- Research article
- Open Access
- Open Peer Review
HLA-DQ2 and -DQ8 genotype frequency in Syrian celiac disease children: HLA-DQ relative risks evaluation
BMC Gastroenterologyvolume 18, Article number: 70 (2018)
Celiac disease (CD) is a common autoimmune disease in Syria which manifesting with inflammation of the small intestine and with various extra intestinal symptoms. The disease is associated with human HLA-DQ genes encoding HLA-DQ2 and DQ8 proteins.
In this study, 49 children patients of CD and 58 healthy control samples were genotyped for HLA-DQ genes using SSP-PCR technique. Relative risks for different genotypes were also evaluated.
The DQB1*0201 allele was the most common in the patients (77.6%) followed by DQB1*0302 allele (10.2%). The highest HLA-DQB risk for CD development was found in patients carriers a DQ2.5/DQ8 genotype (1/10), followed by the patients carriers DQ2.5/DQ2.5 (1/12).
The significant differences in the frequency of HLA-DQ2 and HLA-DQ8 in Syrian patients in compared with controls and relative risks predicted demonstrated the importance role of these alleles in the development of CD in Syrian children patients.
Celiac disease is an immune-mediated disorder with a strong genetic predisposition and dietary gluten as an environmental trigger . This disease causes inflammation in the small intestinal mucosa. The developing villus atrophy can impede the absorption of nutrients .
Several ways to diagnosis celiac disease using anti-tissue transglutaminase (anti-TG2) test, anti-endomysium (EMA) auto-antibodies and small bowel biopsy . This disease is widespread in most Mediterranean countries , and it estimated that about more than 5 million patients will be affected in the next 10 years in the Mediterranean region . The only effective therapy is gluten-free diet for life-long, which leads to complete remission of all clinical signs [6, 7]. Celia disease characterized by an interaction of a certain genes, gluten, and environmental influences . The majority of patients with CD carrying the HLA class II genes: HLA-DQ2.5 (DQA1*05-DQB1*02) and HLA-DQ8 (DQA1*03-DQB1*0302) . Strong association was observed between HLA-DQ2.5 and predisposition to CD, explained by its affinity to binding gluten proteins . While, HLA-DQ2.2 contributes less to the risk of CD . Moreover, the risk of CD is shown to be higher in individuals homozygous for the HLA-DQ2.5 or HLA-DQ2.5/DQ2.2 genotypes compared with those homozygous for HLA-DQ2.2 or heterozygous for HLA-DQ2.5 or DQ2.2 [12, 13].
In Syria, previous reports indicated that Syrian population has high prevalence of the CD (1/62) in healthy blood donors [16, 17], but no published study about the link of HLA-DQ alleles and Syrian celiac patients; so, we aim to study the distribution of the CD genes: DQ2 and DQ8 in 49 children patients (age from 1 to 18 years old) in comparable with 58 control samples of the same ages.
Patients and controls
Forty nine CD patients (35 males and 14 females; mean age, 9.5 years; range (1–18) and fifty eight healthy controls (30 males and 28 females; mean age, 29 years; range, (18–40) with no history of CD, cancer or autoimmune diseases and were negative for CD serological screening, were selected from the blood transfusion center of Damascus University in Syria. All patients had positive tTGA and/or EMA antibodies and histology according to Rostami Marsh classification [3, 18].
This study was approved by the Ethics Committee of Atomic Energy Commission of Syria, and written informed consent was obtained from all participants. If participant was a minor, parental consent was taken.
HLA DQ2 and DQ8 genotyping
Peripheral blood was drawn on BD Vacutainer plus Blood Collection tubes K2 EDTA 18.0 mg (BD Biosciences, USA). Genomic DNA was extracted from peripheral blood cells using the salting-out method . HLA-DQB1 genotype was performed by sequence-specific primer-polymerase chain reaction (SSP-PCR) method using published primers  and for HLA-DQA1 genotype .
Data were analyzed using SPSS software (version 22, SPSS Inc., Chicago, Illinois, USA). Odds ratios was applied to establish statistical significance with 95% confidence (P value ≤0.05). Relative risk of developing disease for specific genotypes were calculated as (% of allele in patients / % of allele in controls) x probability to be affected in general population .
In this study, forty nine definite CD patients (25 males and 24 females) and 58 controls of Syrian origin were genotyped for HLA-DQ2 and DQ8 genes. The allele and genotype frequencies of DQ2 and DQ8 genes in CD were compared with the controls. The frequency of the DQB1*02:01 allele was very significantly increased in patients compared with the controls (77.6% vs. 58.6%; p value: 0.00). The frequency of the DQB1*03:02 allele was also elevated in patients versus controls, but uncorrected P value was borderline significant (10.2% vs. 8.6%; p value: 0.09), Table 1).
Genotype analysis of DQ2 and DQ8 genes shows that the 24 of 49 patients carrying DQ2.5 in homozygous genotype with high frequency 49%, whereas, this genotype was found in 6 controls (10.3%). The frequency of the DQ2.5/DQ2.2 or DQ2.5/DQ8 were increased in patients versus controls (10.2% vs. 3.4%), (10.2% vs. 1.7%) respectively. More genotype analysis demonstrated that 30.6% of patients carried another DQ genotypes, while 84.5% of controls had these genotypes (Table 2).
The highest HLA-DQB relative risk for CD development was found in patients carriers DQ2.5/DQ8 genotype (1/10), while, the patients carriers of DQ2.5/DQ2.5 or DQ2.5/DQ2.2 genotype had a relative risk about 1/12.5 and 1/20 respectively (Table 2).
Based on the Marsh Score, forty-two of 49 CD patients with biopsy result were classified into Marsh I, Marsh II and Marsh III as indicated in Table 3.
The frequency of the patients with mild form of CD (Marsh I) carrying DQ2 allele was 9.5%, whereas, the frequency was 23.8% for the patients with Marsh II having DQ2. While, 26 patients from 42 (52.4%) were classified into Marsh III, 22 out of them carrying DQ2 allele, all this results indicated the involvement of DQ2 allele in the severity of mucosal damage (Table 3).
Celiac disease is a systemic immune mediated disorder caused by the intolerance of gluten containing grains in genetically susceptible person . It was found that there is a significant relation between the wheat consumption and the frequency of HLA alleles DQ2 and DQ8 worldwide . The history of CD is backed to the spreading of wheat cultivation after the agricultural revolution.
The pathogenesis of CD is dependent on the presence of HLA class II genes: HLA- DQ2 (DQA1*05-DQB1*02) and HLA-DQ8 (HLA-DQA1*0301-DQB1*0302) which are specific to gluten . In contrast, the absence of either HLA-DQ2 or HLA-DQ8 has a negative predictive value of nearly 100% in excluding the diagnosis of celiac .
This study demonstrated that, among 49 Syrian children patients from the Children’s Hospital, Damascus, Syria there were 77.6% carried the DQB1*0201 allele and 10.2% carrying the DQB1*0302 allele. All patients studied that carried two DQ2 or DQ8 alleles had a common severe symptoms (fatigue, weight loss, stunted growth, iron deficiency anemia and chronic abdominal pain) and they had high value of anti-transglutaminase antibody, and most of them had classified according to the March classification and villous atrophy of variable severity .
Our results indicated a presence of high significant association between DQB1*02;01 allele and CD development, while, it was a borderline significant association with DQB1*03:02 allele. The results also shown that 87.8% of CD patients carried at least one of DQB1*02:01 or DQB1*03:02 alleles. All these results indicated that there were a high statistically significant association between these two alleles and the Syrians CD patients. This data is in concordance with the results obtained for CD patients from several Arab countries regarding HLA-DQ2 variants: in Egyptian (77.42%); Gaza strip patients (84.6%); Jordanian (100%) and Moroccan CD patients (45.2%) [27,28,29,30]. In the other hand, this HLA-DQ2 variants had also high frequented in French (87%), Italian (84%), and in UK (88%) CD patients .
We have evaluated the relative risk for different HLA genotypes (Table 2). The highest HLA-DQ genotype relative risk for CD in our patients was associated with DQ2.5/DQ8 genotype carriers (1/10). Also, carriers of DQ2.5/DQ2.5 genotype were in group with high relative risk (1/12.5). Recent published studies also confirmed that DQ2.5/DQ2.5 genotype conferred the highest risk for CD [32,33,34]. In the other hands, in our group, the patients carried DQ2.5/DQ2.2 or DQ8/DQX or DQ2.5/DQ7 genotypes showed medium risks for CD (1/20 and 1/25) respectively. The lowest risk was detected in patients of DQ2.2/DQ8 or DQ2.2/DQ2.2 genotype carriers. So, patients with a one dose or double dose of HLA-DQ2.5 have high risk for CD, this data is largely consistent with published data about the dose effect for DQB1*02 allele for development of CD .
In our study, the frequency of CD patients with Marsh I and Marsh II carried DQ2 allele was 9.5 and 23.8% respectively, while, it reached 4.8% in the CD patients carried DQ8. In the other hand, Marsh III score was given more than fivefold higher in the CD patients carried DQ2 (52.4%) in compare with CD patients carried DQ8 (9.5%). These results presenting high positive association of DQ2 allele with Marsh III of CD patients. Several previous studies have demonstrated the role of the DQ2 allele in the severity of mucosal damage [35, 36], where, Zamani et al., revealed that 02:01 allele had a significant association with Marsh IIIc patients (pc: 0.02, OR: 14.55) and it represented involvement of this allele in the severity of mucosal damage .
We studied for the first time the distribution of HLA-DQ genotypes in the children celiac patients and we estimated the risk for CD development. The significant differences in the frequency of HLA-DQ2 and HLA-DQ8 alleles in Syrian patients compared with controls demonstrated the importance role of these alleles in the development of CD and support the possibility of using HLA-DQ typing in confirmation of the disease.
Human leukocyte antigen
Single Specific Primer-Polymerase Chain Reaction
Kurppa K, Taavela J, Saavalainen P, Kaukinen K, Lindfors K. Novel diagnostic techniques for celiac disease. Expert Rev Gastroenterol Hepatol. 2016;10:795–805.
Holopainen P. Genetic susceptibility to celiac disease: HLA-unlinked candidate genes: University of Helsinki, Faculty of Science, Department of Biosciences. Helsinki: 2005.
Villanacci V, Ceppa P, Tavani E, Vindigni C, Volta U. Coeliac disease: the histology report. Dig Liver Dis. 2011;43(Suppl 4):S385–95.
Tucci F, Astarita L, Abkari A, Abu-Zekry M, Attard T, Ben Hariz M, Bilbao JR, Boudraa G, Boukthir S, Costa S, et al. Celiac disease in the Mediterranean area. BMC Gastroenterol. 2014;14:24.
Greco L, Timpone L, Abkari A, Abu-Zekry M, Attard T, Bouguerra F, Cullufi P, Kansu A, Micetic-Turk D, Misak Z, et al. Burden of celiac disease in the Mediterranean area. World J Gastroenterol. 2011;17:4971–8.
Schuppan D. Current concepts of celiac disease pathogenesis. Gastroenterology. 2000;119:234–42.
Schuppan D, Junker Y, Barisani D. Celiac disease: from pathogenesis to novel therapies. Gastroenterology. 2009;137:1912–33.
Green PH, Lebwohl B, Greywoode R. Celiac disease. J Allergy Clin Immunol. 2015;135:1099–106. quiz 1107
Sollid LM. The roles of MHC class II genes and post-translational modification in celiac disease. Immunogenetics. 2017;69:605–16.
Romanos J, Wijmenga C. Predicting susceptibility to celiac disease by genetic risk profiling. Ann Gastroenterol Hepatol. 2010;1(1):1–8.
Fallang LE, Bergseng E, Hotta K, Berg-Larsen A, Kim CY, Sollid LM. Differences in the risk of celiac disease associated with HLA-DQ2.5 or HLA-DQ2.2 are related to sustained gluten antigen presentation. Nat Immunol. 2009;10:1096–101.
Vader W, Stepniak D, Kooy Y, Mearin L, Thompson A, van Rood JJ, Spaenij L, Koning F. The HLA-DQ2 gene dose effect in celiac disease is directly related to the magnitude and breadth of gluten-specific T cell responses. Proc Natl Acad Sci U S A. 2003;100:12390–5.
Congia M, Cucca F, Frau F, Lampis R, Melis L, Clemente MG, Cao A, De Virgiliis SA. Gene dosage effect of the DQA1*0501/DQB1*0201 allelic combination influences the clinical heterogeneity of celiac disease. Hum Immunol. 1994;40:138–42.
Husby S, Koletzko S, Korponay-Szabo IR, Mearin ML, Phillips A, Shamir R, Troncone R, Giersiepen K, Branski D, Catassi C, et al. European Society for Pediatric Gastroenterology, hepatology, and nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr. 2012;54:136–60.
Ribes-Koninckx C, Mearin ML, Korponay-Szabo IR, Shamir R, Husby S, Ventura A, Branski D, Catassi C, Koletzko S, Maki M, et al. Coeliac disease diagnosis: ESPGHAN 1990 criteria or need for a change? Results of a questionnaire. J Pediatr Gastroenterol Nutr. 2012;54:15–9.
Cummins AG, Roberts-Thomson IC. Prevalence of celiac disease in the Asia-Pacific region. J Gastroenterol Hepatol. 2009;24:1347–51.
Challar MH, Jouma M, Sitzmann FC, Seferian V, Shahin E. Prevalence of asymptomatic celiac disease in a Syrian population sample. JABMS. 2004;6:155–160E.
Marsh MN. Gluten, major histocompatibility complex, and the small intestine. A molecular and immunobiologic approach to the spectrum of gluten sensitivity (‘celiac sprue’). Gastroenterology. 1992;102:330–54.
Miller SA, Dykes DD, Polesky HFA. Simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.
Bunce M, O'Neill CM, Barnardo MC, Krausa P, Browning MJ, Morris PJ, Welsh KI. Phototyping: comprehensive DNA typing for HLA-A, B, C, DRB1, DRB3, DRB4, DRB5 & DQB1 by PCR with 144 primer mixes utilizing sequence-specific primers (PCR-SSP). Tissue Antigens. 1995;46:355–67.
Scola L, Lio D, Candore G, Forte GI, Crivello A, Colonna-Romano G, Pes MG, Carru C, Ferrucci L, Deiana L, et al. Analysis of HLA-DRB1, DQA1, DQB1 haplotypes in Sardinian centenarians. Exp Gerontol. 2008;43:114–8.
Megiorni F, Mora B, Bonamico M, Barbato M, Nenna R, Maiella G, Lulli P, Mazzilli MC. HLA-DQ and risk gradient for celiac disease. Hum Immunol. 2009;70:55–9.
Leonard MM, Sapone A, Catassi C, Fasano A. Celiac disease and nonceliac gluten sensitivity: a review. JAMA. 2017;318:647–56.
Lionetti E, Gatti S, Pulvirenti A, Catassi C. Celiac disease from a global perspective. Best Pract Res Clin Gastroenterol. 2015;29:365–79.
Megiorni F, Pizzuti A. HLA-DQA1 and HLA-DQB1 in celiac disease predisposition: practical implications of the HLA molecular typing. J Biomed Sci. 2012;19:88.
Malamut G, Cellier C. Complications of coeliac disease. Best Pract Res Clin Gastroenterol. 2015;29:451–8.
Mohammed MA, Omar NM, Shebl AM, Mansour AH, Elmasry E, Othman G. Celiac disease prevalence and its HLA-genotypic profile in Egyptian patients with type 1 diabetes mellitus. Trends in Medical Research. 2014;9:81–97.
Ayesh BM, Zaqout EK, Yassin MM. HLA-DQ2 and -DQ8 haplotypes frequency and diagnostic utility in celiac disease patients of Gaza strip, Palestine. Auto Immun Highlights. 2017;8:11.
El-Akawi ZJ, Al-Hattab DM, Migdady MA. Frequency of HLA-DQA1*0501 and DQB1*0201 alleles in patients with coeliac disease, their first-degree relatives and controls in Jordan. Ann Trop Paediatr. 2010;30:305–9.
Piancatelli D, Ben El Barhdadi I, Oumhani K, Sebastiani P, Colanardi A, Essaid A. HLA typing and celiac disease in Moroccans. Med Sci (Basel). 2017;5:1–11.
Margaritte-Jeannin P, Babron MC, Bourgey M, Louka AS, Clot F, Percopo S, Coto I, Hugot JP, Ascher H, Sollid LM, et al. HLA-DQ relative risks for coeliac disease in European populations: a study of the European genetics cluster on coeliac disease. Tissue Antigens. 2004;63:562–7.
Liu E, Lee HS, Aronsson CA, Hagopian WA, Koletzko S, Rewers MJ, Eisenbarth GS, Bingley PJ, Bonifacio E, Simell V, et al. Risk of pediatric celiac disease according to HLA haplotype and country. N Engl J Med. 2014;371:42–9.
Stankovic B, Radlovic N, Lekovic Z, Ristic D, Radlovic V, Nikcevic G, Kotur N, Vucicevic K, Kostic T, Pavlovic S, Zukic B. HLA genotyping in pediatric celiac disease patients. Bosn J Basic Med Sci. 2014;14:171–6.
Zamani M, Modares-Sadegi M, Shirvani F, Zamani H, Emami MH. The involvement of the HLA-DQB1 alleles in the risk and the severity of Iranian coeliac disease patients. Int J Immunogenet. 2014;41:312–7.
Murray JA, Moore SB, Van Dyke CT, Lahr BD, Dierkhising RA, Zinsmeister AR, Melton LJ 3rd, Kroning CM, El-Yousseff M, Czaja AJ. HLA DQ gene dosage and risk and severity of celiac disease. Clin Gastroenterol Hepatol. 2007;5:1406–12.
Hernandez-Charro B, Donat E, Miner I, Aranburu E, Sanchez-Valverde F, Ramos-Arroyo MA. Modifying effect of HLA haplotypes located trans to DQB1*02-DRB1*03 in celiac patients of southern Europe. Tissue Antigens. 2008;71:213–8.
We would like to thank Prof. Ibrahim Othman, the Director General of AECS, and the head of Molecular Biology & Biotechnology department, for their support. And we would like also to thanks Dr. Mahmoud Bozo and Dr. Jamal Jaradat for this helping in patients sample collection.
Availability of data and materials
The datasets during and/or analyzed during the current study available from the corresponding author on reasonable request.
Ethics approval and consent to participate
The study was reviewed and approved by the Ethics Committee of Atomic Energy Commission of Syria. Written informed consent was obtained from all participants. If participant was a minor, parental consent was taken.
Consent for publication
Written consent for publication was obtained from all the participants.
The authors declare that they have no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.