IL-17F gene polymorphism is associated with susceptibility to acute myeloid leukemia

Tomasz Wrbel 0 1 2 Katarzyna Gebura 0 1 2 Barbara Wysoczanska 0 1 2 Bozena Jazwiec 0 1 2 Olga Dobrzynska 0 1 2 Grzegorz Mazur 0 1 2 Kazimierz Kuliczkowski 0 1 2 Katarzyna BoguniaKubik 0 1 2 0 g. Mazur Department of Internal, Occupational Diseases and Hypertension, Wrocaw Medical University , Borowska 213, 50-556 Wrocaw, Poland 1 K. gebura B. Wysoczanska K. Bogunia-Kubik ( 2 t . Wrbel B. Jazwiec O. Dobrzynska K. Kuliczkowski Department of Haematology , Blood neoplasms and Bone Marrow t ransplantation, Wrocaw Medical University , Wybrzeze l. Pasteura 4, 50-367 Wrocaw, Poland 3 ) laboratory of Clinical Immunogenetics and Pharmacogenetics, l. Hirszfeld Institute of Immunology and experimental therapy, Polish academy of Sciences , R. Weigla 12, 53-114 Wrocaw, Poland Purpose Recent studies have suggested that th17 cells may play a role in the pathogenesis of acute myeloid leukemia (aMl). this subset of CD4+ cells is characterized by interleukin (Il)-17a and Il-17F production, which share strong homology, and surface expression of the Il-23 receptor (Il-23R). the present study aimed to determine the association between the polymorphic features located within the IL-17A, IL-17F and IL-23R genes and disease susceptibility, progression and response to therapy. In addition, the relationship between the polymorphic variants and the plasma Il-17 levels in patients was analyzed. Methods For this purpose, 187 individuals of Polish origin including 62 aMl patients and 125 healthy controls were typed for IL-17A (rs2275913; g-197a), IL-17F (rs763780; a7488g; His161arg) and IL-23R (rs11209026, g1142a; arg381gln) alleles. Results the rs763780 IL-17F polymorphism appeared to be associated with susceptibility to the disease. the presence of the minor (G) variant (RR = 4.76, p < 0.001) and its homozygosity (RR = 23.02, p < 0.005) was more frequent among patients than healthy individuals. no significant association was observed for either other polymorphisms studied or Il-17 levels. Conclusions thus, the rs763780 IL-17F polymorphism was found to be associated with predisposition to aMl in the Polish population. - acute myeloid leukemia (aMl) is a life-threatening hematopoietic stem cell neoplasm characterized by bone marrow infiltration by leukemic cells that suppress normal hematopoiesis, frequently resulting in fatal infection, bleeding or organ infiltration, with or without leukocytosis (lowenberg et al. 1999; estey and Dohner 2006). the etiology of aMl is heterogeneous and complex, but it is widely accepted that both environmental and genetic factors play significant roles in the development of aMl. Recent studies have suggested that th17 cells may play an important role in patients with aMl [discussed by li et al. (2012)]. It has been reported that the th17 cell frequencies or levels of Il-17 and its related cytokines were different between normal cells and malignant aMl cells, suggesting that th17 cells might be involved in aMl pathogenesis (Wu et al. 2009; abousamra et al. 2013). It has also been observed that the increased th17 cell frequencies were reduced when patients achieved complete remission (CR) after chemotherapy, suggesting that measurement of th17 cell frequencies may have clinical value in evaluation of the therapeutic effect (Wu et al. 2009). the hallmark of the th17 subset is the production of interleukin Il-17a and Il-17F, which share strong homology, and surface expression of the Il-23 receptor (Il-23R) (Hot et al. 2011). Il-23 is essential for the differentiation of th17 cells and plays a key role in the development of pathogenic th17 cells that produce the cytokine Il-17, which induces the production of several pro-inflammatory cytokines, such as tnF-a and Il-6, and chemokines (aggarwal et al. 2003; Bettelli et al. 2008; McKenzie et al. 2006). Il-23 and Il-21 induce the orphan nuclear receptor RORt, which in synergy with Stat3 promotes Il-17 expression (nurieva et al. 2007). the present study aimed to determine the association between the polymorphic features located within the IL17A, IL-17F and IL-23R genes and disease susceptibility, progression and response to therapy. For this purpose, patients with aMl and healthy individuals were typed for the IL-17A (rs2275913; g-197a), IL-17F (rs763780; a7488g; His161arg) and IL-23R (rs11209026, g1142a; arg381gln) alleles. In addition, the relationship between the polymorphic variants of the Il-17 genes and plasma Il-17 levels were analyzed. Materials and methods Patients and controls Sixty-two adult patients (24 females and 38 males, median age 52 years, range 1980 years) with aMl were investigated. Patients with acute promyelocytic leukemia were excluded. In addition 125 Polish healthy individuals of both sexes (female/male: 63/62) served as controls. IL-17A, IL-17F and IL-23R genotyping three biallelic polymorphisms were studied: IL-17A (rs2275913; g-197a), IL-17F (rs763780; a7488g; His161arg) and IL-23R (rs11209026, g1142a). the IL-17F (rs763780; a7488g) polymorphism was analyzed using a polymerase chain reaction restriction fragment length polymorphism (PCRRFlP) assay, which amplified a fragment of the promoter region of the gene using primers as previously described [15] (forward: 5-gtt CCC atC Cag Caa gag a C-3 and reverse: 5-agC tgg gaa tgC aaa Caa a C-3). the PCR conditions were as follows: 94 C for 3 min; 35 cycles at 94 C for 30 s, 60 C for 30 s and 72 C for 30 s; and a final elongation step at 72 C for 7 min. the PCR products were analyzed by electrophoresis in 2 % agarose gel stained with ethidium bromide and visualized under UV light (Uvitec). the PCR products were digested with the NlaIII restriction endonuclease (new england Biolabs Inc.) and analyzed in 2 % agarose gel. three patterns were observed following digestion and electrophoresis: a single 412 bp fragment (individuals homozygous for the IL-17F G allele, lacking the NlaIII site), three fragments of 412, 288 and 124 bp in length (heterozygous individuals) or two fragments of 288 and 124 bp (individuals homozygous for the IL-17F A allele). PCR amplifications for the IL-17F gene polymorphism studies were carried out in the 2720 thermal Cycler (applied Biosystems, Foster City, USa). the IL-17A (rs2275913; g-197a) and IL- 23R (rs11209026, g1142a) alleles were determined by realtime PCR amplifications, and analysis of the typing results was performed using a Roche lightCycler 480 instrument. the lightSniP (rs2275913) assay designed by tIB MOlBIOl (gmbH, Berlin, germany) or t aqMan SnP genotyping assay (rs11209026) (life t echnologies) was used for detection of IL-17A and IL-23R alleles, respectively. Plasma samples were taken from all the patients before chemotherapy was administered. In addition, 20 out of 62 patients were analyzed again after achieving CR. Il-17 levels were measured by enzyme-linked immunoassay (elISa) (R&D Systems, USa) following the manufactur ers instruction. analyses and calibrations were carried out in duplicate. Int (...truncated)