Objective.To assess the possible association between the PTPN22 gene 1858C3 T polymorphism and the predisposition and clinical expression of 2 systemic autoimmune diseases, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE).Methods. Our study population consisted of 826 RA patients, 338 SLE patients, and 1,036 healthy subjects. All subjects were of Spanish Caucasian origin. Genotyping of the PTPN22 gene 1858C3 T polymorphism was performed by real-time polymerase chain reaction technology, using the TaqMan 5-allele discrimination assay.Results. The overall distribution of genotypes in the RA patients was significantly different from that in the controls (P ‫؍‬ 0.005, by chi-square test with 2 ؋ 3 contingency tables). We observed a statistically significant difference in the distribution of the PTPN22 1858T allele between healthy subjects (7.4%), and RA patients Conclusion. These results suggest that the PTPN22 1858T allele may confer differential susceptibility to RA and SLE in the Spanish population.

Interleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in chronic inflammation and autoimmune disorders. In this study, we aimed to determine the potential role of the IL18 gene in SLE. To define the genetic association of the IL18 and SLE, we have genotyped nine SNPs in an independent set of Spanish cases and controls. The IL18 polymorphisms were genotyped by PCR, using a predeveloped TaqMan allele discrimination assay. Two SNPs were still significant after fine mapping of the IL18 gene. The SNP (rs360719) surviving correction for multiple tests was genotyped in two replication cohorts from Italy and Argentina. After the analysis, a significance with rs360719 C-allele remained across the sets and after the meta-analysis (Pooled OR = 1.37, 95% CI 1.21-1.54, combined P = 3.8E-07, Pc = 1.16E-06). Quantitative real-time PCR was performed to assess IL18 mRNA expression in PBMC from subjects with different IL18 rs360719 genotypes. We tested the effect of the IL18 rs360719 polymorphism on the transcription of IL18 by electrophoretic mobility shift assay and western blot. We found a significant increase in the relative expression of IL18 mRNA in individuals carrying the rs360719 C-risk allele; in addition we show that the polymorphism creates a binding site for the transcriptional factor OCT-1. These findings suggest that the novel IL18 rs360719 variant may play an important role in determining the susceptibility to SLE and it could be a key factor in the expression of the IL18 gene.

Objective. To investigate the role of the HO-1 gene as a novel functional candidate gene for rheumatoid arthritis (RA).Methods. We performed a case-control study including 736 RA patients and 846 healthy controls of Spanish Caucasian origin. Two putative functional HO-1 promoter polymorphisms, a (GT) n microsatellite and a -413 A/T single-nucleotide polymorphism (SNP), were selected as genetic markers and genotyped using polymerase chain reaction-based methods. In addition, the intracellular expression of heme oxygenase 1 (HO-1) was determined in healthy individuals with different (GT) n genotypes.Results. The distribution of HO-1 (GT) n short (S) alleles (<25 GT repeats) and long (L) alleles (>25 GT repeats) revealed a significant protective effect of S (GT) n alleles (P ‫؍‬ 0.019) (odds ratio [OR] 0.8, 95% confidence interval [95% CI] 0.7-0.9) and the SS (GT) n genotype (P ‫؍‬ 0.002) (OR 0.6, 95% CI 0.4-0.9). In contrast, the -413 HO-1 promoter SNP did not yield any statistically significant deviation between RA patients and controls, considering either allele or genotype frequencies. The haplotype analysis showed a strong protective effect of the S/A haplotype (P ‫؍‬ 7 ؋ 10 ؊7 , corrected P [P corr ] ‫؍‬ 3 ؋ 10 ؊6 ) (OR 0.4, 95% CI 0.3-0.6), whereas the L/A haplotype showed the opposite tendency (P ‫؍‬ 0.008, P corr ‫؍‬ 0.03) (OR 1.2, 95% CI 1.0-1.4). In addition, we demonstrated that monocytes from individuals carrying the SS (GT) n genotype showed a significantly higher percentage of HO-1 expression than did cells from LL homozygous individuals (P ‫؍‬ 0.0003). Conclusion. In this study, we identified the HO-1 (GT) n microsatellite as a new genetic marker involved in RA genetics in our population.

HLA class I loss or down-regulation is a widespread mechanism used by tumor cells to avoid tumor recognition by cytotoxic T lymphocytes, and thus favor tumor immune escape. Multiple mechanisms are responsible for these HLA class I alterations. In different epithelial tumors, loss of heterozygosity (LOH) at chromosome region 6p21.3, leading to HLA haplotype loss, occurs in 6-50% of all cases depending on the tumor entity. In this paper we report the frequency of LOH at 6p21 in 95 colorectal carcinomas (CRC) previously analyzed for altered HLA class I expression with immunohistological techniques. We used PCR microsatellite amplification of selected STR markers located on Chromosome 6 to identify LOH with DNA from microdissected tumor tissues and the surrounding stroma. Sequence-specific oligonucleotide analysis was performed in microdissected stroma and tumor cells for HLA typing, and to detect HLA haplotype loss. A high frequency (40%) of HLA haplotype loss was found in CRC. Eight tumors showed microsatellite instability. We sometimes observed two or more mechanisms responsible for HLA alteration within the same HLA-altered phenotype, such as LOH and HLA class I total loss. In 25 tumors (26%) no HLA class I alteration could be identified. These data are potentially relevant for CRC patients undergoing T-cell-based immunotherapy.

Monoclonal TCR␣␤ ؉ /CD4 ؉ T-large granular lymphocyte (T-LGL) lymphocytosis is a T-cell disorder with a restricted TCR-V␤ repertoire. In the present study we explored the potential association between the expanded TCR-V␤ families, the CDR3 sequences of the TCR-V␤ gene, and the HLA genotype of patients with monoclonal TCR␣␤ ؉ /CD4 ؉ T-LGL lymphocytosis.

Major histocompatibility complex (MHC) class I loss or downregulation in cancer cells is a major immune escape route used by a large variety of human tumors to evade anti-tumor immune responses mediated by cytotoxic T lymphocytes. Multiple mechanisms are responsible for such HLA class I alterations. However, the precise frequency of these molecular defects has not been clearly determined in tumors derived from specific tissues. To analyze such defects we aim to define the major HLA class I-altered phenotypes in different tumor types. In this paper we report on HLA class I expression in 70 laryngeal carcinomas. We used immunohistological techniques with a highly selective panel of anti-HLA monoclonal antibodies (mAb), and polymerase chain reaction (PCR) microsatellite amplification of previously selected microsatellite markers (STR) located in chromosome 6 and 15. DNA was obtained from microdissected tumor tissues and surrounding stroma to define the loss of heterozygosity (LOH) associated with chromosome 6p21. Our results showed that LOH in chromosome 6 produced HLA haplotype loss (phenotype II) in 36% of the tumors. In addition, HLA class I total loss (phenotype I) was found in 11%; HLA A or B locus downregulation (phenotype III) was detected in 20%; and HLA class I allelic loss (phenotype IV) in 10% of all cases. We sometimes observed two or more associated mechanisms in the same HLA-altered phenotype, such as LOH and HLA total loss in phenotype I. In only 23% of tumors it was not possible to identify any HLA class I alteration. We conclude that the combination of immunohistological techniques and molecular analysis of tumor DNA obtained from microdissected tumor tissues provides a means for the first time of determining the actual frequency of the major HLA class I-altered phenotypes in laryngeal carcinomas.

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Downregulation or total loss of HLA class I expression on tumor cells is known as a mechanism of cancer immune escape. Alterations of the HLA phenotype are frequently due to mutations affecting genes encoding the HLA class I heavy chains located on chromosome 6p21 or the β2-microglobulin (β2m) gene encoding the light chain of the HLA complex located on chromosome 15q21. Frequently irreversible total loss of HLA class I molecules is due to the coincidence of two molecular events, the mutation of one β2m gene and the loss of the second copy. The latter is detectable as loss of heterozygosity (LOH) of microsatellite markers in the β2m region on chromosome 15q21 (LOH-15q21). Thus, LOH-15q21 might be an important event in the processes of HLA class I downregulation and total loss. Here we studied the frequency of LOH-15q21 in tumor tissues of different entities. By determining the status of heterozygosity of two microsatellite markers we detected LOH-15q21 in 44% of bladder carcinomas (n = 69), in 35% of colon carcinomas (n = 95), in 16% of melanomas (n = 70) but only in 7% of renal cancers (n = 45). Moreover, we observed a frequent coincidence of LOH-15q21 and LOH-6p21 in colorectal carcinoma, bladder carcinoma and melanoma, but not for renal carcinoma. We believe that the high incidence of LOH-15q21 in some malignancies and especially the coincidence of LOH-15q21 and LOH-6p21 might have a strong impact on tumor immunogenicity and on the efficiency of cancer immunotherapy.