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Amaç: Çölyak hastalığı yaygın, ailesel, otoimmün bir gastrointestinal hastalıktır. Bu durum, buğday, çavdar ve arpada bulunan diyetteki protein glutene karşı hassasiyetten kaynaklanır. Çölyak hastalığı insan lökosit antijenleriyle (HLA) güçlü bir genetik ilişkiye sahiptir. Çölyak hastalığı için birincil duyarlılık genotipi HLA-DQA1*05 ve DQB1*02'den oluşan HLA-DQ2'dir, vakaların geri kalan kısmı HLA-DQA1*03 ve DQB1*03:02'den oluşan HLA-DQ8 ile ilişkilidir. Bu çalışmanın amacı, Malatya ilinde çölyak ön tanısı almış çocuk hastalarda DQ2 ve DQ8 genotiplerinin sıklığını araştırmaktır. Gereç ve Yöntem: 578 bireyden (338 kadın/240 erkek) DNA örnekleri HLA-DQA1 ve DQB1 için SSP-PCR yöntemine göre genotiplenmiştir. Bulgular: 578 olgunun 304'ünde (% 52.59) HLA-DQ2, 86'sında (% 14.87) HLA-DQ8, 28'inde (% 4.84) HLA-DQ2 /DQ8 tespit edilmiştir ve 160 hastanın ise (% 27.68) DQ2 veya DQ8 genotiplerinden herhangi birine sahip olmadığı görülmüştür. En sık rastlanan alleller DQA1*05 (% 76.64) ve DQB1*02 (% 61.41) olarak belirlenmiştir. Sonuç: Sonuç olarak; çalışmamızdaki DQ2/DQ8 genotiplerinin sıklıklarının, literatür ile benzer olduğu tespit edilmiştir.

HLA-linked genes are relevant to celiac disease (CD), the potential genetic differences present worldwide are not fully understood. Previous results suggest that the distribution of HLA-DQ2/DQ7/DQ8 in Chile may differ from that in Europe and North America. In celiac patients and their first-degree relatives (FDRS), we assessed their clinical, serological and histological characteristics, determined HLA-DQ2, HLA-DQ7 and HLA-DQ8 alleles and genotypes, and evaluated the relations between them. A total of 222 individuals were assessed (56 cases, 166 FDRs). 16.9% of FDRs were tTG positive, 53.6% of them showed overweight/obesity and 3% undernourishment, they spontaneously declared being asymptomatic, but detailed questioning revealed that 60.7% experienced symptoms, which had not been investigated. DQ2 was present in 53.9% and 43.9.0% of cases and FDRs (p &lt, 0.05). The most frequent genotype distribution was DQ2/DQ7 (fr 0.392 (cases) and 0.248 (FDRs), respectively, p &lt, 0.02). The next most common genotypes were HLA-DQ2/DQ8 (fr 0.236 in FDRs and 0.176 in cases, p &lt, 0.05). 3.92% cases were not HLA-DQ2/DQ8 carriers. Among tTG positive FDRs, 57.4%, 22.3% and 20.2% carried DQ2, DQ7 and DQ8, respectively. In cases, 72.7% of the biopsies classified Marsh ≥3 carried at least one DQ2, 91.7% of DQ2/DQ2 and 88.3% of DQ2/DQ7 were Marsh ≥3. Thus, DQ2 frequency is lower than reported, the higher frequency found for DQ8 and DQ7 concur with recent publications from Argentine and Brazil. These results suggest that although CD may manifest clinically in ways similar to those described in other populations, some genetic peculiarities in this region deserve further study.

Celiac disease is an autoimmune disease, which is characterised by intolerance of gliadin creating a part of gluten. Its intolerance leads to a chronic inflammatory response in the mucous membrane of small intestine, followed by malabsorption characterized by chronic diarrhoea, greasy stool and failure to thrive. Celiac disease is a multifactorial disease, caused by external environment factors and genetic factors. Genetic predisposition is given by HLA alleles HLA-DQ2 (DQA1*05/DQB1*02) or DQ8 (DQA1*0301/DQB1*0302) or HLA-DRB1*04. This disease has got a set of unspecified clinical symptoms, that is why most of the patients are not diagnosed at all. Estimated count of the ill people is up to 50 000, however real patients are only 15 % of them. The aim of the theoretical part of the bachelor thesis was to sum up the findings about the celiac disease and molecular genetic methods based on PCR. In the theoretical part about the celiac disease, I deal mainly with the description, types, diagnostics, pathogenesis and also with the diseases associated with celiac disease. Moreover, I dealt with the description and the nomenclature of HLA system and its association with the diseases. In the end of the theoretical part, I dealt with molecular genetic methods based on PCR (PCR-SSP and RT-PCR). I carried out the practical part of the thesis in a genetic laboratory at the Faculty of Health and Social Sciences, at the Faculty of Agriculture and at GENLABS, s.r.o. in Budweis. I dealt with the HLA typisation of alleles associated with the celiac disease. I did the examination by means of two methods - PCR-SSP method with the help of HISTO TYPE Celiac Disease kit and RT-PCR method with the help of EliGene? Coeliac RT kit (DQ2, DQ8, DRB4). The aim of the work was to embrace correct laboratory practice in a genetic laboratory. Then also set and optimize a commercially sold kit for an examination of HLA alleles associated with the celiac disease on the PCR-SSP principle. Examine the already examined patients by means of RT-PCR method. Compare both methods on the terms of accordance and difficulty.

Purpose Celiac disease (CD) diagnosis can be established by serological and small bowel biopsy (SBB), while absence of HLA-DQ2 and -DQ8 haplotypes excludes the disease. The present study aims at evaluating the diagnosis of a representative sample of pediatric and adult CD patients of Gaza strip in light of DQ2 and DQ8 haplotypes expression. Methods Unrelated CD patients (n = 101) and matched healthy controls (n = 97) were genotyped for DQA1*05, DQB1*02 and DQB1*03:02 alleles by allele-specific real-time PCR. The diagnosis was re-evaluated according to the patient laboratory tests and HLA-DQ genotype. Results The diagnosis of 35 patients who have been managed for CD could not be confirmed. Twenty-five of them were diagnosed upon their clinical presentation only. The remaining were either negative for serological and SBB tests or negative for HLA-DQ haplotypes. The HLA-DQ alleles were negative in 4 SBB and one Anti-EMA positive patients. The frequency of DQ2 and DQ8 haplotypes among the remaining 65 confirmed cases was 70.8 and 15.4%, respectively, compared to 17.5 and 27.8% in the controls. The DQB1*02 allele was the most common in the cases (84.6%) followed by DQA1*05 allele (80%) and DQB1*03:02 allele (20%). The DQA1*05 allele was commonest in the control group (54.6%) followed by DQB1*02 allele (42.3%) and DQB1*03:02 allele (28.9%). Conclusions Absence of HLA-DQ2 and HLA-DQ8 genotyping in the workup of patients may result in CD misdiagnosis, particularly in a setting with poor histopathological diagnostic capacity.

Background: no systematic studies on the prevalence of coeliac disease (CD) have been reported from China. In western populations CD is more common in patients with insulin depen dent diabetes mellitus (IDDM) and in diarrhoea-predominant irrita ble bowel syndrome (D-IBS). We have screened patients with these conditions presenting to the outpatient department of a large hospital of “Traditional Chinese Medicine” (TCM) in Nan jing, Jiangsu province, P.R. China. Methods: we tested sera of 78 unrelated Han Chinese pa tients (5 IDDM and 73 D-IBS), using ELISA serological tests for IgG anti-gliadin antibodies (IgG-AGA) and IgA anti-tissue transglu taminase antibodies (IgA-tTG). Results: six out of 78 patients (7.7%) were positive for IgGAGA (two men and four women) and two (2.6%) were positive for IgA-tTGs. One of the latter patients was negative for IgG-AGA. Besides, one patient had a dubious IgA-tTG antibody and a posi tive IgG-AGA. None of the six patients agreed to undergo duode nal biopsy. Two out of these six patients followed a gluten-free diet for one year. In one patient the diarrhoea ceased and his body weight increased. Another stopped losing weight. Conclusions: this study previously published as a letter in GUT (Wu J, Xia B, von Blomberg BME, Zhao C, Yang XW, Cru sius JBA, Pena AS. Coeliac disease: emerging in China? Gut 2010; 59(3): 418-9) demonstrated that CD may exist in the Jiangsu province of P.R. China. The present article draws atten tion to the difficulties of following a standard protocol in China such as established in western countries and highlights important factors less well known in the west in relation to the development of CD in China. Wheat production became significant in China between 1600 and 1300 B.C. After the Han dynasty (500-200 B.C.), wheat was one of the main cereals in China. One the major wheat fields in China is located in the Jiangsu province where the research for this article was performed. A review of Chinese liter ature shows that the predominant HLA-DQ CD risk alleles and haplotypes are present in the Jiangsu province. Genetic back ground, food consumption, and the results of our study suggest that CD should actively be investigated in P.R. China.

Cilj: Utvrditi učestalost alela i genotipova HLA-DQA1 i HLA-DQB1 gena u oboljelih od celijakije, kao i njihov utjecaj na kliničku ekspresiju bolesti. Ispitanici i metode: U ispitivanje je uključeno 110 pacijenata (60 žena i 50 muškaraca) s dijagnosticiranom celijakijom prema revidiranim ESPGHAN kriterijima (The European Society for Pediatric Gastroenterology Hepatology and Nutrition) koji su klinički obrađeni na Klinici za internu medicinu i Klinici za pedijatriju KBC-a Rijeka. Genotipizacija HLA alela klase II u pacijenata izvršena je metodom lančane reakcije polimerazom (PCR, engl. Polymerase Chain Reaction) s alel specifičnim početnicama (engl. Sequence Specific Primer – SSP), pri čemu su korišteni komercijalni kitovi niske i srednje rezolucije GenoVision SSP. Rezultati: Svi pacijenti imali su barem jedan rizični HLA-DQA1 ili HLA–DQB1 alel. Homozigota za rizične HLA-DQB1 alele bilo je 36,3 %, a za HLA-DQA1 34,5 %. Genotipovi za HLA-DQ2 i/ili DQ8 heterodimere prisutni su u 92,7 % pacijenata. HLA-DQ2 prisutan je u 79,1 % pacijenata, a HLA-DQ8 u 20,9 %. Nije utvrđena značajna razlika (P < 0,05) u učestalosti nosioca HLA-DQ2 i/ili DQ8 ovisno o tipu celijakije. Opažen je trend ranijeg postavljanja dijagnoze kao i veća pojavnost drugih autoimunosnih bolesti u homozigota u odnosu na ostale genotipove. Zaključci: Dobiveni rezultati ukazuju da je prisutnost HLA-DQ2/DQ8 heterodimera u hrvatskih pacijenata s celijakijom sukladna postojećem gradijentu sjever-jug u europskim populacijama. HLA genotipizacija pacijenata ima nesumnjiv značaj u kliničkoj praksi pri postavljanju dijagnoze., Objective: To determine the frequency of alleles and genotypes of HLA-DQA1 and HLA-DQB1 genes in patients with celiac disease and their impact on the clinical expression of the disease. Subjects and Methods: The study involved 110 patients (60 women and 50 men) with celiac disease diagnosed according to the revised ESPGHAN (The European Society for Pediatric Gastroenterology Hepatology and Nutrition) criteria at the Clinic of Internal Medicine and Pediatric Clinic, University Hospital Centre Rijeka. The genotyping of HLA class II alleles was performed by polymerase chain reaction method (PCR) with allele-specific primers (Sequence Specific Primer-SSA) using commercial kits of low and medium resolution GenoVision SSP. Results: All patients had at least one risk HLA-DQA1 or HLA-DQB1 allele. Homozygotes for the HLA-DQB1 alleles were 36.3 %, and for HLA-DQA1 34.5 %. Genotypes for the HLA-DQ2 and/or HLA-DQ8 heterodimers were present in 92.7 % of patients. HLA-DQ2 was present in 79.1 % of patients while HLA-DQ8 in 20.9 %. There are no significant difference (P > 0.05) in the frequency of the HLA-DQ2 and/or HLA-DQ8 carriers depending on the type of celiac disease. An earlier diagnosis and greater incidence of other autoimmune diseases in homozygotes, compared to other genotypes, was observed. Conclusions: The results have shown that the presence of HLA-DQ2/DQ8 heterodimers in Croatian celiac patients is in range with the existing north-south gradient in European populations. HLA genotyping in celiac patients has explicit effects in clinical practice when setting up the diagnosis.

Aims/hypothesisThe aim of our study was to test the hypothesis that HLA genotypes conferring risk of diabetes, cord blood autoantibodies, or both are associated with increased birthweight. Methods: HLA genotypes were determined in dried blood spots of cord blood from a total of 16,709 children born to healthy mothers in the Diabetes Prediction in Skane (DiPiS) study, a population-based observational clinical investigation of newborn children. Children born to mothers with diabetes or gestational diabetes were excluded. Autoantibodies to glutamic acid decarboxylase (GAD65Ab) and insulinoma-associated protein 2 were determined in standard radioligand binding assays. Birthweight was adjusted for gestational age and divided into quartiles. The upper quartile was defined as high relative birthweight (HrBW) and the lower quartile as low relative birthweight (LrBW). Results: Genotypes conferring risk of type 1 diabetes were strongly associated with relative birthweight (rBW) (p=0.01). The high-risk HLA-DQ2/8, DQ8/0604 and DQ8/X genotypes were associated with HrBW (odds ratio [OR] [95% CI]=1.20 [1.08-1.33], p=0.0006). The HLA-DQB1*0603 allele, which is negatively associated with type 1 diabetes, was also associated with HrBW (p=0.025), confirming a previous report on DQB1*0603-linked HLA-DR13. GAD65Ab were negatively associated with HrBW (OR [95% CI]=0.72 [0.56-0.93], p=0.01). Regression analysis showed that the HLA-associated increase in rBW was independent of confounding factors. Conclusions/Interpreation: HLA genotypes may be associated with intrauterine growth independent of type 1 diabetes risk. The epidemiological observation that high birthweight is a risk factor for type 1 diabetes could possibly result from a moderating effect on intrauterine growth of HLA genotypes conferring a high risk of diabetes.

Celiac disease is caused by an abnormal intestinal T-cell response to gluten proteins of wheat, barley and rye. Over the last few years, a number of gluten T-cell epitopes restricted by celiac disease associated HLA-DQ molecules have been characterized. In this work, we give an overview of these epitopes and suggest a comprehensive, new nomenclature.

The basis of proper recognition of pathogens and tumours is provided by adaptive immunity. This immunological reaction of the recognition function of T-cell receptors on T lymphocytes detects antigenic peptides bound to major histocompatibility complex (MHC) molecules. Structural insight into this process has few grown considerably in the last years. In some of the cases, antigens are self-protein fragments causing autoimmunity diseases. Type 1 diabetes is such a disease connected with the human leukocyte antigen-DQ8 molecule, a class II MHC glycoprotein. Its crystal structure, complexed with LVEALYLVCGERGG peptide (insulin B peptide), has been solved, and important information about the significance of P1, P4 and P9 binding pockets has been discovered. The complex structure also revealed an unusual large number of intermolecular hydrogen bonds between insulin B peptide and MHC molecule. To further investigate the dynamics of peptide/MHC interactions, we perform molecular dynamic simulations in explicit water. Analysis of the results provided useful information of the binding of the peptide antigen to MHC molecule, which is supported by numerous hydrogen bonds besides the electrostatic (P1 and P9 pockets) or hydrophobic interactions (P4). Results also allowed some implications to be drawn for the role of residues located outside of the binding groove. Molecular Simulation

Background: no systematic studies on the prevalence of coeliac disease (CD) have been reported from China. In western populations CD is more common in patients with insulin dependent diabetes mellitus (IDDM) and in diarrhoea-predominant irritable bowel syndrome (D-IBS). We have screened patients with these conditions presenting to the outpatient department of a large hospital of "Traditional Chinese Medicine" (TCM) in Nanjing, Jiangsu province, P.R. China. Methods: we tested sera of 78 unrelated Han Chinese patients (5 IDDM and 73 D-IBS), using ELISA serological tests for IgG anti-gliadin antibodies (IgG-AGA) and IgA anti-tissue transglutaminase antibodies (IgA-tTG). Results: six out of 78 patients (7.7%) were positive for IgG-AGA (two men and four women) and two (2.6%) were positive for IgA-tTGs. One of the latter patients was negative for IgG-AGA. Besides, one patient had a dubious IgA-tTG antibody and a positive IgG-AGA. None of the six patients agreed to undergo duodenal biopsy. Two out of these six patients followed a gluten-free diet for one year. In one patient the diarrhoea ceased and his body weight increased. Another stopped losing weight. Conclusions: this study previously published as a letter in GUT (Wu J, Xia B, von Blomberg BME, Zhao C, Yang XW, Crusius JBA, Peña AS. Coeliac disease: emerging in China? Gut 2010; 59(3): 418-9) demonstrated that CD may exist in the Jiangsu province of P.R. China. The present article draws attention to the difficulties of following a standard protocol in China such as established in western countries and highlights important factors less well known in the west in relation to the development of CD in China. Wheat production became significant in China between 1600 and 1300 B.C. After the Han dynasty (500-200 B.C.), wheat was one of the main cereals in China. One the major wheat fields in China is located in the Jiangsu province where the research for this article was performed. A review of Chinese literature shows that the predominant HLA-DQ CD risk alleles and haplotypes are present in the Jiangsu province. Genetic background, food consumption, and the results of our study suggest that CD should actively be investigated in P.R. China.

Die vorliegende Arbeit untersucht die Ausprägung der humoralen Immunreaktion bei Typ 1 Diabetespatienten mit Zöliakie. Dabei wurde die Verteilung von IgA und IgG Anti-tTG-AK im Kontext Typ 1 Diabetes fokussiert. Dabei soll geprüft werden, ob die Ergebnisse eventuell Rückschlüsse auf das immunologische Geschehen ermöglichen und ob daraus eine Verbesserung der Diagnostik zu gewinnen ist.

The goal was to identify HLA-DQ8-bound beta cell epitopes important in the T cell response in autoimmune diabetes. We first identified HLA-DQ8 (DQA1*0301/DQB1*0302) beta cell epitopes using a computational approach and then related their identification to CD4 T cell responses. The computational program (TEA-DQ8) was adapted from one previously developed for identifying peptides bound to the I-A(g7) molecule and based on a library of naturally processed peptides bound to HLA-DQ8 molecules of antigen-presenting cells. We then examined experimentally the response of NOD.DQ8 mice immunized with peptides derived from the Zinc transporter 8 protein. Log-of-odds scores on peptides were experimentally validated as an indicator of peptide binding to HLA-DQ8 molecules. We also examined previously published data on diabetic autoantigens, including glutamic acid decarboxylase-65, insulin and insulinoma-associated antigen-2, all tested in NOD.DQ8 transgenic mice. In all examples, many peptides identified with a favorable binding motif generated an autoimmune T cell response, but importantly many did not. Moreover, some peptides with weak-binding motifs were immunogenic. These results indicate the benefits and limitations in predicting autoimmune T cell responses strictly from MHC-binding data. TEA-DQ8 performed significantly better than other prediction programs.

BACKGROUND: Studies on intestinal T cell clones from the mucosa of patients with coeliac disease have led to the identification of immunogenic gliadin epitopes. One is HLA-DQ8 restricted, its recognition by T cells being increased by introduction of negatively charged residues operated by tissue transglutaminase. AIM: To test HLA-DQ8 restricted epitope in both native (QYPSGQGSFQPSQQNPQA) and deamidated (QYPSGEGSFQPSQENPQA) forms in an organ culture system of treated coeliac mucosa from HLA-DQ8 positive and HLA-DQ8 negative patients. PATIENTS AND METHODS: Jejunal biopsies obtained from 10 patients with coeliac disease (six HLA-DQ8 positive and four HLA-DQ8 negative) were cultured in vitro with a peptic-tryptic digest (PT) of gliadin, or with the native (peptide A) or deamidated (peptide B) peptide. Intraepithelial CD3(+) and lamina propria total CD25(+) and CD3(+)CD25(+) cells were counted, lamina propria intercellular adhesion molecule 1 (ICAM-1) expression was evaluated, as well as that of Fas molecules on epithelial cells. RESULTS: In HLA-DQ8 positive, but not in HLA-DQ8 negative, coeliacs the density of intraepithelial CD3(+) cells, lamina propria total CD25(+), and CD3(+)CD25(+) cells, as well as expression of ICAM-1 and Fas molecules were significantly increased in biopsies cultured with PT, peptide A, or peptide B compared with biopsies cultured in medium alone. CONCLUSION: These data show that the DQ8 restricted gliadin peptide is immunogenic only in the intestinal mucosa of HLA-DQ8 positive coeliac patients in both native and deamidated forms.