Julio Sánchez-Román

The STAT4 gene influences the genetic predisposition to systemic sclerosis phenotype

The aim of this study was to investigate the possible role of STAT4 gene in the genetic predisposition to systemic sclerosis (SSc) susceptibility or clinical phenotype. A total of 1317 SSc patients [896 with limited cutaneous SSc (lcSSc) and 421 with diffuse cutaneous SSc (dcSSc)] and 3113 healthy controls, from an initial case-control set of Spanish Caucasian ancestry and five independent cohorts of European ancestry (The Netherlands, Germany, Sweden, Italy and USA), were included in the study. The rs7574865 polymorphism was selected as STAT4 genetic marker. We observed that the rs7574865 T allele was significantly associated with susceptibility to lcSSc in the Spanish population [P = 1.9 x 10(-5) odds ratio (OR) 1.61 95% confidence intervals (CI) 1.29-1.99], but not with dcSSc (P = 0.41 OR 0.84 95% CI 0.59-1.21). Additionally, a dosage effect was observed showing individuals with rs7574865 TT genotype higher risk for lcSSc (OR 3.34, P = 1.02 x 10(-7) 95% CI 2.11-5.31). The association of the rs7574865 T allele with lcSSc was confirmed in all the replication cohorts with different effect sizes (OR ranging between 1.15 and 1.86), as well as the lack of association of STAT4 with dcSSc. A meta-analysis to test the overall effect of the rs7574865 polymorphism showed a strong risk effect of the T allele for lcSSc susceptibility (pooled OR 1.54 95% CI 1.36-1.74; P < 0.0001). Our data show a strong and reproducible association of the STAT4 gene with the genetic predisposition to lcSSc suggesting that this gene seems to be one of the genetic markers influencing SSc phenotype.

CTLA4 polymorphism in Spanish patients with systemic lupus erythematosus.

The cytotoxic T-lymphocyte antigen 4 (CTLA4, CD152) gene is a positional and functional candidate gene to susceptibility to systemic lupus erythematosus (SLE) because CTLA4 gene maps in the described SLE risk region 2q33 and CTLA4 molecule has an inhibitory effect on T-cell activation. Several polymorphisms have been described in CTLA4 gene, among them, a T/C change at position -1722, a C/T transition at position -319, and another A/G transition at position +49. The aim of this study was to investigate the possible association of these polymorphisms with the susceptibility to SLE in 276 Spanish autochthonous patients using a healthy control group composed of 194 ethnically matched volunteer bone marrow donors. Genotyping of these CTLA4 positions was performed in SLE patients and controls using a polymerase chain reaction amplification refractory mutation system. The genotypic frequencies were in Hardy-Weinberg equilibrium in all patients. No differences in the distribution of the genotype frequencies between patients and controls were found in any case. Our results from the Spanish autochthonous population differ from those found in the Korean population regarding the involvement of the polymorphism located at -1722 in the susceptibility to SLE.

Analysis of autosomal genes reveals gene-sex interactions and higher total genetic risk in men with systemic lupus erythematosus

Objectives Systemic lupus erythematosus (SLE) is a sexually dimorphic autoimmune disease which is more common in women, but affected men often experience a more severe disease. The genetic basis of sexual dimorphism in SLE is not clearly defined. A study was undertaken to examine sex-specific genetic effects among SLE susceptibility loci. Methods A total of 18 autosomal genetic susceptibility loci for SLE were genotyped in a large set of patients with SLE and controls of European descent, consisting of 5932 female and 1495 male samples. Sex-specific genetic association analyses were performed. The sex–gene interaction was further validated using parametric and non-parametric methods. Aggregate differences in sex-specific genetic risk were examined by calculating a cumulative genetic risk score for SLE in each individual and comparing the average genetic risk between male and female patients. Results A significantly higher cumulative genetic risk for SLE was observed in men than in women. (P=4.52x10-8) A significant sex–gene interaction was seen primarily in the human leucocyte antigen (HLA) region but also in IRF5, whereby men with SLE possess a significantly higher frequency of risk alleles than women. The genetic effect observed in KIAA1542 is specific to women with SLE and does not seem to have a role in men. Conclusions The data indicate that men require a higher cumulative genetic load than women to develop SLE. These observations suggest that sex bias in autoimmunity could be influenced by autosomal genetic susceptibility loci.

Identification of a new putative functional IL18 gene variant through an association study in systemic lupus erythematosus

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.

The interleukin 23 receptor gene does not confer risk to systemic sclerosis and is not associated with systemic sclerosis disease phenotype

OBJECTIVES Multiple studies indicate the role of the interleukin (IL)-17/IL-23 axis in autoimmune diseases, including systemic sclerosis (SSc). The aim of the current study was to investigate the possible implication of the IL23R gene in SSc susceptibility and/or clinical phenotype. METHODS An initial case-control study in 143 Dutch patients with SSc and geographically matched healthy individuals (n = 246) was carried out and followed by a replication study in a cohort of 365 Spanish patients with SSc and 515 healthy individuals. Seven single nucleotide polymorphisms (SNPs) spanning the IL23R gene were selected and genotyped using a Taqman assay. RESULTS Using a Dutch cohort of patients with SSc and controls we observed an association between two (rs11209032, rs1495965) of the seven tested SNPs and disease susceptibility (allelic p values: p = 0.02 and p = 0.01 respectively). However, a replication study in an independent Spanish cohort did not confirm these findings and reveal no association of any of the IL23R-tested SNP with disease susceptibility or clinical phenotype. Similarly, a meta-analysis considering both populations did not reveal any significant association. In addition, no association was observed between IL23R genetic variants and SSc clinical phenotypes. CONCLUSIONS Our results suggest that the IL23R gene is not associated with SSc susceptibility or clinical phenotype.

Salivary and serum β2-microglobulin and gamma-glutamyl-transferase in patients with primary Sjögren syndrome and Sjögren syndrome secondary to systemic lupus erythematosus

BACKGROUND Sialochemistry has been proposed as a simple and useful tool for the diagnosis of Sjögren syndrome (SS). Although many changes have been detected in several constituents of saliva from patients with SS, none are individually sensitive or specific enough for diagnosing SS. The aim of this study was to assess the value of the combined determination of beta2-microglobulin (beta2m) and gamma-glutamyl-transferase (GGT) activity in serum and saliva as a diagnostic instrument for differentiating primary and secondary [to systemic lupus erythematosus (SLE)] SS patients from normal subjects. METHODS Nineteen primary SS (pSS) patients, 15 patients with SS secondary to SLE, and 25 SLE patients without SS were studied. Thirty healthy subjects were included in the study as control group. RESULTS By means of a mathematical model, (a) 84.1%, (b) 85.7%, and (c) 87.0% of patients were correctly classified as SS or normal when (a) salivary beta2m and GGT values, (b) serum beta2m and salivary GGT values, and (c) salivary beta2m and GGT along with serum beta2m values, respectively, were considered. To differentiate between pSS and sSS by means of the mathematical model, the combination of serum beta2m and salivary GGT values achieved that 81.8% of the patients were correctly classified. CONCLUSION Since sialochemistry is an easy, safe and reliable test, the combined determination of beta2m and GGT in saliva and serum was useful for differentiating SS patients from normal subjects, but not excessively good for differentiating pSS from sSS patients.

A large multicentre analysis of CTGF −945 promoter polymorphism does not confirm association with systemic sclerosis susceptibility or phenotype

Objective: To conduct a replication study to investigate whether the −945 CTGF genetic variant is associated with systemic sclerosis (SSc) susceptibility or specific SSc phenotype. Methods: The study population comprised 1180 patients with SSc and 1784 healthy controls from seven independent case–control sets of European ancestry (Spanish, French, Dutch, German, British, Swedish and North American). The −945 CTGF genetic variant was genotyped using a Taqman 5′ allelic discrimination assay. Results: An independent association study showed in all the case–control cohorts no association of the CTGF −945 polymorphism with SSc susceptibility. These findings were confirmed by a meta-analysis giving a pooled OR = 1.12 (95% CI 0.99 to 1.25), p = 0.06. Investigation of the possible contribution of the −945 CTGF genetic variant to SSc phenotype showed that stratification according to SSc subtypes (limited or diffuse), selective autoantibodies (anti-topoisomerase I or anticentromere) or pulmonary involvement reached no statistically significant skewing. Conclusion: The results do not confirm previous findings and suggest that the CTGF −945 promoter polymorphism does not play a major role in SSc susceptibility or clinical phenotype.

TAP polymorphism in patients with Behçet's disease.

OBJECTIVE--To determine if susceptibility to Behçets disease (BD) is associated with polymorphism of HLA-DRB1, HLA-DQB1, DQB1, and TAP1 and TAP2 genes. METHODS--Fifty eight Spanish BD patients and 116 ethnically matched unrelated healthy subjects were typed at the HLA-DRB1 and HLA-DQB1 loci using polymerase chain reaction/sequence specific oligotyping (PCR/SSO). TAP1 and TAP2 alleles were assigned using amplification refractory mutation system-PCR. RESULTS--TAP1C was absent in BD patients, but was found in 12.1% of control subjects (pcorr < 0.05; relative risk = 0.06). Additionally, a linkage disequilibrium between HLA-DQB1*0501 and TAP2B was observed in BD patients (delta = 0.095, pcorr < 0.02), but not in the control group (delta = -0.0031, p > 0.05). CONCLUSIONS--The complete absence of TAP1C alleles in BD patients may indicate that TAP1 polymorphism is not without some significance in the development of BD. Furthermore, the existence of a linkage disequilibrium between HLA-DQB1*0501 and TAP2B in our patients suggests that the gene conferring susceptibility for BD is inherited as an extended haplotype in the population studied.

Autoantibodies to DEK oncoprotein in systemic lupus erythematosus (SLE)

Autoantibodies against the transcriptional DEK protein have been considered characteristic of the pauciarticular onset subtype of juvenile rheumatoid arthritis (JRA) associated with iridocyclitis in young girls. In this study we investigated the presence of anti‐DEK autoantibodies in the sera of 288 patients with SLE using a recombinant DEK protein as autoantigenic target. Thirty sera (10·4%) were positive against DEK protein by immunoblotting. Patients with anti‐DEK autoantibodies show a lower frequency of cutaneous manifestation, exhibit more frequently certain markers of a chronic inflammatory status like anaemia and positivity for C‐reactive protein, as well as a higher frequency of anti‐double‐stranded DNA autoantibodies. In contrast to JRA patients positive for anti‐DEK autoantibodies, no association with erosive arthritis nor iridocyclitis were found in SLE. In conclusion, our results show that 10·4% of SLE patients from our area show antibodies against DEK protein, although this feature did not clearly establish a clinical subset of the disease.

Antimyeloperoxidase antibodies in individuals with occupational exposure to silica.

OBJECTIVE: To determine the prevalence of autoantibodies to myeloperoxidase (MPO) in a series of patients exposed to silica. METHODS: The study included 52 patients with occupational exposure to silica (mean exposure time seven years) and a control group comprising seven patients with progressive systemic sclerosis (PSS), six patients with systemic lupus erythematosus (SLE), and 15 healthy individuals. Antibodies to MPO were detected using commercial enzyme linked immunosorbent assay (ELISA) plates coated with MPO. Indirect immunoflurescence studies for antineutrophil cytoplasmic antibodies were performed using ethanol and formol fixed neutrophils. Clinical and biological data of individuals exposed to silica were recorded (published previously). RESULTS: Antibodies to MPO were detected in 14 individuals exposed to silica (27%). There was a statistically significant difference in anti-MPO ELISA units between the healthy subjects and patients (SLE, PSS, silica exposed individuals) (p < 0.01), but no difference between the different disease groups. CONCLUSIONS: Individuals chronically exposed to silica, whether or not they have a connective tissue disease, have levels of antibodies to MPO (as detected by ELISA) that are greater than those found in the normal population, but similar to those in patients with systemic diseases not induced by silica (SLE/PSS).