophylaxis or diagnostic strategies.

Lack of association of ACP1 gene with inflammatory bowel disease: a case-control study.

The red cell acid phosphatease (ACP1) gene, which encodes a low molecular weight phosphotyrosine phosphatase (LMW-PTP), has been suggested as a common genetic factor of autoimmunity. In the present study, we aimed to investigate the possible influence of ACP1 polymorphisms in the susceptibility of inflammatory bowel disease (IBD). A total of 1271 IBD Spanish patients [720 Crohn's disease (CD) and 551 ulcerative colitis (UC)] and 1877 healthy subjects were included. Four single-nucleotide polymorphisms (SNPs), rs10167992, rs11553742, rs7576247 and rs3828329, were genotyped using TaqMan SNP genotyping assays. Common ACP1 alleles (i.e. ACP1*A, ACP1*B and ACP1*C) were determined by two of these SNPs. After the analysis, no evidence of association of the ACP1 genetic variants was found with CD or UC. Therefore, our results suggest that the ACP1 gene may not play a relevant role in the development of IBD.

Association of ATG16L1 and IRGM genes polymorphisms with inflammatory bowel disease: a meta-analysis approach.

The aim of this study was to determine the role of the ATG16L1 (rs2241880) and IRGM (rs13361189 and rs4958847) genes polymorphism in Crohn's disease (CD) and ulcerative colitis (UC). Our study included 557 CD and 425 UC patients and 672 ethnically matched Spanish controls and a meta-analysis with the data published to date. The polymorphisms were genotyped using predesigned TaqMan single nucleotide polymorphism genotyping assays. There was a statistically significant difference in the distribution of the ATG16L1 rs2241880 G allele between CD patients and controls in the Spanish population: P=6.5 x 10(-9), odds ratio (OR)=1.62. Although no differences were observed between UC patients and controls in the Spanish cohort, a meta-analysis demonstrated that the ATG16L1 G allele increase significantly risk for UC (P=0.0003, pooled OR=1.08). In addition, our meta-analysis data showed that IRGM rs13361189 and rs4958847 polymorphisms were associated with CD (rs13361189 C allele P=1.07 x 10(-19), pooled OR=1.34; rs4958847 A allele P=2.78 x 10(-17), pooled OR=1.31) and UC (rs13361189 P=0.0069, pooled OR=1.16; rs4958847 P=0.014, pooled OR=1.13). In conclusion, our results confirm the ATG16L1 rs2241880 and IRGM rs13361189 and rs4958847 polymorphisms as important markers for CD susceptibility and indicate that these variants are also associated with UC.

Analysis of KIR gene frequencies in HLA class I characterised bladder, colorectal and laryngeal tumours.

Three cohorts of patients with laryngeal, bladder or colorectal tumours were investigated for frequency of killer immunoglobulin-like receptor (KIR) genes compared with a normal control population. The frequency of KIR3DL1 and KIR2DS4 was significantly increased (but not after correction for number of comparisons made) in patients with bladder tumour compared with controls. No other significant differences were found in gene frequencies or in the frequencies of those KIR genes with and without their human leucocyte antigen (HLA) ligands. Furthermore, no significant differences were found in KIR gene frequencies, taking into consideration the type of loss of HLA expression in the individual tumours. Finally, in the group of colorectal carcinomas, there was an overall significant difference in the frequencies of C group heterozygosity and homozygosity with HLA alterations on the tumour.

Analysis of HLA class I alterations in tumors: choosing a strategy based on known patterns of underlying molecular mechanisms.

The application of peptide-based immunotherapy in the treatment of cancer has known limitations in patients with loss or downregulation of human leukocyte antigen (HLA) class I expression on tumor cells. These alterations diminish the ability of cancer cells to present tumor peptides to T cells and therefore lead to failure of peptide-based cancer vaccination. Abnormal expression of HLA class I molecules in malignant cells is a frequent event that ranges from total loss of class I molecules to partial loss of HLA-specific haplotypes or alleles. Different mechanisms underlie these alterations and might require different therapeutic approaches. A complete characterization of molecular defects may suggest strategies for the selection and follow-up of patients undergoing T-cell based immunotherapy. Moreover, a precise identification of the mechanism leading to HLA class I defects in patients with cancer will help develop new, personalized patient-tailored treatment protocols. Here, we describe several examples showing the necessity and feasibility of making detailed individual analysis of HLA alteration mechanisms based on previously described molecular patterns in different types of malignancy. We recommend using this approach, at least in some patients, to enhance the therapeutic benefit of cancer immunotherapy.

Contribution of MHC class I region to genetic susceptibility for giant cell arteritis.

OBJECTIVE: The aim of this study was to assess the potential contribution of HLA-class I MICA and HLA-B gene polymorphisms towards the pathogenesis of giant cell arteritis (GCA). METHODS: Ninety-eight biopsy-proven GCA patients and 225 ethnically matched controls from Lugo, Northwest Spain, were genotyped for the MICA-TM microsatellite polymorphism using a polymerase chain reaction (PCR)-based method. Genotyping of HLA-B was performed using PCR and detection with a reverse sequence-specific oligonucleotide (SSO) probes system. RESULTS: A significant difference in the distribution of the alleles of MICA between patient and control groups (P = 0.005) was found. This was due to an increased frequency of the MICA A5 allele in GCA patients compared with controls (26 vs 13.6%; P = 0.0001; P(C) = 0.0005; OR 2.2, 95% CI 1.4-3.4). In addition, the HLA-B*15 allele showed a higher frequency in GCA patients compared with controls (P = 0.004; P(C) = 0.04; OR 2.7, 95% CI 1.3-5.7). Interestingly, the association observed with the MICA A5 allele seems to be independent of linkage disequilibrium with HLA-B, as well as independent of that previously described with HLA-DRB1*04. Remarkably, simultaneous presence of MICA A5 and HLA-B*15 or HLA-DRB1*04 genetic markers leads to an increase in the OR obtained for each individual genetic marker (MICA A5 + B*15 OR 3.2; MICA A5 + DRB1*04 OR 5.8). CONCLUSIONS: Our results provide the first evidence that the MICA and HLA-B genes are independently associated with the genetic susceptibility to GCA, and suggest that several genes within the MHC might have independent effects in the susceptibility to this systemic vasculitis.

Study of a functional polymorphism in the p53 gene in systemic lupus erythematosus: lack of replication in a Spanish population.

The aim of this study was to assess the possible association between the p53 suppressor gene codon 72 polymorphism and systemic lupus erythematosus (SLE). Our study population consisted of 513 SLE patients and 567 healthy controls. All the individuals were of Spanish Caucasian origin. Genotyping of the p53 codon 72 polymorphism was performed by allele-specific PCR. No statistically significant differences were observed between SLE patients and healthy controls when p53 codon 72 genotype and allele frequencies were compared. In addition, no evidence for association with clinical subfeatures of SLE was found. In conclusion, the p53 codon 72 polymorphism associated with SLE in a Korean population does not appear to play a major role in the susceptibility or severity of SLE in the Spanish population.

Inducible and endothelial nitric oxide synthase genes polymorphism in inflammatory bowel disease.

To assess the influence of inducible and endothelial nitric oxide synthase gene (NOS2A and NOS3) polymorphisms in susceptibility to Crohn's disease (CD) and ulcerative colitis (UC). A total of 505 inflammatory bowel disease (IBD) patients (221 with UC and 284 with CD) and 332 ethnically matched controls were studied. Patients and controls were genotyped by polymerase chain reaction -based techniques for a multiallelic (CCTTT)(n) repeat and biallelic marker (TAAA)(n) in the promoter region of the NOS2A gene and for a T/C polymorphism at position -786 in the promoter region and a polymorphism in exon 7(298Glu/Asp) of the NOS3 gene. There was not association between NOS2A and NOS3 genotypes, alleles or haplotypes frequencies with UC, CD and controls. Our data suggest that NOS2A and NOS3 polymorphisms do not play a major role in IBD predisposition.

Study of the role of functional variants of SLC22A4, RUNX1 and SUMO4 in systemic lupus erythematosus.

BACKGROUND: Functional polymorphisms of the solute carrier family 22, member 4 (SLC22A4), runt related transcription factor 1 (RUNX1) and small ubiquitin-like modifier 4 (SUMO4) genes have been shown to be associated with several autoimmune diseases. OBJECTIVE: To test the possible role of these variants in susceptibility to or severity of systemic lupus erythematosus (SLE), on the basis that common genetic bases are shared by autoimmune disorders. METHODS: 597 SLE patients and 987 healthy controls of white Spanish origin were studied. Two additional cohorts of 228 SLE patients from Sweden and 122 SLE patients from Colombia were included. A case-control association study was carried out with six single nucleotide polymorphisms (SNP) spanning the SLC22A4 gene, one SNP in RUNX1 gene, and one additional SNP in SUM04 gene. RESULTS: No significant differences were observed between SLE patients and healthy controls when comparing the distribution of the genotypes or alleles of any of the SLC22A4, RUNX1, or SUMO4 polymorphisms tested. Significant differences were found in the distribution of the SUMO4 genotypes and alleles among SLE patients with and without nephritis, but after multiple testing correction, the significance of the association was lost. The association of SUMO4 with nephritis could not be verified in two independent SLE cohorts from Sweden and Colombia. CONCLUSIONS: These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analysed do not play a role in the susceptibility to or severity of SLE.

APC and chromosome instability in colorectal cancer.

Colon cancer is a common disease that can be sporadic or familial. An inactivated adenomatous polyposis coli (APC) suppressor gene is found in over 80% of colorectal tumors, this being an early alteration in the development of adenomatous polyps. APC function is not only critical for tumor initiation and progression, and chromosome instability (CIN) is another characteristic dependent at least partly on APC mutations.

The functional genetic variation in the PTPN22 gene has a negligible effect on the susceptibility to develop inflammatory bowel disease.

The aim of this study was to assess the possible association between the protein tyrosine phosphatase non-receptor 22 (PTPN22) gene 1858C-->T (rs2476601, encoding R620W) polymorphism and inflammatory bowel disease (IBD). Our study population consisted of 1113 IBD [544 ulcerative colitis (UC) and 569 Crohn's disease (CD)] patients and 812 healthy subjects. All the individuals were of Spanish white origin. Genotyping of the PTPN22 gene 1858C-->T polymorphism was performed by real time polymerase chain reaction technology, using TaqMan 5'-allelic discrimination assay. The frequency of the PTPN22 1858T allele in healthy subjects was 6.2% compared with 6.7% in the UC patients and 5.1% in Crohn's patients. No statistically significant differences were observed when the PTPN22 1858C-->T allele and genotype distribution among CD patients, UC patients and healthy controls were compared. These results indicate that the PTPN22 1858C-->T polymorphism does not appear to play a major role in IBD predisposition in our population.

CTLA4/CT60 polymorphism is not relevant in susceptibility to autoimmune inflammatory intestinal disorders.

The aim of this work was to investigate the possible influence of the recently described CT60 A/G dimorphism of the CTLA4 (cytotoxic T-lymphocyte antigen 4) gene in the susceptibility to two different autoimmune inflammatory intestinal disorders, inflammatory bowel disease (IBD) and celiac disease. We analyzed a case-control cohort composed of 528 Spanish patients with IBD (284 with Crohn disease and 244 with ulcerative colitis) and 454 unrelated healthy individuals, and additionally a group of 90 celiac disease families. CT60 genotyping was performed with a TaqMan 5' allelic discrimination assay. After comparing patients with IBD with the control population, we found no significant deviation in the distribution of the alleles or genotypes of CTLA4/CT60 dimorphism. In addition, by means of familial and case-control analysis, no evidence for a statistically significant association was observed between CTLA4/CT60 and celiac disease susceptibility. Therefore, our results suggest that the CTLA4/CT60 polymorphism does not play a major role in inflammatory intestinal disorders.

Molecular strategies to define HLA haplotype loss in microdissected tumor cells.

Loss of heterozygosity (LOH) of chromosome 6p21 is an important mechanism that generates HLA haplotype loss in various human tumors. This mechanism produces non-reversible HLA-deficient tumor cells that can escape T cell immune responses in peptide-vaccinated cancer patients. However, the exact frequency of this mechanism is still unknown, because contaminating stroma in solid tumor tissues masks the tumor DNA obtained from solid samples. A microdissection technique was applied to 4-8 microm sections of cryopreserved tumor tissues from a group of colorectal and laryngeal carcinomas. Fifteen patients were analyzed for the presence of LOH associated with the beta(2)-microglobulin gene in chromosome 15, and five patients for LOH associated with HLA genes in chromosome 6. In two cases, autologous metastasis tissue samples were also available. The patients were selected for showing an altered HLA class I tumor phenotype as determined by immunohistological techniques. DNA was obtained from this microdissected material and amplified in order to detect the presence or absence of nine previously selected microsatellite markers. HLA sequence based typing (SBT) was also applied to these microdissected DNA samples to define the HLA genotype. Microdissection greatly improved the definition of LOH, with nearly 100% signal reduction in one of the alleles. In addition, this procedure allowed us to detect beta(2)-microglobulin LOH in tumors that expressed some HLA molecules. Our data indicate that this procedure can be successfully applied to microdissected samples from solid tumors, thus enhancing the power and sensitivity of LOH detection.

Beta2-microglobulin gene mutation is not a common mechanism of HLA class I total loss in human tumors.

One hundred and sixty-two tumor samples were analyzed for HLA class I expression using immunohistological techniques. HLA class I total loss (phenotype no. I) was detected in 31 cases (19%), comprising 20 colorectal, 3 laryngeal, and 2 bladder carcinomas and 6 melanomas. Twenty-one cases were selected for molecular analysis due to a higher proportion of tumor cells versus stroma cells (75%). We investigated whether beta2-microglobulin mutation was responsible for HLA downregulation. Single-strand conformation polymorphism and sequencing analysis of DNA samples was performed. Alterations were detected only in melanomas M78 (a point mutation in the initiation ATG sequence), M79 (a mutation in codon 31 producing a stop codon), and M34 (a TTCT deletion introducing a termination codon signal). We found no beta2-microglobulin gene mutation in the other 18 samples. Loss of heterozygosity in 15q close to the beta2-microglobulin gene was found in 5 cases. We conclude that HLA class I total loss can frequently occur without beta2-microglobulin gene mutations.

Criteria to define HLA haplotype loss in human solid tumors.

Short tandem repeat (STR) markers are currently used to define loss of heterozygosity (LOH) of genes and chromosomes in tumors. Chromosome 6 and chromosome 15 STR markers are applied to define loss of HLA and related genes (e.g. TAP and beta2m). The number of STR identified in the HLA region is still increasing. In this study, seven representative STR markers covering the 6p/6q arms of chromosome 6 including the HLA region and two for chromosome 15 flanking the beta2m gene, were selected as minimally required for reliable LOH studies. A multiplex polymerase chain reaction (PCR) strategy is proposed when small number of cells are available in microdissected tumor samples.