Andrea Latini

Polymorphisms in MIR122, MIR196A2, and MIR124A Genes are Associated with Clinical Phenotypes in Inflammatory Bowel Diseases

BackgroundInflammatory bowel diseases (IBDs), including Crohn’s disease (CD) and ulcerative colitis (UC), are multifactorial disorders that result from a dysregulated inflammatory response to environmental factors in genetically predisposed individuals. Recently, microRNAs (miRNAs) have been shown to be involved in the development of IBDs.AimsWe investigated common variants in five miRNA genes in a cohort of Italian IBD patients, to evaluate their possible role in the disease’s susceptibility and phenotype manifestations.MethodsThe analysis included 267 CD patients, 207 UC patients, and 298 matched healthy controls. Polymorphisms in the MIR122, MIR499, MIR146A, MIR196A2, and MIR124A genes were evaluated by allelic discrimination assay.ResultsWe did not find associations between mir polymorphisms and IBD susceptibility. In both diseases, rs17669 and rs11614913 (MIR122 and MIR196A2) seem to contribute to clinical phenotypes: ileal location in CD (odds ratio [OR] = 1.82, p = 0.03; OR = 0.51, p = 0.01), and left-sided extent in UC (OR = 0.43, p = 0.05; OR = 0.28, p = 0.002). In CD, the MIR124A polymorphism (rs531564) contributed to colon location (p = 0.03, OR = 2.74). Finally, the variant allele of rs11614913 was associated with early age at onset in both diseases (p = 0.05 and p = 0.02).ConclusionsWe showed for the first time that polymorphisms in MIR122, MIR196A2, and MIR124A could play a role in clinical phenotype modulation in IBD.

Polymorphisms in miRNA genes and their involvement in autoimmune diseases susceptibility

MicroRNAs (miRNAs) are small non-coding RNA molecules that negatively regulate the expression of multiple protein-encoding genes at the post-transcriptional level. MicroRNAs are involved in different pathways, such as cellular proliferation and differentiation, signal transduction and inflammation, and play crucial roles in the development of several diseases, such as cancer, diabetes, and cardiovascular diseases. They have recently been recognized to play a role also in the pathogenesis of autoimmune diseases. Although the majority of studies are focused on miRNA expression profiles investigation, a growing number of studies have been investigating the role of polymorphisms in miRNA genes in the autoimmune diseases development. Indeed, polymorphisms affecting the miRNA genes can modify the set of targets they regulate or the maturation efficiency. This review is aimed to give an overview about the available studies that have investigated the association of miRNA gene polymorphisms with the susceptibility to various autoimmune diseases and to their clinical phenotypes.

Polymorphisms in STAT4, PTPN2, PSORS1C1 and TRAF3IP2 genes are associated with the response to TNF inhibitors in patients with rheumatoid arthritis

Objective Rheumatoid Arthritis (RA) is a progressive autoimmune disease characterized by chronic joint inflammation and structural damage. Remission or at least low disease activity (LDA) represent potentially desirable goals of RA treatment. Single nucleotide polymorphisms (SNPs) in several genes might be useful for prediction of response to therapy. We aimed at exploring 4 SNPs in candidate genes (STAT4, PTPN2, PSORS1C1 and TRAF3IP2) in order to investigate their potential role in the response to therapy with tumor necrosis factor inhibitors (TNF-i) in RA patients. Methods In 171 RA patients we investigated the following SNPs: rs7574865 (STAT4), rs2233945 (PSORS1C1), rs7234029 (PTPN2) and rs33980500 (TRAF3IP2). Remission, LDA, and EULAR response were registered at 6 months and 2 years after initiation of first line TNF-i [Adalimumab (ADA) and Etanercept (ETN)]. Results STAT4 variant allele was associated with the absence of a good/moderate EULAR response at 2 years of treatment in the whole RA group and in ETN treated patients. The PTPN2 SNP was associated with no good/moderate EULAR response at 6 months in ADA treated patients. Patients carrying PSORS1C1 variant allele did not reach LDA at 6 months in both the whole RA group and ETN treated patients. TRAF3IP2 variant allele was associated with the lack of LDA and remission achievement at 6 months in all RA cohort while an association with no EULAR response at 2 years of treatment occurred only in ETN treated patients. Conclusions For the first time, we reported that SNPs in STAT4, PTPN2, PSORS1C1, and TRAF3IP2 are associated with response to TNF-i treatment in RA patients; however, these findings should be validated in a larger population.

Association between a MIR499A polymorphism and diabetic neuropathy in type 2 diabetes

AIMS Diabetic polyneuropathy (DPN) and cardiovascular autonomic neuropathy (CAN) affect a large percentage of diabetic people and impact severely on quality of life. As it seems that miRNAs and their variations might play a role in these complications, we investigated whether the rs3746444 SNP in the MIR499A gene could be associated with susceptibility to DPN and/or CAN. METHODS We analyzed 150 participants with type 2 diabetes. DNA was extracted from peripheral blood samples and genotyping was performed by TaqMan genotyping assay. Cardiovascular tests, MNSI-Q and MDNS for neuropathic symptoms and signs, VPT, and thermal thresholds were used for CAN and DPN assessment. We performed a genotype-phenotype correlation analysis. RESULTS We observed that the GG genotype was associated with a higher risk of developing CAN (P=0.002 and OR=16.08, P=0.0005 and OR=35.02, for early and confirmed CAN, respectively) and DPN (P=0.037 and OR=6.56), after correction for BMI, sex, age, HbA1c and disease duration. Moreover, the GG genotype was associated with worse values of MDNS (P=0.017), VPT (P=0.01), thermal thresholds (P=0.01), and CAN score (P<0.001). A logistic multivariate analysis confirmed that MIR499A GG genotype, disease duration and HbA1c contributed to early CAN (R2=0.26), while the same variables and age contributed to DPN (R2=0.21). With a multiple linear regression, we observed that GG genotype (P=0.001) and disease duration (P=0.035) were the main variables contributing to the CAN score (R2=0.35). CONCLUSIONS We described for the first time that the MIR499A genetic variation could be involved in diabetic neuropathies susceptibility. In particular, patients carrying the rs3746444 GG genotype had a higher risk of CAN development, together with a more severe form of CAN.

A polymorphism upstream MIR1279 gene is associated with pericarditis development in Systemic Lupus Erythematosus and contributes to definition of a genetic risk profile for this complication

Recently, a study has shown that a polymorphism in the region of MIR1279 modulates the expression of the TRAF3IP2 gene. Since polymorphisms in the TRAF3IP2 gene have been described in association with systemic lupus erithematosus (SLE) susceptibility and with the development of pericarditis, our aim is to verify if the MIR1279 gene variability could also be involved. The rs1463335 SNP, located upstream MIR1279 gene, was analyzed by allelic discrimination assay in 315 Italian SLE patients and 201 healthy controls. Moreover, the MIR1279 gene was full sequenced in 50 patients. A case/control association study and a genotype/phenotype correlation analysis were performed. We also constructed a pericarditis genetic risk profile for patients with SLE. The full sequencing of the MIR1279 gene in patients with SLE did not reveal any novel or known variation. The variant allele of the rs1463335 SNP was significantly associated with susceptibility to pericarditis (P = 0.017 and OR = 1.67). A risk profile model for pericarditis considering the risk alleles of MIR1279 and three other genes (STAT4, PTPN2 and TRAF3IP2) showed that patients with 4 or 5 risk alleles have a higher risk of developing pericarditis (OR = 4.09 with P = 0.001 and OR = 6.04 with P = 0.04 respectively). In conclusion, we describe for the first time the contribution of a MIR1279 SNP in pericarditis development in patients with SLE and a genetic risk profile model that could be useful to identify patients more susceptible to developing pericarditis in SLE. This approach could help to improve the prediction and the management of this complication.

AB0004 Polymorphisms in Genes in The IL-17 Pathway and B Cell Mediated Immune Response Modulate The Development of Specific Autoimmune Manifestations in Systemic Lupus Erythematosus

Background Systemic Lupus Erythematosus (SLE) is a chronic autoimmune disease in which a complex interaction between genetic, environmental and immunological factors determines the susceptibility and the phenotype. Dysregulations in IL-17 pathway as well as in B-cell mediated immune response have been observed in SLE. We have recently demonstrated that polymorphisms in TRAF3IP2 gene are associated with susceptibility for SLE and can predispose for the development of pericarditis (1). TRAF3IP2 encodes for Act1, a molecule that acts as a negative regulator of B cell by inhibiting CD40-mediated signaling and as a positive signaling adapter in IL-17-mediated cellular responses. TRAF6 is essential for the IL-17/Act1-mediated activation of NF-kB, while CD40 is the target for the Act1 mediated inhibition of B cell response. Objectives The primary goal of our study was to evaluate the association of polymorphisms in CD40, TRAF6 and TNFSF4 (OX40) genes with susceptibility to SLE. Secondary objectives were to assess the possible association of these polymorphisms with the clinical and laboratory features. Methods We recruited 315 Italian SLE patients and 278 healthy controls. Genotyping of rs4810485 in CD40, rs4755453 and rs5030437 in TRAF6, and rs2205960 and rs10489265 in TNFSF4 (OX40) SNPs was performed by allelic discrimination assay. A case/control association study and a genotype/phenotype correlation analysis were performed. Results Deviations from Hardy–Weinberg equilibrium for the three SNPs were not observed. None of the studies polymorphisms was associated with susceptibility for SLE. Only CD40 rs4810485 was associated at genotypic (P=0.034) but not at the allelic level. Nonetheless, these polymorphisms seem to contribute to define disease phenotype. TRAF6 was associated with the presence of anemia (P=0.019, OR=1.96), rs2205960 of TNFSF4 was associated with the pericarditis (P=0.013, OR=2.14), and rs4810485 of CD40 with the presence of anti-SSB/La (P=0.014, OR=2.26) and lupus nephritis (P=0.024, OR=1.8). Conclusions We were not able to confirm previous association of TRAF6 and CD40 polymorphisms with susceptibility for SLE. However, such SNPs seem to influence the disease phenotype. In particular, it is of interest the association of rs4810485 in CD40 with lupus nephritis and the presence of anti-SSB/La. Indeed, the modulation of CD40-mediated T cell-dependent antibody response has already been associated with the development of anti-SSB/La in association with SLE-like nephritis in a mouse model (2), probably due to an uncontrolled B cell activation signal. References Perricone C, Ciccacci C, Ceccarelli F, et al. TRAF3IP2 gene and systemic lupus erythematosus: association with disease susceptibility and pericarditis development. Immunogenetics. 2013 Oct;65(10):703–9 Qian Y, Giltiay N, Xiao J, et al. Deficiency of Act1, a critical modulator of B cell function, leads to development of Sjögrens syndrome. Eur J Immunol. 2008 Aug;38(8):2219–28. Disclosure of Interest None declared

Evaluation of ATG5 polymorphisms in Italian patients with systemic lupus erythematosus: contribution to disease susceptibility and clinical phenotypes:

Systemic lupus erythematosus (SLE) is a common heterogeneous autoimmune disease that is caused by the involvement both of genetic and environmental factors. There is evidence that autophagy is involved in several aspects of SLE pathogenesis. In particular, polymorphisms in the ATG5 gene have been observed to be associated with disease susceptibility. Our aim was to verify if ATG5 polymorphisms are involved in the susceptibility to disease and its clinical phenotypes in an Italian cohort of SLE patients. This study involved 315 SLE patients and 265 healthy controls. Three polymorphisms in the ATG5 gene (rs573775, rs6568431 and rs2245214) were investigated by allelic discrimination assay. A case-control association study, a genotype/phenotype correlation analysis and a haplotype study were performed. Moreover, an expression study was conducted in peripheral blood mononuclear cells from 15 SLE patients to verify a possible effect of the three SNPs on the expression of ATG5. Among the three investigated SNPs, only the rs573775 SNP was significantly associated with disease susceptibility with the variant allele conferring a higher risk of developing SLE (OR = 1.50, p = 0.018 and OR = 1.48, p = 0.007 at the genotypic and allelic level, respectively). The variant allele of rs6568431 SNP was more present in patients with anemia (OR = 1.86, p = 0.009) and renal involvement (OR = 1.63, p = 0.06), while the variant allele of rs2245214 SNP was significantly associated with a higher risk of producing anti-DNA autoantibodies (OR = 1.66, p = 0.04). Carriers of the rs6568431 variant allele showed higher messenger RNA levels compared to the carriers of the wild-type allele, suggesting also a potential variant allele dose-dependent effect on gene expression. In conclusion, our study confirms a role for ATG5 polymorphisms both in disease susceptibility and in the modulation of clinical phenotypes in an Italian SLE cohort. These results further suggest that genetic variations in autophagy genes could play a role in autoimmune diseases susceptibility and are worth further investigation.

THU0013 Towards the definition of a risk model profile of pericarditis in systemic lupus erythematosus: a genetic study

Background We have recently demonstrated that polymorphisms (SNPs) in TRAF3IP2 gene are associated with susceptibility for systemic lupus erythematosus (SLE) and can predispose to the development of pericarditis (1). Moreover, we found that other genes previously associated with susceptibility to SLE (MIR1279, STAT4, PTPN2) seem to be involved in the development of pericarditis (2). At this purpose, we built a genetic risk model for the development of pericarditis in SLE (3). Objectives We aimed to expand the knowledge on the genetic risk of pericarditis in SLE by studying the role of rs2205960 (TNFSF4) and rs2233945 (ATG16L1) SNPs, previously associated with SLE susceptibility, improving our genetic risk model. Methods We recruited SLE patients (diagnosed according to 1997 revised ACR criteria) and healthy subjects served as controls. Study protocol included complete physical examination and blood drawing. The clinical and laboratory data were collected in a standardized, computerized, electronically-filled form (3). Clinical and laboratory features were assessed with a dichotomous score (present=1; absent=0). SNPs genotyping was performed by allelic discrimination assay. A case/control association study and a genotype/phenotype correlation analysis were performed and a risk profile model for pericarditis in SLE was built. Results Three-hundred fifteen SLE patients [285 F (90.5%), 30 M (9.5%), mean age 43.11±11.28 years, mean age at onset 32.19±11.84 years] and 278 healthy controls were enrolled. Pericarditis was present in 56 (17.8%) SLE patients. Deviations from Hardy–Weinberg equilibrium for the studied SNPs were not observed. The variant alleles of the rs2205960 (TNFSF4, P=0.013, OR=2.14) and of the rs2233945 (ATG16L1 P=0.009, OR=2.32) were significantly associated with susceptibility to pericarditis. A risk profile model for pericarditis considering the risk alleles of TRAF3IP2, MIR1279, STAT4, PTPN2, TNFSF4 and ATG16L1 showed that patients with more than 5 risk alleles have a significantly higher risk to develop pericarditis (P<0.001, OR=8.01). Anti-Sm antibodies were the only laboratory parameter associated with the development of pericarditis. Thus, a multivariate analysis by binary regression analysis, considering as dependent variable the presence or absence of pericarditis and as independent variables all the studied SNPs associated with pericarditis, was performed. In a stepwise approach including anti-Sm, TRAF3IP2, MIR1279, STAT4, PTPN2, TNFSF4 and ATG16L1 SNPs, the model explains about 25% (R2 Cox &Snell) of the variability involved in the susceptibility to pericarditis. Conclusions We describe for the first time the contribution of TNFSF4 and ATG16L1 SNPs in pericarditis development in SLE patients. We improved our genetic risk profile model to better and earlier identify SLE patients more susceptible to develop this complication. References Perricone C, Ciccacci C, et al. Immunogenetics. 2013 Oct;65(10):703–9. Ciccacci C, Perricone C, et al. PLoS One. 2014 Nov 4;9(11):e111991. Ciccacci C, Perricone C, et al. Lupus. 2016. doi: 10.1177/0961203316679528. Disclosure of Interest None declared

SAT0006 Polymorphisms in STAT4, IL10, PSORS1C1, PTPN2 and MIR146A Genes Are Differently Associated with Negative Prognostic Factors in Italian Patients Affected by Rheumatoid Arthritis

Background Rheumatoid Arthritis (RA) is a systemic autoimmune disease resulting in chronic inflammation of the synovium and consequent cartilage and bone erosion. RA is strongly associated with the presence of rheumatoid factor (RF) and consists of clinical subsets of anti-citrullinated protein antibody (ACPA)-positive and negative patients. Several genes have been proven to contribute to the disease susceptibility and they may be associated with a particular pattern of disease and with the response or toxicity to therapy. It is important to identify novel genomic biomarkers associated not only with disease susceptibility but also able to detect early those patients with negative prognostic factors who may benefit from a more aggressive therapeutic approach. Objectives This study was designed to evaluate whether the most relevant single nucleotide polymorphisms (SNPs) associated with RA and other autoimmune disorders are related to RF, ACPA, and clinical phenotype in a cohort of biologic-naïve Italian RA patients. Methods A total of 192 RA patients [female n=147 (76.5%), age 54.1 ± 13.2 years, disease duration 7.8 ± 9 years, positive RF n=130 (67.7%), positive ACPA n=137 (71.7%), 28 joint count disease activity score (DAS28) 5.2 ± 1.3, bone erosions at diagnosis n=69 (60%), disease modifying anti-rheumatic drugs (DMARDs) n=148 (77.1%)] were enrolled. 278 age-matched healthy controls were included. We analyzed a total of 12 SNPs in STAT4, IL10, PSORS1C1, PTPN2, ERAP1, TRAF3IP2 and MIR146A genes by allelic discrimination assays. Case-control association studies and genotype/phenotype correlation analyses were performed (Figure 1A-B). Results A higher risk to develop RA was observed for rs7574865 in STAT4 gene (P=0.035, OR=1.41). We observed a significant association between the variant alleles of rs1800872 in IL-10 gene and RA susceptibility. Subjects carrying the variant alleles (in heterozygous and homozygous status) showed a lower risk to develop RA in comparison with wildtype individuals (P=0.014, OR=0.63). The presence of RF resulted significantly associated with rs1800872 variant in IL10 (P=0.032, OR=2) while rs2910164 in MIR146A was protective (P=0.05, OR=0.55). ACPA were significantly associated with rs7574865 in STAT4 (P=0.004, OR=2.64). The SNP rs2233945 in PSORS1C1 gene was protective regarding the presence of bone erosions (P=0.042, OR=0.44) while rs2542151 in PTPN2 gene was associated with joint damage (P=0.044, OR=3.26). Conclusions Our results confirm that polymorphisms in STAT4 and IL10 genes confer susceptibility to RA. For the first time we described that SNPs in PSORS1C1, PTPN2 and MIR146A genes were differently associated with a severe disease phenotype in terms of autoantibody status and radiographic damage in an Italian RA population. Disclosure of Interest None declared

THU0277 Evaluation of Polymorphisms of STAT4, TRAF31P2 and HCP5 in Sjögren Syndrome: Association with Disease Susceptibility and Clinical Aspects

Background Primary Sjögren syndrome (pSS) is a systemic inflammatory condition characterized by lymphocytic infiltrates in exocrine glands. STAT4 is one of the genes involved in “interferon (IFN) signature” and a single nuclear polymorphism (SNP), rs7574865 has been found associated with pSS. Other SNPs in TRAF3IP2 (rs33980500 and rs13193677) and HCP5 (rs3099844) genes, have been previously associated with the susceptibility to systemic lupus erythematosus (SLE), and seem to influence the disease phenotype. Objectives To evaluate the association of different SNPs [STAT4 (rs7574865), TRAF3IP2 (rs33980500), HCP5 (rs3099844)] with the susceptibility to pSS and their role in modulation of clinical and laboratory features. Methods One hundred thirty-five consecutive patients with pSS (AECC) were enrolled and clinical and laboratory data collected. Three hundred age and sex-matched healthy subjects were used as controls. Genomic DNA was isolated from peripheral blood mononuclear cells (Qiagen blood DNA mini kit). Genotyping was performed by allelic discrimination assays (Applied Biosystems, Foster City, CA, USA). Results Clinical and laboratory features are reported in table. Clinical features Number (%) Laboratory features Number (%) Sex (M/F) 5/130 ANA 116/135 (85.9) Age (mean ± SD), yrs 59±12.1 Anti-Ro/SSA 81/135 (60) Age at diagnosis (mean ± SD), yrs 53,2±12,2 Anti-La/SSB 56/135 (41.4) Xerophthalmia 124/135 (91.8) Hypergammaglobulinemia 44/135 (32.5) Xerostomia 124/135 (91.8) Rheumatoid factor 46/135 (34) Glandular swelling 27/135 (20) Leucopenia 28/135 (20.7) Arhritis 16/135 (11.8) Hypocomplementemia 11/135 (8.1) Lymphoma 4/135 (2.9) Monoclonal component 13/135 (9.6) Cryoglobulinemia 6/135 (4.4) Deviations from Hardy–Weinberg equilibrium were not observed. The SNPs rs7574865 of STAT4 and rs3099844 of HCP5 were found associated with pSS at both the genotypic and allelic level (p<0.001, OR=1.84, 95%CI=1.28–2.65 and p<0.05, OR=2.28, 95%CI=1–3.7, respectively) and the data was confirmed after adjustment for other variables. STAT4 was found associated with the presence of monoclonal component (p=0.021). HCP5 was associated with the presence of leukopenia (p=0.021, OR=2.91, 95%CI=1.15–7.38) and lymphoma (p<0.001). Conclusions This study confirms the association between the rs7574865 polymorphism of STAT4 and the susceptibility for pSS, and shows for the first time the association with a variant of HCP5. STAT4 was associated with monoclonal component supporting the role of IFN signature in autoantibodies production. Furthermore, HCP5 seem to be able to influence the disease phenotype predisposing to the development of leukopenia and lymphoma. These data need to be validated on larger cohorts of patients. Disclosure of Interest None declared