Katarzyna Gębura

Analysis of associations between polymorphisms within genes coding for tumour necrosis factor (TNF)-alpha and TNF receptors and responsiveness to TNF-alpha blockers in patients with rheumatoid arthritis

INTRODUCTION Despite the fact that therapy with TNF-α inhibitors constitutes a breakthrough in rheumatoid arthritis management, no improvement is still achieved in approximately 30% of cases. The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. METHODS Five SNPs within the TNF-α and TNF receptor encoding genes (TNFA: G-308A, G-238A, C-857T; TNFR1A G36A; TNFR1B T676G) were determined in 280 RA patients who had been treated with TNF-α inhibitors for at least 6 months or they stop therapy because of adverse events. The association between the relative change in DAS28 and SNP genotypes was tested by linear regression. RESULTS At week 24, low disease activity or remission was achieved by 45% of the patients. After 6 months remission of the disease or low disease activity were more frequently observed among patients homozygous for the TNFR1A 36A allele than among those who were GG homozygotes (52% vs. 34%, P=0.04). At week 24 DAS28 was significantly lower in the subgroup of patients homozygous for the TNFA-857T variant compared to the C allele carriers (P=0.045). The other polymorphisms were not found to be significantly associated with EULAR response at week 12 and 24 of the anti-TNF treatment. CONCLUSIONS Homozygosity for the TNFR1A 36A allele and the TNFA-875T variant could act as a genetic factor associated with better response to anti-TNF treatment.

VEGF and bFGF gene polymorphisms in patients with non-Hodgkin's lymphoma.

Angiogenesis and lymphangiogenesis are important in the proliferation and survival of the malignant hematopoietic neoplasms, including non-Hodgkins lymphomas (NHLs). Vascular endothelial growth factor (VEGF) and basic fibroblast growth factor (bFGF) play an important role in the initiation of angiogenesis. Both VEGF and bFGF have been reported to have prognostic significance in NHL. The present study aimed to determine an association between the VEGF and bFGF gene polymorphisms and disease susceptibility and progression. VEGF (rs3025039; 936 C>T) and bFGF (rs308395, −921 G>C) variants were determined in 78 NHL patients and 122 healthy individuals by PCR-RFLP technique. The presence of the VEGF 936T allele was found to significantly associate with worse prognosis of the disease (expressed by the highest International Prognostic Index (IPI)) (0.41 versus 0.20, P = 0.044 for IPI 4 among patients having and lacking the T allele). The VEGF 936T variant was also more frequent among patients with IPI 4 than in controls (OR = 3.37, P = 0.029). The bFGF −921G variant was more frequently detected among patients with aggressive as compared to those with indolent histological subtype (0.37 versus 0.18, P = 0.095) and healthy individuals (0.37 versus 0.19, OR = 2.51, P = 0.038). These results imply that VEGF and bFGF gene polymorphisms have prognostic significance in patients with NHL.

Genetic variant of the G-CSF receptor gene is associated with lower mobilization potential and slower recovery of granulocytes after transplantation of autologous peripheral blood progenitor cells

Peripheral blood mobilized by cytokines (i.e. granulocyte colony stimulating factor, G-CSF) and chemotherapy has become a major source of hematopoietic stem and progenitor cells for transplantation (PBPCT). In this study the effect of the G-CSF receptor (CSF3R) gene polymorphism was investigated. The presence of the CSF3R variant (T allele, rs3917924) was related to CD34(+) mobilization yield and the pace of granulocyte recovery after autologous PBPCT. The mobilization yield was higher in patients lacking the CSF3R variant (OR=4.756, p=0.046) and those with multiple myeloma (OR=10.534, p=0.019). The pace of granulocyte recovery was found to be associated with the CSF3R polymorphism and was significantly slower in patients carrying the CSF3R-T variant than in CC homozygotes (median of 17 vs. 13 days, p<0.001). This association was confirmed (OR=4.445, p=0.014) by multiple regression analysis considering patient age and sex, the number of transplanted CD34(+) cells, diagnosis and CSF3R polymorphism. These results imply that CSF3R gene polymorphism plays a significant role in PBPCT.

Polymorphisms within Genes Involved in Regulation of the NF-κB Pathway in Patients with Rheumatoid Arthritis

Genes involved in regulation of the nuclear factor-κB (NF-κB)—pathway are suggested to play a role in pathogenesis of rheumatoid arthritis (RA). In the present study, genetic polymorphisms of TLR2, TLR4, TLR9 and NF-κB1 genes were investigated to assess their associations with RA susceptibility, progression and response to anti-TNF-α therapy. A group of 110 RA patients and 126 healthy individuals were genotyped for TLR2 (rs111200466), TLR4 (rs4986790, rs4986791), TLR9 (rs5743836, rs187084) and NF-κB1 (rs28362491) alleles. The presence of the TLR9 −1486 T variant (p < 0.0001) and its homozygosity (p < 0.0001) were found to be associated with disease susceptibility. The TLR9 −1237 C allele was associated with predisposition to RA in females only (p = 0.005). Moreover, the TLR4 rs4986791 G (rs4986790 T) alleles were more frequently detected among patients with the stage IV disease (p = 0.045), and were associated with more effective response to anti-TNF-α therapy (p = 0.012). More efficient response to anti-TNF-α treatment was also observed in patients with del within the NF-κB1 gene (p = 0.047), while for the TLR9 −1486 T homozygotes, the treatment was ineffective (p = 0.018). TLR polymorphisms affect disease susceptibility and response to therapy with TNF-α inhibitors in RA patients of Caucasian origin.

Significance of association of HLA-C and HLA-E with psoriatic arthritis

Psoriatic arthritis (PsA) is a complex genetic disorder that results from an interplay between multiple genetic and environmental factors. The aim of the study was to assess the significance of the association between the HLA-C and HLA-E allelic groups and PsA. Our results confirm the association between HLA-C(∗)06 and PsA (OR=5.16, p<0.0001). Furthermore, HLA-C(∗)06-positive patients develop more severe disease (p<0.01) and more frequently present with polyarticular pattern of PsA (p=0.08). Additionally our study revealed that the HLA-C(∗)02 allele was more frequently observed in PsA patients (OR=5.40, p<0.0005) and also that the HLA-E(∗)01:01 allele was significantly over-represented among HLA-C(∗)02-negative patients in comparison to healthy individuals (OR=6.44, p=0.045). Therefore these results suggest that the HLA-E and HLA-C(∗)02 molecules may also play an important role in determination immune response contributing to the PsA development.

Variations in genes involved in regulation of the nuclear factor – κB pathway and the risk of acute myeloid leukaemia

Genes involved in regulation of the nuclear factor – kappa B (NF‐κB) pathway are suggested to play a role in the pathogenesis of acute myeloid leukaemia (AML). The present study aimed to assess the association between the NF‐κB1, TRAF3 and TLRs genes single nucleotide polymorphisms (SNPs) and disease susceptibility as well as progression in patients with AML. For this purpose 62 patients and 126 healthy individuals were genotyped for NF‐κB1 (rs28362491), TRAF3 (rs11160707; rs12147254), TLR2 (rs201786064), TLR4 (rs4986790; rs4986791) and TLR9 (rs5743836; rs187084) alleles. Three SNPs were found to be associated with the risk for the AML development. The TRAF3 (rs12147254) AA homozygosity (RR = 2.770, P = 0.0392), TLR9 (rs5743836) C wild‐type allele (RR = 2.542, P = 0.0096) as well as TLR9 (rs187084) T allele (RR = 13.396, P < 0.0001) and its homozygosity (RR = 11.805, P < 0.0001) were more frequent among patients with AML than healthy individuals. The associations of the rs187084 SNP were significant for both sexes. Moreover, patients who relapsed were more frequently characterized with the presence of the rs187084 TLR9 TT genotype (P = 0.045) or the rs12147254 TRAF3 A variant (P = 0.066). In conclusion, polymorphisms within the TLR9 and TRAF3 genes are associated with predisposition to AML and may affect the progression of the disease in the Polish population.

Polymorphisms within beta-catenin encoding gene affect multiple myeloma development and treatment

Recent studies have suggested that cereblon (CRBN) is essential for the anti-myeloma (MM) activity of immunomodulatory drugs (IMiDs), such as thalidomide and lenalidomide, and that dysregulation of Wnt/β-catenin pathway may be one of possible reasons of lenalidomide resistance. This prompted us to analyze the effect of polymorphisms within the genes coding for cereblon (CRBN (rs121918368 C>T)) and β-catenin (CTNNB1 (rs4135385 A>G; rs4533622 A>C)). MM patients (n=142) and healthy individuals (n=123) were genotyped using the Light SNiP assays. The presence of the CTNNB1 (rs4533622) A allele was more frequently detected in patients presented with stage II-III disease according to International Staging System (63/82 vs. 26/44, p=0.043) and Durie-Salmon criteria (75/99 vs. 14/26, p=0.049). The CTNNB1 (rs4135385) AA homozygosity was more frequent among patients with better response to CTD, i.e., cyclophosphamide-thalidomide-dexamethasone (18/23 vs. 32/60, p=0.047). Patients carrying the CTNNB1 (rs4533622) AA genotype were better responders to the first line therapy with thalidomide containing regimens (p<0.05). No significant association was observed between the effect of lenalidomide therapy and polymorphisms studied. However, the occurrence of neutropenia during lenalidomide therapy was more frequent among the CTNNB1 (rs4135385) AA carriers (p=0.019), while the CTNNB1 (rs4533622) AA homozygosity characterized patients with high grade (3-4) neutropenia (p=0.044). No association was found for the CRBN polymorphism. These results suggest that the CTNNB1 polymorphisms may affect the clinical course and response to chemotherapy in patients with multiple myeloma.

OP0071 Tnf-Alpha, TNF Receptor, HLA-E and NKG2A Gene Polymorphisms and Response to Anti-TNF-Alpha Treatement in Rheumatoid Arthritis

Background Despite the fact that therapy with TNF-alpha inhibitors constitutes a breakthrough in rheumatoid arthritis (RA) management, no improvement is still achieved in approximately 30% of cases. Objectives The aim of the study was to evaluate whether single nucleotide polymorphisms (SNPs) within the TNF-α and TNF receptor, and HLA-E and NKG2A receptor encoding genes affect the efficacy of therapy with TNF-α inhibitors in patients with RA. Methods For these purpose 280 RA patients who had been treated with TNF-alpha inhibitors for at least 6 months or they stopped therapy because of adverse events were investigated and genotyped for 9 SNPs within the TNFA promoter (rs1800629 G>A; rs361525 G>A; rs1799724 C>T); TNF receptors (TNFR1A: rs767455 G>A; TNFR1B: rs1061622; T>G) while 89 patients were studied for the HLA-E (rs1264457 C>T; HLA-E*01:01, HLA-E*01:03, A>G) and NKG2A (rs7301582 C>T; rs2734440 A>G) genes using LightSNiP typing or Custom TaqMan® SNP Genotyping Assays. Results Among polymorphisms located within TNF-alpha and receptors genes only the TNFR1A (rs767455, G>A) and one of the TNFA (rs1799724, C>T) promoter polymorphisms were found to be associated with response to anti-TNF therapy. Significantly more patients with the homozygous TNFR1A AA genotype achieved a good EULAR response at 3 months compared to patients carrying the G allele (p=0.011). At week 24 DAS28 was significantly lower in patients homozygous for the TNFA T variant (DAS28 – 2.05) compared to the C allele carriers (p=0.045). As for HLA-E and NKG2A genes, after 3 months of anti-TNF treatment the significantly worse (EULAR DAS28) response was observed in patients carrying the HLA-E C allele (20/45 vs. 9/28, p=0.030), HLA-E*01:03/01:03 genotype (8/10 vs. 30/73, p=0.038), NKG2A-(rs7301582)-CC genotype (28/51 vs. 10/33, p=0.043) or NKG2A-(rs2734440)-AA genotype (15/26 vs. 15/50, p=0.026). At week 12 low disease activity or remission was not observed in any of the patients with the HLA-E CC genotype (p=0.09). Also treatment failure (inefficiency or loss of effectiveness of therapy) was more frequently observed in the HLA-E CC homozygous patients (5/8 vs. 12/61, p=0.018) as well as in those with the NKG2A-(rs2734440)-AA genotype (15/37 vs. 3/33, p=0.005). Conclusions These results imply that the polymorphisms within genes coding for TNF-alpha and its TNFR1 receptor as well as HLA-E and NKG2A affect the response to anti-TNF therapy in patients with RA. Acknowledgements Supported by the UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367 grants from the National Science Center. Disclosure of Interest J. Swierkot Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, M. Iwaszko Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, K. Gebura Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, B. Nowak Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, L. Korman Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367, K. Kolossa: None declared, S. Jeka: None declared, P. Wiland: None declared, K. Bogunia-Kubik Grant/research support: UMO-2012/05/N/NZ5/02607 and 2011/01/B/NZ5/05367 DOI 10.1136/annrheumdis-2014-eular.5416

Dual role of the CXCL12 polymorphism in patients with chronic lymphocytic leukemia.

The CXCL12 [chemokine (C–X–C motif) ligand 12] is a member of the CXC family of chemokines and interacts with its CXCR4 receptor. The CXCL12/CXCR4 axis is involved in regulation of proliferation, survival and trafficking of hematopoietic stem cells, including B lymphocytes and disruption within this signaling pathway has been implicated in pathogenesis of chronic lymphocytic leukemia (CLL). The aim of this study was to determine a potential association of the CXCL12 rs1801157 G > A polymorphism with susceptibility to CLL, the disease course and efficacy of therapy. Also, expression of the CD74 and CD38 proteins on B cells was analyzed in relation to clinical parameters and genotyping results. A total of 124 patients with CLL and 75 healthy controls were studied. CXCL12 genotyping was performed using polymerase chain reaction ‐ restriction fragment length polymorphism (PCR‐RFLP) method. The CD74 and CD38 surface expression was determined using flow cytometry. There was a significantly increased frequency of the A allele and AA genotype in CLL patients compared with control group (P < 0.001 in both cases). In addition, the A allele was overrepresented among patients with worse response to therapy in comparison to other genotypes (P < 0.001). On the contrary, patients carrying the A allele displayed lower grade of the disease at diagnosis more frequently than patients homozygous for the G allele (P = 0.037). Moreover, the AA homozygosity correlated with lower CD74 expression on B cells (P = 0.007). In conclusion, data from this study indicate that the CXCL12 rs1801157 G > A polymorphism may affect CLL development, disease progression as well as response to treatment.

FRI0474 Serum Level of IL-23 and IL-23R Polymorphisms in Patients with Psoriatic Arthritis

Background Interleukin (IL) - 23 is one of the of cytokines involved in systemic inflammation. Interaction between this cytokine and its receptor (IL-23R) that plays an important role in pathogenesis of psoriatic arthritis (PsA). Objectives The present study aimed to assess the associations between polymorphisms within gene coding IL-23R, IL-23 serum levels and disease activity in patients with PsA. Methods Fifty-two PsA patients (diagnosed by the criteria recommended by CASPAR group) were genotyped for the IL-23R (rs11209026 and rs7530511) polymorphisms. The nuclear factor kappa (NF-kB1 (rs28362491; ins/del)) polymorphism (associated with the promoter activity of this gene and cytokine gene expressions, including IL-23) was also analyzed in PsA patients group. IL-23 serum levels were assessed by ELISA in patients with PsA, and for comparison, 10 healthy individuals. These laboratory data were further related with clinical characteristics of the patients. Disease Activity Score was measured (swollen and tender joints, ESR, CRP) in addition to BASDAI, BASFI, VAS, and PASI scores. Results Significantly (p<0.05) elevated levels of IL-23 cytokine were observed in PsA patients (126.5 pg/ml) when compared to control group (24.9 pg/ml). Moreover, IL-23 serum levels were associated with the IL-23R rs7530511 polymorphism. Patients carrying the IL-23R T allele characterized with higher concentrations of IL-23 in serum (299.1 vs 86.8, p<0.05). Interestingly, patients with the IL-23R T allele were also more frequently carrying the ins/ins homozygous NF-kB1 genotype (associated with a better promoter activity and higher expression of cytokines) (7/17 vs 4/34, distribution of the T allele among ins/ins vs del allele positive patients, p=0.03). No association was found between for IL-23 levels or IL-23R polymorphisms and disease activity. Conclusions Patients with PsA characterize with higher serum levels of IL-23 than healthy individuals. IL-23 concentrations in serum of PsA patients are associated with the polymorphism (rs7530511) of IL-23 receptor encoding gene. Disclosure of Interest None declared