Genetic association study of systemic lupus erythematosus and disease subphenotypes in European populations.

Epidemiological studies suggest a strong contribution of genetic factors in the pathogenesis of systemic lupus erythematosus (SLE). In the last decades, many risk loci have been identified in several genetic association studies following both candidate gene and genome-wide approaches. The present work was conducted by GAPAID (Genes And Proteins for AutoImmunity Diagnostics) consortium with a dual aim: to replicate the association of several previously reported SLE susceptibility loci in an independent European sample and to explore their relation with some disease subphenotypes. A total of 48 single nucleotide polymorphisms (SNP) from 40 associated loci were typed in a cohort of 208 SLE patients and 152 controls from Rheumatology Units of the University Hospital of Pisa (Italy) and University of Pécs Medical Center (Hungary). Regression analyses were performed to detect disease susceptibility loci and to identify genes affecting specific disease manifestations (renal, neurological, or skin involvement; arthritis; secondary Sjögren syndrome; and secondary antiphospholipid syndrome). Association of previously described risk alleles from HLA locus has been replicated, while IRF5, BLK, ITGAM, and IRF8 loci have been found to be consistent with previous published results. In addition, two new subphenotype-specific associations have been detected: SNP rs5754217 (UBE2L3) with skin involvement and rs3093030 (ICAM1-ICAM4-ICAM5) with hematological disorders. Overall, results from GAPAID project are consistent with previously established associations for HLA, IRF5, BLK, ITGAM, and IRF8 SLE susceptibility loci and report for the first time two subphenotype-specific associations.

Serological and Genetic Evidence for Altered Complement System Functionality in Systemic Lupus Erythematosus: Findings of the GAPAID Consortium.

Systemic lupus erythematosus is a chronic autoimmune disease with multifactorial ethiopathogenesis. The complement system is involved in both the early and late stages of disease development and organ damage. To better understand autoantibody mediated complement consumption we examined ex vivo immune complex formation on autoantigen arrays. We recruited patients with SLE (n = 211), with other systemic autoimmune diseases (n = 65) and non-autoimmune control subjects (n = 149). Standard clinical and laboratory data were collected and serum complement levels were determined. The genotype of SNP rs1143679 in the ITGAM gene was also determined. Ex vivo formation of immune complexes, with respect to IgM, IgG, complement C4 and C3 binding, was examined using a functional immunoassay on autoantigen microarray comprising nucleic acids, proteins and lipids. Complement consumption of nucleic acids increased upon binding of IgM and IgG even when serum complement levels were decreased due to consumption in SLE patients. A negative correlation between serum complement levels and ex vivo complement deposition on nucleic acid autoantigens is demonstrated. On the contrary, complement deposition on tested protein and lipid autoantigens showed positive correlation with C4 levels. Genetic analysis revealed that the non-synonymous variant rs1143679 in complement receptor type 3 is associated with an increased production of anti-dsDNA IgG antibodies. Notwithstanding, homozygous carriers of the previously reported susceptible allele (AA) had lower levels of dsDNA specific IgM among SLE patients. Both the non-synonymous variant rs1143679 and the high ratio of nucleic acid specific IgG/IgM were associated with multiple organ involvement. In summary, secondary complement deficiency in SLE does not impair opsonization of nucleic-acid-containing autoantigens but does affect other antigens and potentially other complement dependent processes. Dysfunction of the receptor recognizing complement opsonized immune complexes promotes the development of class-switched autoantibodies targeting nucleic acids.

Genetic variants associated with rheumatoid arthritis patients and serotypes in European populations.

OBJECTIVES: To replicate the association of rheumatoid arthritis (RA) susceptibility loci in an independent European sample and to assess their specificity with anti-citrullinated protein antibodies (ACPA) status. METHODS: A selection of 64 SNP previously associated with RA have been typed in a cohort of 267 RA patients (169 ACPA-positive and 98 ACPA-negative) and 152 controls from the Rheumatology Units of the University Hospital of Pisa (Italy) and the University of Pécs Medical Center (Hungary). Regression analyses were performed first considering overall RA patients and secondly, taking both serotype subgroups as different disease entities. The results have been adjusted for age, gender and origin of individuals. RESULTS: The well-known CD2, REL, TNFAIP3, IRF5, PTPRC, and CCR6 have been confirmed as RA disease associated loci together with recently discovered BACH2, RASGRP1, and IKZF3 loci, taking all RA patients as a unique phenotype. Results from both serological subgroups separately reflect the specificity of these susceptibility loci and show additional ACPA-positive specific associations for variants at IL6R, IL2RA, BLK, DDX6, IL6, and TLE3 genes. CONCLUSIONS: The results from GAPAID project are consistent with previously established RA disease associations for CD2, PTPRC, REL, CCR6, TNFAIP3, IRF5, BLK, IL2RA, and DDX6 loci. In addition, IL6R, BACH2, RASGRP1, TLE3, and IKZF3 are replicated for the first time in an independent European population and IL6 appears to be a suggestive new RA associated locus. The stratified analysis based on ACPA status provides further support for distinct genetic aetiologies of RA subsets, which might have therapeutic implications.

Influence of MTHFR C677T polymorphism on methotrexate monotherapy discontinuation in rheumatoid arthritis patients: results from the GAPAID European project.

OBJECTIVES: Methotrexate (MTX) is the most widely prescribed drug for rheumatoid arthritis (RA) patients, but 45% of them discontinue therapy within two years, either due to inefficacy or toxicity. Several authors have reported contradictory results related to C677T polymorphism in the MTHFR gene and response to MTX in RA. The purpose of this study was to further explore this genotype-response association in a European RA population. METHODS: This retrospective longitudinal study included a total of 269 RA patients from Italy and Hungary, of whom 73.2% had available data on MTX treatment (197 patients). C677T polymorphism (rs1801133) was genotyped by quantitative PCR using TaqMan assays. Genotype association analysis and Kaplan-Meier method were used for statistical comparisons between patients continuing and patients who abandoned MTX treatment. RESULTS: A total of 85 out of the 197 RA patients (43%) abandoned MTX treatment by the time of analysis. No significant genotype-MTX discontinuation association was found for the overall population, either at the end of the study (p=0.375), or during the follow-up (p=0.324). When the analysis was restricted to the 68 patients on MTX monotherapy, a borderline association (OR 3.15, 95% CI 0.93-10.67, p=0.057) was noted with the recessive genetic model. In agreement with that, a Kaplan-Meier analysis showed a significantly shorter time-to-discontinuation of MTX monotherapy for homozygous carriers of the T-allele (p=0.042). CONCLUSIONS: These results demonstrate that the C677T polymorphism in the MTHFR gene is involved in MTX monotherapy discontinuation in a multicentre European patient cohort, confirming previous results.

Overexpression of proCOL11A1 as a stromal marker of breast cancer.

BACKGROUND: Our previous studies demonstrated the expression of procollagen11A1 in fibroblasts of pancreatic cancer desmoplasia and the lack of expression in fibroblasts of pancreatitis by means of the polyclonal antibody (anti-proCOL11A1 pAb) we generated. In a similar way, we decided to compare the expression of procollagen11A1 in fibroblasts of infiltrating ductal carcinoma of the breast and fibroblasts of benign sclerosing lesions of the breast, in order to validate the anti-proCOL11A1 pAb in this setting and to study how proCOL11A1 expression relates to other prognostic and predictive factors, as well as to survival. METHODS: 45 core biopsies of sclerosing adenosis and 50 core biopsies of infiltrating ductal carcinoma of the breast were stained with anti-proCOL11A1 pAb, a polyclonal antibody highly specific to the less homologous fraction of proCOL11A1 (in comparison with proCOL5A1 and proCOL11A2). In addition, the expression of the proCOL11A1 gene was measured by RT-qPCR. On the other hand, the expression of proCOL11A1 was compared to the expression of estrogenic receptors, progestagen receptors, the state of the epidermal growth factor receptor 2 (HER2), the histologic grade and the stage of the disease. We also compared the immunohistochemical expression of proCol11A1 to the disease-free interval, and to overall survival. RESULTS: The immunohistochemical analysis showed that proCOL11A1 was expressed in 100% of infiltrating ductal carcinomas, but only focally expressed in 2.2% (1 case) of sclerosing adenosis, in agreement with RT-qPCR results. ProCOL11A1 expression did not prove to have a prognostic value in relation to the disease-free interval or to overall survival in infiltrating ductal carcinoma. CONCLUSION: The anti-proCOL11A1 pAb is a stromal marker for breast cancer and the expression of proCOL11A1 does not seem to have a prognostic value in infiltrating ductal carcinoma of the breast.

Genetic predisposition to early recurrence in clinically localized prostate cancer.

UNLABELLED: WHAT'S KNOWN ON THE SUBJECT? AND WHAT DOES THE STUDY ADD?: Currently available nomograms to predict preoperative risk of early biochemical recurrence (EBCR) after radical prostatectomy are solely based on classic clinicopathological variables. Despite providing useful predictions, these models are not perfect. Indeed, most researchers agree that nomograms can be improved by incorporating novel biomarkers. In the last few years, several single nucleotide polymorphisms (SNPs) have been associated with prostate cancer, but little is known about their impact on disease recurrence. We have identified four SNPs associated with EBCR. The addition of SNPs to classic nomograms resulted in a significant improvement in terms of discrimination and calibration. The new nomogram, which combines clinicopathological and genetic variables, will help to improve prediction of prostate cancer recurrence. OBJECTIVES: To evaluate genetic susceptibility to early biochemical recurrence (EBCR) after radical prostatectomy (RP), as a prognostic factor for early systemic dissemination. To build a preoperative nomogram to predict EBCR combining genetic and clinicopathological factors. PATIENTS AND METHODS: We evaluated 670 patients from six University Hospitals who underwent RP for clinically localized prostate cancer (PCa), and were followed-up for at least 5 years or until biochemical recurrence. EBCR was defined as a level prostate-specific antigen >0.4 ng/mL within 1 year of RP; preoperative variables studied were: age, prostate-specific antigen, clinical stage, biopsy Gleason score, and the genotype of 83 PCa-related single nucleotide polymorphisms (SNPs). Univariate allele association tests and multivariate logistic regression were used to generate predictive models for EBCR, with clinicopathological factors and adding SNPs. We internally validated the models by bootstrapping and compared their accuracy using the area under the curve (AUC), net reclassification improvement, integrated discrimination improvement, calibration plots and Vickers' decision curves. RESULTS: Four common SNPs at KLK3, KLK2, SULT1A1 and BGLAP genes were independently associated with EBCR. A significant increase in AUC was observed when SNPs were added to the model: AUC (95% confidence interval) 0.728 (0.674-0.784) vs 0.763 (0.708-0.817). Net reclassification improvement showed a significant increase in probability for events of 60.7% and a decrease for non-events of 63.5%. Integrated discrimination improvement and decision curves confirmed the superiority of the new model. CONCLUSIONS: Four SNPs associated with EBCR significantly improved the accuracy of clinicopathological factors. We present a nomogram for preoperative prediction of EBCR after RP.

Improved prediction of biochemical recurrence after radical prostatectomy by genetic polymorphisms.

PURPOSE: Single nucleotide polymorphisms are inherited genetic variations that can predispose or protect individuals against clinical events. We hypothesized that single nucleotide polymorphism profiling may improve the prediction of biochemical recurrence after radical prostatectomy. MATERIALS AND METHODS: We performed a retrospective, multi-institutional study of 703 patients treated with radical prostatectomy for clinically localized prostate cancer who had at least 5 years of followup after surgery. All patients were genotyped for 83 prostate cancer related single nucleotide polymorphisms using a low density oligonucleotide microarray. Baseline clinicopathological variables and single nucleotide polymorphisms were analyzed to predict biochemical recurrence within 5 years using stepwise logistic regression. Discrimination was measured by ROC curve AUC, specificity, sensitivity, predictive values, net reclassification improvement and integrated discrimination index. RESULTS: The overall biochemical recurrence rate was 35%. The model with the best fit combined 8 covariates, including the 5 clinicopathological variables prostate specific antigen, Gleason score, pathological stage, lymph node involvement and margin status, and 3 single nucleotide polymorphisms at the KLK2, SULT1A1 and TLR4 genes. Model predictive power was defined by 80% positive predictive value, 74% negative predictive value and an AUC of 0.78. The model based on clinicopathological variables plus single nucleotide polymorphisms showed significant improvement over the model without single nucleotide polymorphisms, as indicated by 23.3% net reclassification improvement (p = 0.003), integrated discrimination index (p <0.001) and likelihood ratio test (p <0.001). Internal validation proved model robustness (bootstrap corrected AUC 0.78, range 0.74 to 0.82). The calibration plot showed close agreement between biochemical recurrence observed and predicted probabilities. CONCLUSIONS: Predicting biochemical recurrence after radical prostatectomy based on clinicopathological data can be significantly improved by including patient genetic information.

Prediction of functional impairment and remission in rheumatoid arthritis patients by biochemical variables and genetic polymorphisms.

OBJECTIVE: To develop a model to predict RA outcome based on biochemical variables and single nucleotide polymorphisms (SNPs). METHODS: We collected baseline data from RA patients. SNP genotyping was performed using an oligonucleotide microarray. Remission and severe disability were investigated as outcomes of the study. Logistic regression models and receiver operating characteristic (ROC) curves were used to determine sensitivity (S), specificity (Sp) and likelihood ratio (LR). RESULTS: Six hundred and thirty-two patients (375 in the study and 257 in the validation) were included. Twenty-two out of 152, and 19 out of 208 patients had an HAQ > 2. The model obtained to predict disability included levels of the anti-cyclic citrullinated peptide (anti-CCP) antibodies, ESR and SNP rs2070874 in the IL-4 gene. Homozygous and heterozygous carriers of the IL-4 33T allele had a decreased risk of severe disability. The discriminative power had an area under the curve (AUC) of 0.792 (95% CI 0.694, 0.889), with S 41%, Sp 95% and LR +7.6. Twenty-one out of 268 and 17 out of 211 patients were in remission in the study and validation cohorts, respectively. The model included absence of anti-CCP antibodies and the SNP rs2476601 on the PTPN22 gene. Homozygous and heterozygous carriers of the PTPN22 1858T allele had a decreased probability of remission. The discriminative power had an AUC of 0.842 (95% CI 0.756, 0.928), with S 76%, Sp 86% and LR + 5.4. Predictive ability was confirmed on the validation cohort. CONCLUSIONS: We have developed two models based on laboratory variables that are associated with relevant outcomes for RA patients at disease onset.

Identification of chitotriosidase isoforms in plasma of Gaucher disease patients by two dimensional gel electrophoresis.

Chitotriosidase protein (ChT) is the most important biochemical marker described for Gaucher disease (GD). ChT activity is increased several hundred-fold in plasma of GD patients and shows a strong positive correlation with the severity of the disease. However, a recessively inherited enzyme deficiency, with an incidence of about 6% in the Caucasian population, means that not all patients with GD can be monitored by measuring ChT activity. Applying two-dimensional gel electrophoresis (2-DE) technology this study describes the localization and identification of five ChT isoforms in 2-DE images obtained from plasma of GD patients. All these isoforms were unequivocally identified using MALDI-TOF mass spectrometry (MS) and validated by western blot analysis. The features of each ChT isoform separated by 2-DE in plasma from GD patients homozygous for the wild-type ChT allele, carriers of one defective allele and patients homozygous for the mutant allele are presented. We also show the correlation between each ChT isoform and the plasma ChT enzymatic activity of the GD patients sampled in this study.