/2015/107/ Revista Española de Enfermedades Digestivas Vol. 107, N.º 9, pp , 2015 ORIGINAL PAPERS

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1 113-18/215/17/ Revista Española de Enfermedades Digestivas Copyright 215 Arán Ediciones, S. L. Rev Esp Enferm Dig (Madrid Vol. 17, N.º 9, pp , 215 ORIGINAL PAPERS Contribution of KIR (killer immunoglobulin-like receptor) genes, HLA class I ligands, and KIR/HLA class I ligand combinations on the genetic predisposition to celiac disease and coexisting celiac disease and type 1 diabetes mellitus H. Haluk Akar 1, Turkan Patiroglu 1, Eylem Sevinc 2, Duran Aslan 2, Deniz Okdemir 3 and Selim Kurtoglu 3 Departments of 1 Pediatric Immunology, 2 Pediatric Gastroenterology, 3 Pediatric Endocrinology. Erciyes University. Medical Faculty. Kayseri, Turkey ABSTRACT Backgound and aim: There are some common genetic features between celiac disease (CD) and diabetes mellitus type 1 (DM). However, the genetic risk factors have not been fully clarified for CD and the co-occurrence of CD and DM. KIR (killer immunoglobulin-like receptor) genes regulate the cytolitic activity of NK-cells and T lymphocytes. The aim of this study is to evaluate the contribution of KIR genes, KIR ligands, and combinations of KIR/ KIR ligands on the genetic predisposition to CD and co-occurrence of CD and DM. Material and methods: Forty six patients with CD (n = 46), 2 patients with CD+DM (n = 2), and 6 healthy controls (n = 6) were included in this study. KIR genes and KIR ligands were investigated with PCR-SSOP and PCR-SSP in all subjects, respectively. Results: This study showed that while the telomeric KIR genes (2DS5 and 3DS1), and combinations of 3DS1+HLA-BBw4-Thrand 3DS1+HLA-BBw4-Iso- (p <.1, p <.1, p <.1, and p <.1, respectively) were observed more frequently in patients with CD than in controls, the 2DS5, 3DS1 KIR genes, C1 ligand, and combinations of 3DS1+HLA-BBw4-Thr- and 3DS1+HLA-BBw4-Iso- (p =.2, p =.4, p =.36, p <.1, and p =.7, respectively) were observed more frequently in patients with CD+DM than in controls. Conclusions: The results of this study indicated that some KIR genes, KIR ligands, and KIR/KIR ligand interactions may be responsible for a predisposition to CD and the coexistence of CD and DM. For development of coexisting CD and DM, the 2DS5 and 3DS1 genes, C1 ligand, and combinations of 3DS1+HLA-BBw4- Thr- and 3DS1+HLA-BBw4-Iso- were found to be risk factors. Key words: Celiac disease. KIR genes. KIR ligands. KIR/KIR ligand combinations. Type 1 diabetes mellitus. INTRODUCTION Celiac disease (CD) is a chronic autoimmune disorder which is caused by both environmental (gluten intolerance) and genetic factors such as human leukocyte antigen (HLA) genes. The prevalence of CD has been accepted as almost.5-2% worldwide (1,2). Type 1 diabetes mellitus (DM) results from an autoimmune process against pancreatic beta cells. As in CD patients, DM is a multifactorial autoimmune disease that shows strong genetic associations (3). It has been proposed that CD is a T-cell-mediated disorder. Gliadin derived peptides lead to T lymphocyte infiltration in the lamina propria which causes effector T-cell responses (both of T H 2 and T H 17-cells) (4). However, gliadin derived peptides can also trigger non-t lymphocyte responses. Different innate immune gene families such as killer immunoglobulin-like receptor (KIR) genes have been reported as susceptible genes in CD patients in recent years (5). As compared to the general population, patients with a single autoimmune disease (e.g. CD, DM) have increased risk for the development of another autoimmune disease. For example, approximately 3% of CD patients have another autoimmune disease (6,7). The togetherness of CD and DM is the result of a complex interaction between genetic and environmental factors which have not been fully clarified (8). This togetherness between CD and DM can be explained to a certain extent by common genetic factors. For example, both CD and DM are related to DQ2 and DQ8 (9-12). KIRs, which are expressed on natural killer (NK) cells and some subsets of T-cells, have an important role in innate immune responses. The KIR genes are located on chromosome 19q13.4 within the leukocyte receptor complex (13). The family of KIR genes includes 16 identified genes and 2 pseudogenes (PGs). Some KIRs have specific HLA class I ligands. KIRs and HLA class I molecules interact as receptor and ligand. Some combinations of KIR/HLA class I ligands have activating or inhibitory signal effects on the NK cells (14). The influence of KIR genes, HLA class I ligands, and KIR/ HLA ligand combinations in patients with CD and coexist- Received: Accepted: Correspondence: H. Haluk Akar. Department of Pediatric Immunology. Erciyes University. Medical Faculty. Kayseri, Turkey himmetakar@gmail.com Akar HH, Patiroglu T, Sevinc E, Aslan D, Okdemir D, Kurtoglu S. Contribution of KIR genes, HLA class I ligands, and KIR (killer immunoglobulin-like receptor)/hla class I ligand combinations on the genetic predisposition to celiac disease and coexisting celiac disease and type 1 diabetes mellitus. Rev Esp Enferm Dig 215;17:

2 548 H. H. AKAR ET AL. Rev Esp Enferm Dig (Madrid) ing CD and DM has rarely been investigated to date. The aim of the presented study is to evaluate the contribution of KIR genes, HLA class I ligands, and KIR/HLA class I ligand combinations on the genetic predisposition to CD and coexisting CD and DM. MATERIAL AND METHODS Subjects Forty-six patients with CD (n = 46, aged 5-17 years), 2 patients with CD+DM (n = 2, aged 7-16 years), and 6 healthy unrelated Turkish subjects (n = 6, aged 5-18 years) were enrolled in this study. The patients are followed-up in the Pediatric Gastroenterology and Pediatric Endocrinology Departments of Erciyes University, Medical Faculty, in Kayseri, Turkey. All participants were Turkish and residing in Kayseri and the surrounding cities, in the Middle Anatolia region of Turkey. CD was diagnosed according to the European Society for Pediatric Gastroenterology, Hepatology and Nutrition (ESPGHAN) criteria in all patients (12,15). In the coexisting CD and DM group, while 7 patients were diagnosed with CD before the onset of DM, 13 patients were diagnosed with DM before the onset of CD. The presented study was approved by the Erciyes University Medical Faculty Ethics Committee, and written informed consent was obtained from subjects parents. Laboratory methods The genomic DNA of all participants was isolated from peripheral venous blood samples by using Bio-robot EZ1 (Qiagen, Hilden, Germany). For KIR genes, KIR genotyping was studied in all subjects by polymerase chain reaction with the sequence oligonucleotide probe (PCR-SSOP) method by using Luminex technology (Immucor Gamma-Lifecodes, Stamford, CA). Fourteen KIR genes (2DL1, 2DL2, 2DL3, 2DL4, 2DL5, 2DS1, 2DS2, 2DS3, 2DS4, 2DS5, 3DL1, 3DL2, 3DL3, 3DS1) and 2 PGs (2DP1, 3DP1) were investigated. For HLA class I ligands, a KIR/HLA ligand kit was used (Olerup SSP-KIR HLA Ligand, Stockholm, Sweden). HLA class I ligands were classified as follows: HLA-CwAsparagin8 (HLA-C1), HLA- CwLysine8 (HLA-C2), HLA-BBw4-Threonine8 (HLA-BBw4- Thr), HLA-BBw4-Isoleucine8 (HLA-BBw4-Iso), and HLA-ABw4. KIRs interact with their specific HLA class I ligands on their target cells as follows: (2DL2, 2DL3, and 2DS2 bind to HLA-C1), (2DL1 and 2DS1 bind to HLA-C2), and (3DL1 and 3DS1 bind to HLA- BBw4-Thr and HLA-BBw4-Iso) (16). All kits were used according to the manufacturer s instructions. Statistical analysis Statistical analyses were carried out using the IBM SPSS statistical package, version 22. After classification of the HLA class I ligands, 2 combinations of KIR/HLA class I ligand were formed according to the presence or absence of specific HLA class I ligands (17-19). The frequencies of KIR genes, HLA class I ligands, and KIR/HLA class I ligand combinations were calculated with descriptive statistic methods. The two tailed Fisher exact test was used for comparison among controls and both groups of patients (CD and CD+DM). A p value of less than.5 was considered significant. Odds ratios (ORs) were determined at 95% confidence intervals (95% CI). The Bonferroni correction test was applied in the multiple comparison of between the centromeric and telomeric genes. RESULTS KIR genes The frequencies of 14 KIR genes and 2 PGs were studied in all groups. Tables I and II show the frequencies of all KIR genes and their comparisons among controls and patients with CD and CD+DM. Some statistical significances were observed in both groups. In the CD group, while the 2DL2, 2DS2, 2DS4, 3DL1, and 2DP1 KIR genes [(p =.46, OR: 2.35, 95% CI: ), (p =.31, OR: 2.56, 95% CI: ), (p =.3, OR: 3.55, 95% CI: ), (p <.1, OR: 4.13, 95% CI: ), (p =.1, OR: 4.28, 95% CI: ), respectively] were less common, the 2DS5 and 3DS1 KIR genes [(p <.1, OR:.8, 95% CI: ), (p <.1, OR: 2.15, 95% CI: ), respectively] were more frequent (Table I). In the coexisting CD and DM group, although the 2DS2 gene (p =.42, OR: 3.4, 95% CI: ) was less common, the 2DS5 and 3DS1 KIR genes [(p =.2, OR: 1.53, 95% CI: ) and (p =.4, OR: 1.5, 95% CI: ), respectively] were more frequent (Table II). In the comparison of 3 centromeric and 3 telomeric KIR genes, the telomeric KIR genes (2DS5, 3DS1) were more frequent in both groups of patients (CD and CD+DM) than in controls (for both groups of patients, p <.1, Table III). HLA class I ligands The frequencies of the HLA class I ligand are shown in tables IV and V. In the CD group, the frequency of the HLA-ABw4 ligand was less common than in the controls (p =.1, OR = 3.46, 95% CI: ) (Table IV). In the coexisting CD and DM group, the frequency of the HLA-C1 ligand was higher than in the controls (p =.36, OR = 1.44, 95% CI: ) (Table V). Combinations of KIR/HLA class I ligand In order to evaluate whether patients and controls carried both molecules required for the interaction of KIR/ HLA class I ligand, the presence or absence of HLA class I ligands was evaluated in patients and controls who carried the KIR gene (Tables VI and VII). The inhibitory combinations of 2DL2+HLA-C1+, 3DL1+HLA-BBw4- Thr-, 3DL1+HLA-BBw4-Iso-,and 3DL1+HLA-BBw4-Iso Rev Esp Enferm Dig 215; 17 (9):

3 Vol. 17, N.º 9, 215 CONTRIBUTION OF KIR (KILLER IMMUNOGLOBULIN-LIKE RECEPTOR) GENES, HLA CLASS I LIGANDS, AND KIR/HLA 549 CLASS I LIGAND COMBINATIO ON THE GENETIC PREDISPOSITION TO CELIAC DISEASE AND COEXISTING CELIAC DISEASE AND TYPE 1 DIABETES MELLITUS Table I. The frequencies of KIR genes in CD group and controls CD (n = 46) n (%) Control (n = 6) n (%) p OR (95% CI) 2DL1 44 (95.7) 57 (95) 2DL2 22 (47.8) 41 (68.3) ( ) 2DL3 37 (8.4) 5 (83.3) 2DL4 46 (1) 6 (1) 2DL5 24 (52.2) 41 (68.3) 2DS1 24 (52.2) 31 (51.7) 2DS2 17 (37) 36 (6) ( ) 2DS3 2 (43.5) 34 (56.7) 2DS4 13 (28.3) 35 (58.3) ( ) 2DS5 44 (95.7) 38 (63.3) <.1.8 ( ) 3DL1 17 (37) 43 (71.7) < ( ) 3DL2 45 (97.8) 6 (1) 3DL3 46 (1) 6 (1) 3DS1 46 (1) 4 (66.7) < ( ) 2DP1 18 (39.1) 44 (73.3) ( ) 3DP1 46 (1) 6 (1) CD: Celiac disease; CI: Confidence interval; N: Number of patients; n: KIR gene frequency; : Not significant; OR: Odds ratio. Table II. The frequencies of KIR genes in coexisting CD and DM group and controls CD + DM (n = 2) n (%) Control (n = 6) n (%) p OR (95% CI) 2DL1 19 (95) 57 (95) 2DL2 14 (7) 41 (68.3) 2DL3 5 (83.3) 2DL4 2 (1) 6(1) 2DL5 16 (8) 41 (68.3) 2DS1 14 (7) 31 (51.7) 2DS2 36 (6) ( ) 2DS3 34 (56.7) 2DS4 9 (45) 35 (58.3) 2DS5 2 (1) 38 (63.3) ( ) 3DL1 1 (5) 43 (71.7) 3DL2 19 (95) 6 (1) 3DL3 2 (1) 6 (1) 3DS1 2 (1) 4 (66.7) ( ) 2DP1 11 (55) 44 (73.3) 3DP1 2 (1) 6 (1) CD: Celiac disease; CI: Confidence interval; DM: Type 1 diabetes mellitus; N: Number of patients; : Not significant; n: KIR gene frequency; : Not significant; OR: Odds ratio. [(p =.46, OR: 2.35, 95% CI: ), (p =.22, OR: 3.33, 95% CI: ), (p =.31, OR: 2.56, 95% CI: ), and (p =.18, OR:.54, 95% CI: ), respectively] were less common in CD group than controls. Also, while the activating combinations of 2DS2+HLA-C1+ and 3DS1+HLA-BBw4-Iso+ Rev Esp Enferm Dig 215; 17 (9):

4 55 H. H. AKAR ET AL. Rev Esp Enferm Dig (Madrid) 3 centromeric genes: 2DL2 2DS2 2DS3 3 telomeric genes: 2DS1 2DS5 3DS1 Table III. The frequencies of centromeric and telomeric KIR genes CD (n = 46) n (%) CD+DM (n = 2) n (%) Control (n = 6) n (%) p 22 (47.8) 17 (37) 2 (43.5) 24 (52.2) 44 (95.7) a 46 (1) a 14 (7) 14 (7) 2 (1) a 2 (1) a 41 (68.3) 36 (6) 34 (56.7) 31 (51.7) 38 (63.3) b 4 (66.7) b <.1 <.1 CD: Celiac disease; CI: Confidence interval; DM: Type 1 diabetes mellitus; N: Number of patients; n: Gene frequency; : Not significant; OR: Odds ratio; a,b Letters show statistically significance. Table IV. The frequencies of HLA class I ligands in CD group and controls HLA lıgand gene CD (n = 46) n (%) Control (n = 6) n (%) p OR (95% CI) HLA-C1 HLA-C2 HLA-BBw4-Thr HLA-BBw4-Iso HLA-ABw4 29 (63) 31 (67.4) 11 (23.9) 25 (54.3) 7 (15.2) 36 (6).1 CD: Celiac disease; CI: Confidence interval; N: Number of patients; n: HLA class I ligand frequency; : Not significant; OR: Odds ratio ( ) Table V. The frequencies of HLA class I ligands in coexisting CD and DM group and controls HLA ligand gene CD+DM (n = 2) n (%) Control (n = 6) n (%) p OR (95% CI) HLA-C1 HLA-C2 HLA-BBw4-Thr HLA-BBw4-Iso HLA-ABw4 2 (1) 9 (45) 36 (6) ( ) CD: Celiac disease; CI: Confidence interval; DM: Type 1 diabetes mellitus; N: Number of patients; n: HLA class I ligand frequency; : Not significant; OR: Odds ratio. [(p =.31, OR: 2.56, 95 % CI: ), (p =.1, OR: 3.46, 95% CI: ), respectively] were less common, 3DS1+HLA-BBw4-Thr- and 3DS1+HLA- BBw4-Iso- [(p <.1, OR:.12, 95% CI:.52-.3) and (p <.1, OR:.7 95% CI: ), respectively] were more frequent in the CD group than in controls. In the coexisting CD and DM group, the activating combinations of 3DS1+HLA-BBw4-Iso-, and 3DS1+H- LA-BBw4-Iso- [(p <.1, OR:.13, 95% CI: ) and (p =.7, OR:.21, 95% CI: )] were more frequent than in controls. DISCUSSION In the pathogenesis of CD, gliadin derived peptides trigger innate immune responses as well as adaptive immune responses (19). Although NK cells have important roles in the innate immune system, a few studies have reported the association among KIR genes, HLA class I ligands, and KIR/HLA class I ligand interactions and CD or coexisting CD and DM patients (3,5,21-23). In this study, we observed that patients with CD and CD+DM had some differences in terms of the frequencies of KIR genes, HLA class I ligands, and combinations of KIR/HLA class I ligands compared to the controls. In the CD group, while the frequencies of 2DL2, 2DS2, 2DS4, 3DL1 and 2DP1 KIR genes, HLA-ABw4 ligand, and combinations of 2DL2+HLA-C1+, 3DL1+HLA-BBw4-Thr-, 3DL1+HLA- BBw4-Iso+, 3DL1+HLA-BBw4-Iso-, 2DS2+HLA-C1+ and 3DS1+HLA-BBw4-Iso+ were less common, the 2DS5 and 3DS1 KIR genes, combinations of 3DS1+H- LA-BBw4-Thr+ and 3DS1+HLA-BBw4-Iso- were more frequent (Tables I, IV, and VI). In the coexisting CD and DM group, while the frequency of the 2DS2 gene was less common, the 2DS5 and 3DS1 genes, HLA-C1 ligand, and combinations of 3DS1+HLA-BBw4-Thr- and 3DS1+H- LA-BBw4-Iso- were more frequent (Tables II, V, VII). In the comparison of centromeric and telomeric KIR genes, the telomeric 2DS5 and 3DS1 KIR genes were more fre- Rev Esp Enferm Dig 215; 17 (9):

5 Vol. 17, N.º 9, 215 CONTRIBUTION OF KIR (KILLER IMMUNOGLOBULIN-LIKE RECEPTOR) GENES, HLA CLASS I LIGANDS, AND KIR/HLA 551 CLASS I LIGAND COMBINATIO ON THE GENETIC PREDISPOSITION TO CELIAC DISEASE AND COEXISTING CELIAC DISEASE AND TYPE 1 DIABETES MELLITUS Table VI. The frequencies of KIR/HLA class I ligand combinations in CD group and controls KIR gene/hla ligand gene CD (n = 46) n (%) Control (n = 6) n (%) p OR (95% CI) 2DL2+HLA-C1+ 2DL2+HLA-C1 2DS2+HLA-C1+ 2DS2+HLA-C1 2DL3+HLA-C1+ 2DL3+HLA-C1 2DS3+HLA-C1+ 2DS3+HLA-C1 2DL1+HLA-C2+ 2DL1+HLA-C2 2DS1+HLA-C2+ 2DS1+HLA-C2 3DL1+HLA-BBw4-Thr+ 3DL1+HLA-BBw4-Thr 3DS1+HLA-BBw4-Thr+ 3DS1+HLA-BBw4-Thr 3DL1+HLA-BBw4-Iso+ 3DL1+HLA-BBw4-Iso 3DS1+HLA-BBw4-Iso+ 3DS1+HLA-BBw4-Iso 22 (47.8) 17 (37) 29 (63) 8 (17.4) 2 (43.5) 31 (67.4) 13 (38.3) 24 (52.2) 11 (23.9) 6 (13) 11 (23.9) 35 (76.1) 17 (37) 7 (15.2) 39 (84.9) 41 (68.3) 36 (6) 4 (6.7) 34 (56.7) 12 (2) 31 (51.7) 2 (33.3) 36 (6) 7 (11,7) < <.1 CD: Celiac disease; CI: Confidence interval; N: Number of patients; n: KIR/HLA ligand frequency; : Not significant; OR: Odds ratio ( ) 2.56 ( ) 3.33 ( ).12 (.52-.3) 2.56 ( ).54 ( ) 3.46 ( ).7 ( ) Table VII. The frequencies of KIR/HLA class I ligand combinations in coexisting CD and DM group and controls KIR gene/hla ligand gene CD+DM (n = 2) n (%) Control (n = 6) n (%) p OR (95% CI) 2DL2+HLA-C1+ 2DL2+HLA-C1 2DS2+HLA-C1+ 2DS2+HLA-C1 2DL3+HLA-C1+ 2DL3+HLA-C1 2DS3+HLA-C1+ 2DS3+HLA-C1 2DL1+HLA-C2+ 2DL1+HLA-C2 2DS1+HLA-C2+ 2DS1+HLA-C2 3DL1+HLA-BBw4-Thr+ 3DL1+HLA-BBw4-Thr 3DS1+HLA-BBw4-Thr+ 3DS1+HLA-BBw4-Thr 3DL1+HLA-BBw4-Iso+ 3DL1+HLA-BBw4-Iso 3DS1+HLA-BBw4-Iso+ 3DS1+HLA-BBw4-Iso 14 (7) 6 (3) 1 (5) 15 (75) 9 (45) 1 (5) 41 (68.3) 36 (6) (6,7) 34 (56.7) 12 (2) 31 (51.7) 2 (33.3) 36 (6) 7 (11,7) < ( ).21 ( ) CD: Celiac disease; CI: Confidence interval; DM: Type 1 diabetes mellitus; N: Number of patients; n: KIR/HLA class I ligand frequency; : Not significant; OR: Odds ratio. quent in both groups of patients (CD and CD+DM) than in controls (Table III). For KIR gene frequencies, some different results were observed in the presented study. As in mentioned above, the CD patients had less common frequent 2DL2, 2DS2, 2DS4, 3DL1, and 2DP1 KIR genes as well as the two more frequent 2DS5 and 3DS1 KIR genes compared to the controls in this study. In the medical literature, only one study by Smigoc Schweiger et al. (3) was reported with only one more frequent KIR gene (2DS3) in the celiac patients than in controls. In other previously reported studies, no significant association has been reported between CD and controls so far (21-23). For Rev Esp Enferm Dig 215; 17 (9):

6 552 H. H. AKAR ET AL. Rev Esp Enferm Dig (Madrid) HLA class I ligand frequencies, in this study, negative and positive associations of HLA-ABw4 and HLA-C1 class I ligands were observed in the CD group and coexisting CD and DM group, respectively. The activities of NK-cells are regulated by inhibitory and activating KIRs which interact with specific HLA class I ligands (24). The HLA-C1 and HLA-C2 ligands are bound predominantly inhibitory receptors 2DL2/2DL3 and 2DL1/2DS1, respectively. The 2DL1/HLA-C2, 2DL2/HLA-C1, 2DL3/HLA-C1 interactions show the strongest, intermediate, and weakest inhibitory effect on NK cells, respectively. As a rule, inhibitory and activating KIRs (e.g. 2DL1 and 2DS1, respectively) have the same or similar HLA class I ligand specificity. Also, the 3DL1 and 3DS1 KIR genes bind to HLA-B and HLA-A class I ligands which include the Bw4 motif (18,25). In the literature, only one study was reported on the topic of HLA class I ligands (3). In contrast to our results, in that study, Smigoc Schweiger et al. (3) reported that the coexisting CD and DM group showed a negative and positive association with HLA-C1 and HLA-BBw4 class I ligands, respectively. In the investigation of KIR/ HLA class I ligand combinations, a positive association of activating 3DS1+HLA-BBw4-Thr- and 3DS1+HLA- BBw4-Iso- combinations was observed in both groups of patients (CD and CD+DM) in the presented study. Also, a negative association was observed in combinations of 2DL2+HLA-C1+, 2DS2+HLA-C1+, 3DL1+H- LA-BBw4-Thr-, 3DL1+HLA-BBw4-Iso+, 3DL1+HLA- BBw4-Iso-, and 3DS1+HLA-BBw4-Iso+ interactions in the CD group. In the medical literature, only 2 studies have been reported about KIR/HLA class I ligand combinations in patients with CD and with CD+DM so far. The results of those studies were different our results. In one of them, a positive association of the inhibitory 2DL3-C1+ combination and a negative association of the inhibitory 2DL3-C1- combination were reported by Smigoc Schweiger et al. (3) in the coexisting CD and DM group. In another study on this topic reported by Caggiari et al. (21), complications of CD were found to be associated with combinations of 2DL2+HLA-C1+, 2DS2+HLA-C1+ and 3DL1+HLA-Bw4+. Another parameter for comparison between groups and controls was the frequency of centromeric and telomeric KIR genes in this study. In the comparison of centromeric and telomeric genes, a positive association was observed in the telomeric KIR genes (2DS5 and 3DS1) with CD and coexisting CD and DM patients. Caggiari et al. (21) reported that centomeric KIR genes were associated with the complications of CD. In all parameters (KIR genes, HLA class I ligands, KIR/ HLA class I ligand combinations), significant differences were observed between groups and controls in this study. Also, the results of the presented study were different from those of previously reported studies (3,5,21-23). It can be speculated that these differences among studies may be associated with the ethnicity and sample sizes of the studies. In conclusion, some contributions of KIR genes, HLA class I ligands, and combinations of KIR/HLA class I ligands were observed in both groups of patients (CD, CD+DM). According to the presented study, activating 2DS5 and 3DS1 KIR genes (telomeric genes) can be risk factors for CD and coexisting CD and DM patients. The HLA-ABw4 class I ligand can be a protective molecule against the development of CD. Also, the HLA-C1 class I ligand can be risk factor for coexisting CD and DM. In addition to these data, we observed that some combinations of KIR/HLA class I ligands can be either risk (3DS1+H- LA-BBw4-Thr- and 3DS1+HLA-BBw4-Iso-) or protective (2DL2+HLA-C1+, 2DS2+HLA-C1+, 3DL1+HLA-BBw4- Thr-, 3DL1+HLA-BBw4-Iso+, 3DL1+HLA-BBw4-Isoand 3DS1+HLA-BBw4-Iso+) factors for the development of CD. Also, for coexisting CD and DM, activating combinations of 3DS1+HLA-BBw4-Thr- and 3DS1+HLA- BBw4-Iso- were observed as risk factors. The presented study is the first study on the combinations of KIR genes, HLA class I ligands, and KIR/HLA class I ligand combinations in CD and coexisting CD and DM patients in Turkey. These results are needed confirmation with further studies which have a larger number of subjects. REFERENCES 1. Gujral N, Freeman HJ, Thomson AB. Celiac disease: Prevalence, diagnosis, pathogenesis and treatment. World J Gastroenterol 212;18: DOI: /wjg.v18.i Lucendo AJ, García-Manzanares A. Bone mineral density in adult coeliac disease: An updated review. 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7 Vol. 17, N.º 9, 215 CONTRIBUTION OF KIR (KILLER IMMUNOGLOBULIN-LIKE RECEPTOR) GENES, HLA CLASS I LIGANDS, AND KIR/HLA 553 CLASS I LIGAND COMBINATIO ON THE GENETIC PREDISPOSITION TO CELIAC DISEASE AND COEXISTING CELIAC DISEASE AND TYPE 1 DIABETES MELLITUS 12. Lorinczy K, Juhász M, Csontos Á, et al. Does dermatitis herpetiformis result in bone loss as coeliac disease does? A cross sectional study. Rev Esp Enferm Dig 213;15: DOI: /S Shen Y, Cao D, Li Y, et al. Distribution of HLA-A, -B, and C alleles and HLA/KIR combinations in Han population in China. Immunol Res 214;214: DOI: /214/ Lanier LL. NK cell recognition. Annu Rev Immunol 25;23: DOI: /annurev.immunol Husby S, Koletzko S, Korponay-Szabó IR, et al. European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr 212;54: DOI: 1.197/MPG.b13e31821a23d 16. Jamil KM, Khakoo SI. KIR/HLA interactions and pathogen immunity. J Biomed Biotechnol 211;211: Thielens A, Vivier E, Romagné F. NK cell MHC class I specific receptors (KIR): From biology to clinical intervention. Curr Opin Immunol 212;24: DOI: 1.116/j.coi Kuśnierczyk P. Killer cell immunoglobulin-like receptor gene associations with autoimmune and allergic diseases, recurrent spontaneous abortion, and neoplasms. Front Immunol 213;4:8. DOI: / fimmu Blum S, Csurhes P, Reddel S, et al. Killer immunoglobulin-like receptor and their HLA ligands in Guillain-Barré syndrome. J Neuroimmunol 214;267:92-6. DOI: 1.116/j.jneuroim Harris KM, Fasano A, Mann DL. Monocytes differentiated with IL-15 support Th17 and Th1 responses to wheat gliadin: Implications for celiac disease. Clin Immunol 21;135:43-9. DOI: 1.116/j.clim Caggiari L, Toffoli G, De Re V, et al. KIR/HLA combination associated with the risk of complications in celiac disease. Int J Biol Markers 211;26: DOI: 1.531/JBM Santin I, Castellanos-Rubio A, Perez de Nanclares G, et al. Association of KIR2DL5B gene with celiac disease supports the susceptibility locus on 19q13.4. Genes Immun 27;8: DOI: 1.138/sj.gene Moodie SJ, Norman PJ, King AL, et al. Analysis of candidate genes on chromosome 19 in coeliac disease: an association study of the KIR and LILR gene clusters. Eur J Immunogenet 22;29: DOI: 1.146/j x 24. Nash WT, Teoh J, Wei H, et al. Know Thyself: NK-Cell inhibitory receptors prompt self-tolerance, education, and viral control. Front Immunol 214;5:175. DOI: /fimmu Moesta AK, Parham P. Diverse functionality among human NK cell receptors for the C1 epitope of HLA-C: KIR2DS2, KIR2DL2, and KIR2DL3. Front Immunol 212;3:336. Rev Esp Enferm Dig 215; 17 (9):