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 Akar¹, Turkan Patiroglu¹, Eylem Sevinc², Duran Aslan², Deniz Okdemir³ and Selim Kurtoglu³

¹ Departments of Pediatric Immunology,
² Pediatric Gastroenterology,
³ Pediatric Endocrinology. Erciyes University. Medical Faculty. Kayseri, Turkey

Correspondence

 

 

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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), 20 patients with CD+DM (n = 20), and 60 healthy controls (n = 60) 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-Thr- and 3DS1+HLA-BBw4-Iso- (p < 0.001, p < 0.001, p < 0.001, and p < 0.001, 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 = 0.002, p = 0.004, p = 0.036, p < 0.001, and p = 0.007, 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.

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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 0.5-2% 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_H2 and T_H17-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 30% 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 coexisting 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), 20 patients with CD+DM (n = 20, aged 7-16 years), and 60 healthy unrelated Turkish subjects (n = 60, 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-CwAsparagin80 (HLA-C1), HLA-CwLysine80 (HLA-C2), HLA-BBw4-Threonine80 (HLA-BBw4-Thr), HLA-BBw4-Isoleucine80 (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, 20 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 0.05 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 = 0.046, OR: 2.35, 95% CI: 1.064-5.208), (p = 0.031, OR: 2.56, 95% CI: 1.161-5.642), (p = 0.003, OR: 3.55, 95% CI: 1.562-8.083), (p < 0.001, OR: 4.13, 95% CI: 1.899-9.805), (p = 0.001, OR: 4.28, 95% CI: 1.878-9.746), respectively] were less common, the 2DS5 and 3DS1 KIR genes [(p < 0.001, OR: 0.08, 95% CI: 0.017-0.356), (p < 0.001, OR: 2.15, 95% CI: 1.714-2.697), respectively] were more frequent (Table I). In the coexisting CD and DM group, although the 2DS2 gene (p = 0.042, OR: 3.04, 95% CI: 1.052-8.780) was less common, the 2DS5 and 3DS1 KIR genes [(p = 0.002, OR: 1.53, 95% CI: 1.266-1.840) and (p = 0.004, OR: 1.50, 95% CI: 1.254-1.794), 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 < 0.001, 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 = 0.010, OR = 3.46, 95% CI: 1.328-9.029) (Table IV). In the coexisting CD and DM group, the frequency of the HLA-C1 ligand was higher than in the controls (p = 0.036, OR = 1.44, 95% CI: 1.224-1.682) (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 [(p = 0.046, OR: 2.35, 95% CI: 1.064-5.208), (p = 0.022, OR: 3.33, 95% CI: 1.211-9.171), (p = 0.031, OR: 2.56, 95% CI:1.161-5.642), and (p = 0.018, OR: 0.54, 95% CI: 0.446-0.643), respectively] were less common in CD group than controls. Also, while the activating combinations of 2DS2+HLA-C1+ and 3DS1+HLA-BBw4-Iso+ [(p = 0.031, OR: 2.56, 95 % CI: 1.161-5.642), (p = 0.010, OR: 3.46, 95% CI: 1.328-9.029), respectively] were less common, 3DS1+HLA-BBw4-Thr- and 3DS1+HLA-BBw4-Iso- [(p < 0.001, OR: 0.12, 95% CI: 0.052-0.300) and (p < 0.001, OR: 0.07 95% CI: 0.027-0.189), 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+HLA-BBw4-Iso- [(p < 0.001, OR: 0.13, 95% CI: 0.041-0.419) and (p = 0.007, OR: 0.21, 95% CI: 0.073-0.625)] 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+HLA-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+HLA-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 frequent 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 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+HLA-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+HLA-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-Iso- and 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.

 

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Correspondence:
H. Haluk Akar.
Department of Pediatric Immunology.
Erciyes University.
Medical Faculty. Kayseri, Turkey
e-mail: himmetakar@gmail.com

Received: 21-04-2015
Accepted: 13-05-2015