Jesús Martínez-Borra

The MICA‐A9 triplet repeat polymorphism in the transmembrane region confers additional susceptibility to the development of psoriatic arthritis and is independent of the association of Cw*0602 in psoriasis

OBJECTIVE To investigate the relative contribution of HLA antigens in the susceptibility to psoriasis and to localize additional genetic factors involved in psoriatic arthritis (PsA). METHODS DNA from 45 patients with psoriasis, 65 with PsA, and 177 healthy control subjects was examined by polymerase chain reaction (PCR) using sequence-specific oligonucleotide probes to determine HLA-C. To examine whether MICA (class I major histocompatibility complex chain-related gene A) confers additional susceptibility, trinucleotide repeat polymorphism in the transmembrane region of the MICA gene was investigated by radioactive PCR. Further analysis of MICA was made by PCR-single-strand conformational polymorphism to determine the allelic variant corresponding to MICA transmembrane polymorphism. RESULTS Our results reveal new findings: 1) the frequency of the Cw*0602 allele was significantly increased in both patient groups: psoriasis (corrected P [Pcorr] < 10(-5), relative risk [RR] 6.2), PsA (Pcorr < 10(-6), RR 6.3), 2) the trinucleotide repeat polymorphism MICA-A9 was present at a significantly higher frequency in PsA patients (Pcorr < 0.00035, RR 3.2), whereas a similar distribution was found in both the control and psoriasis population, 3) this polymorphism corresponds to the MICA-002 allele and was found to be overrepresented in patients with the polyarticular form (Pcorr < 0.0008, RR 9.35), 4) the increase in MICA-A9 in PsA patients is independent of linkage disequilibrium with Cw*0602, 5) this allele confers additional relative risk (RR 3.27, etiologic fraction 0.44; etiologic fraction is the proportion of disease cases among the total population that are attributable to 1 allele when the relative risk is > 1) in PsA patients who carry Cw*0602. CONCLUSION The data obtained in this study are consistent with the polygenic inheritance of psoriasis. Cw*0602 appears to be the stronger genetic susceptibility factor for psoriasis. Independent of the HLA-C association, MICA-A9 polymorphism corresponding to the MICA-002 allele is a possible candidate gene for the development of PsA.

TNF-α− 308A promoter polymorphism is associated with enhanced TNF-α production and inflammatory activity in Crohn's patients with fistulizing disease

OBJECTIVE:Tumor necrosis factor-α (TNF-α) plays a key role in the inflammatory response and pathogenesis of Crohns disease (CD). TNF-α− 308A polymorphism within the TNF-α gene promoter has been associated with enhanced TNF-α production in vitro. The aim of this study was to investigate the effect of TNF-α promoter polymorphism at −308 on the susceptibility and phenotypic expression of fistulizing CD.METHODS:The distribution of −308 TNF-α genotypes was analyzed in 50 patients with fistulizing CD and 100 healthy matched controls. TNF-α, interleukin-1β, and interleukin-6 serum levels were measured by ELISA. Serum amyloid-A, C-reactive protein, α1-antitrypsin, α1-acid glycoprotein, and haptoglobin were measured by nephelometry.RESULTS:No significant differences were found in the allele frequencies of the polymorphism between patients and controls. However, compared with −308GG patients, those carrying −308AG had a significant increase of serum levels of TNF-α (58 ± 79 vs 8 ± 19 pg/ml, p < 0.001), interleukin-1β (36 ± 45 vs 16 ± 20 pg/ml, p = 0.048), and acute phase proteins (APPs). −308A carriers had also a higher frequency of arthritis (66% vs 26%, p = 0.039). The logistic regression model showed that the patients carrying −308A polymorphism had a relative risk for developing arthritis of 5.45 (95% CI = 1.1–25.6). No other clinical or analytical findings were predictive for the risk of development of arthritis.CONCLUSIONS:TNF-α− 308A polymorphism is associated with enhanced TNF-α production, more intense inflammatory activity, and an increased risk for arthritis susceptibility in CD patients with fistulizing disease.

Protective Effect of the HLA-Bw4I80 Epitope and the Killer Cell Immunoglobulin-Like Receptor 3DS1 Gene against the Development of Hepatocellular Carcinoma in Patients with Hepatitis C Virus Infection

The aim of the present study was to investigate, in 152 Spanish patients infected with hepatitis C virus (HCV), the possibility that killer cell immunoglobulin-like receptors (KIRs) influence progression to hepatocellular carcinoma. KIRs are related to the activation and inhibition of natural killer cells and may play an important role in the innate response against infection with such viruses as HCV. We found that the human leukocyte antigen-Bw4I80 epitope and the KIR3DS1 gene were more frequent in HCV carriers than in patients with hepatocellular carcinoma. Moreover, these associations were not independent of each other--the KIR3DS1/Bw4I80 genotype clearly was also more frequent in HCV carriers (odds ratio, 24.22).

Polymorphism in MICA rather than HLA-B/C genes is associated with psoriatic arthritis in the Jewish population

The aim of this study was to examine whether the association of psoriatic arthritis (PsA) with human leukocyte antigen (HLA) class I genes is secondary to linkage disequilibrium with a nearby gene. We examined a sample of the Jewish population to investigate whether HLA-B/C and DR polymorphism is associated with susceptibility, or whether other closely related class I loci, such as the major histocompatibility complex class I chain-related gene A (MICA) and tumor necrosis factor (TNF), might play a role in disease development. Comparisons of different populations with different HLA profiles would be of value in identifying the candidate genes involved in PSA. Fifty-two patients with PsA and 73 random matched controls from a Jewish population were selected and DNA typed by polymerase chain reaction-single-strand oligonucleotide probe (PCR-SSOP) (HLA-C), PCR sequence-specific primers (PCR-SSP) (HLA-B, -DR), radioactive PCR (MICA-TM polymorphism in the transmembrane region), and PCR-RFLP (TNF). Some findings can be concluded from the study: (1) the frequency of HLA-B*5701, B*3801, B*39, B*27, Cw*0602, Cw*07, DRB1*0402, and DRB1*0701 were not found to be significantly increased in PsA; (2) no significant differences of TNFalpha promoter alleles at positions -308 and -238 were found between PsA and healthy controls; (3) the trinucleotide repeat polymorphism MICA-A9 was present at a higher frequency in PsA patients, (p(c) < 0.009, RR = 3.34, EF = 0.39); and (4) MICA-A9 polymorphism was found in linkage disequilibrium with HLA-B alleles (B*5701, B*3801) described to be associated with PsA in Caucasians. These results suggest that the MICA gene or other nearby gene(s) may be involved in the development of PsA, and it would thus appear that psoriasis vulgaris (PsV) and PsA are associated with different MHC susceptibility genes.

High serum tumor necrosis factor-α levels are associated with lack of response to infliximab in fistulizing Crohn’s disease

OBJECTIVES:Infliximab, a chimeric monoclonal antibody directed against tumor necrosis factor-α (anti-TNF-α), has been effective in the treatment of patients with active Crohns disease and with fistulas. We investigated the effect of infliximab on circulating cytokines and acute phase proteins in patients with fistulas to determine the clinical response to anti-TNF-α.METHODS:A total of 36 patients with fistulizing Crohns disease were selected for study. Serum from patients was drawn before the infusion on day 0 and at wk 2, 4, 6, 8, and 10 after completion of treatment. Circulating concentrations of TNF-α, interleukin-1β (IL-1β), and IL-6 were measured by ELISA. The functional activity of circulating TNF-α was assessed by the WEHI 164 TNF-α bioassay. Acute phase proteins were also determined.RESULTS:Elevated TNF-α, IL-1β, IL-6, and acute phase proteins were observed in patients with Crohns disease. Of the patients with fistulas, 22 (61.1%) responded to treatment. Before receiving infliximab, higher levels of serum TNF-α were found in patients who did not respond to infliximab compared with those who did (median interquartile range 26, 0–245 pg/ml; n = 14 vs 0, 0–22 pg/ml, n = 22). Patients showed no change in circulating levels of TNF-α during the course of the study.CONCLUSIONS:This treatment produces a clinical improvement in about two-thirds of CD patients with fistulas. The circulating levels of TNF-α are associated with the response to infliximab and could help to identify patients who would benefit from anti-TNF-α treatment.

Contribution of KIR3DL1/3DS1 to ankylosing spondylitis in human leukocyte antigen-B27 Caucasian populations

Killer cell immunoglobulin-like receptors (KIRs) and humaAn leukocyte antigen (HLA) loci are both highly polymorphic, and some HLA class I molecules bind and trigger cell-surface receptors specified by KIR genes. We examined whether the combination of KIR3DS1/3DL1 genes in concert with HLA-B27 genotypes is associated with susceptibility to ankylosing spondylitis (AS). Two HLA-B27-positive Caucasian populations were selected, one from Spain (71 patients and 105 controls) and another from the Azores (Portugal) (55 patients and 75 controls). All were typed for HLA-B and KIR (3DS1 and 3DL1) genes. Our results show that in addition to B27, the allele 3DS1 is associated with AS compared with B27 controls (p < 0.0001 and p < 0.003 in the Spanish population and Azoreans, respectively). We also observed that the association of KIR3DS1 to AS was found in combination with HLA-B alleles carrying Bw4-I80 in trans position in the Spanish population (30.9% in AS versus 15.2% in B27 controls, p = 0.02, odds ratio (OR) = 2.49) and in Azoreans (27.2% in AS versus 8.7% in B27 controls, p = 0.01, OR = 4.4 in Azoreans). On the other hand, 3DL1 was decreased in patients compared with B27 controls (p < 0.0001 in the Spanish population and p < 0.003 in Azoreans). The presence of this allele in combination with Bw4-I80 had a protective effect against the development of AS in the Spanish population (19.7% in AS, 35.2% in B27 controls; p = 0.03, OR = 0.45). The presence of KIR3DS1 or KIR3DL1 in combination with HLA-B*27s/HLA-B Bw4-I80 genotypes may modulate the development of AS. The susceptibility to AS could be determined by the overall balance of activating and inhibitory composite KIR-HLA genotypes.

HLA-B27 alone rather than B27-related class I haplotypes contributes to ankylosing spondylitis susceptibility

Characterization of non-B27 susceptibility genes will be required to know the pathogenesis of AS. The aim of this study was to examine whether ankylosing spondylitis (AS) susceptibility is controlled by B27 alone rather than B27 haplotypes and, whether other closely related class I loci, such as MICA and TNFA genes might play a role in AS. Three hundred eleven B27-positive samples from Caucasoid, Asian, and African populations were selected and genotypes were carried out by PCR/SSOP (HLA-B27 and HLA-C), PCR/SSP (MICA-TM polymorphism in the transmembrane region), PCR/SSCP (MICA alleles), and PCR-RFLP (TNF-alpha). Of these, 161 were AS patients, chosen in order to investigate the contribution of TNFA and MICA loci to AS in HLA-B27 positive individuals. Some findings can be concluded from the study: (a) No significant differences of TNF-alpha promoter alleles at position -308 and -238 (A/G) were found between AS patients and B27 matched alleles from healthy controls; (b) strong linkage disequilibrium was found between the B27 and the MICA alleles. The MICA-A4 was found to be in association with B*2705,02,03 and 08; MICA-A5 with B*2704 and B*2707 and MICA-A.5.1 with B*2706; (c) no significant differences of MICA alleles were found between AS and controls carrying the B27-associated alleles, and therefore no evidence is provided for an additional role of MICA gene in AS susceptibility; (d) there are a striking correlations between the structure of B27 extended haplotypes (from MICA region to HLA-C) and the ethnic distribution of these subtypes. The results of differential linkage disequilibrium with HLA-B27 subtypes suggest that B27 itself remains the primary gene for AS susceptibility, and TNFA and MICA are not involved in the pathogenesis of the disease.

Transcriptional regulation of MICA and MICB: a novel polymorphism in MICB promoter alters transcriptional regulation by Sp1.

MHC class I‐related genes A/B (MICA/B) are ligands of the NKG2D receptor expressed on T and NK cells. Their expression is highly restricted in normal tissues, but is up‐regulated in tumoral and infected cells. We show that the minimal promoter of both genes contains a CCAAT box, which binds to NF‐Y, and a GC box, which binds to Sp1, Sp3 and Sp4. We also demonstrate that MICB promoter is polymorphic, showing three single nucleotide polymorphisms (C>G at +16, –341, –408) and a deletion of two base pairs at –66 (AG>‐‐) that is located close to the CCAAT box (‐70) and the GC box (‐86). Transcriptional activity associated with MICB promoter variants carrying this deletion, present in the 45.3% of Spanish population, showed a remarkable decrease (18‐fold, p <0.01). By functional analysis, we show that the deletion plays a critical role in MICB promoter activity by diminishing Sp1 transcriptional activation. These important variations in MICB expression among normal individuals could imply a significant difference in the natural immune response against infections or tumor transformation, and might thereby contribute to an increased aberrant immune response against self cells, providing the molecular basis for the associations of the MICB gene to different autoimmune diseases.

HFE gene mutations in alcoholic and virus-related cirrhotic patients with hepatocellular carcinoma

OBJECTIVE:The increased risk of developing hepatocellular carcinoma in hereditary hemochromatosis has been associated with cirrhosis and hepatic iron overload. The aim of this study was to investigate the association between HFE gene mutations (C282Y, H63D) and hepatocellular carcinoma in patients with alcoholic and virus-related cirrhosis.METHODS:Serum markers of iron status and HFE mutations were determined in 179 patients with alcoholic cirrhosis and 98 patients with hepatitis B and/or hepatitis C virus-related cirrhosis. A total of 43 patients with alcoholic cirrhosis and 34 patients with virus-related cirrhosis had hepatocellular carcinoma. The control group consisted of 159 healthy bone marrow donors.RESULTS:No differences were found in the frequencies of mutations among patients with alcoholic cirrhosis, those with virus-related cirrhosis, and the control subjects. However, nine (20.9%) of the 43 patients with alcoholic cirrhosis and hepatocellular carcinoma were heterozygous for the C282Y mutation, compared with six (4.4%) of the 136 patients without tumor (p = 0.002). This difference was not found in patients with virus-related cirrhosis, with or without hepatocellular carcinoma, or the H63D mutation. The transferrin saturation was the only serum iron marker the value of which was significantly higher among C282Y heterozygotes with alcoholic cirrhosis compared to those without mutation.CONCLUSIONS:The high frequency of heterozygosity for the C282Y mutation in patients with alcoholic cirrhosis plus hepatocellular carcinoma suggests that the presence of this mutation could be associated with an increased risk of developing hepatocellular carcinoma in these patients.

The predictive value of soluble major histocompatibility complex class I chain-related molecule A (MICA) levels on heart allograft rejection.

Background. Recently the presence of a soluble form of major histocompatibility complex class I chain-related molecule A (sMICA) has been detected in the sera of patients with tumors. Shedding of sMICA by tumor cells downregulates NKG2D-mediated antitumor immunity. The aim of this investigation was to study the possible involvement of sMICA in the allograft acceptance after heart transplantation (HTX). Methods. We monitored the levels of sMICA by specific enzyme-linked immunosorbent assay (ELISA) in a total of 146 serum samples obtained from 34 heart transplantation patients followed up during the first year post-HTX. Results. The persistence of sMICA expression was correlated with the clinical evolution of these patients. sMICA was detected in the serum of 21 of 34 patients (61.70%) between 15 and 20 days after implantation and was practically absent in pretransplant serum samples. Twenty of these 21 patients (95.24%) with sMICA did not experience episodes of severe rejection during this period (P=0.0001), whereas sMICA was practically absent in patients with manifestations of severe acute rejection. The longitudinal study of these patients revealed that the presence of sMICA was consistently maintained in 75% of the patients with good graft status during the period of observation. Conclusion. This has led us to believe that the presence of levels of sMICA during the first year post-HTX may contribute to allograft acceptance. Additionally, functional studies indicate that sMICA downregulates NKG2D surface expression, which may lead to a functional impairment of cell-mediated cytolysis. These data suggest a significant correlation between the presence of sMICA and a lower incidence of rejection.