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A rapid and sensitive assay to identify HLA-DQ2/8 risk alleles for celiac disease using real-time PCR method

Kazem Mashayekhi, Mohammad Rostami Nejad, Davar Amani, Mostafa Rezaei-Tavirani, Hamid Mohaghegh Shalmani, Mohammad Reza Zali




Aim: To perform a simple, rapid and sensitive Real-time PCR based SYBR Green method to determine the human leukocyte antigen (HLA)-DQ 2/8 alleles in celiac disease (CD) patients.

Background: Many molecular techniques are available to determine the HLA-DQ2 and DQ8 alleles, but they are too expensive and have many steps that make them difficult to use.

Methods: To determine the HLA-DQ 2/8 alleles we have developed a new real-time PCR assay, using SYBR Green technique with melting curve analysis on genomic DNA isolated from 75 CD patients and 94 healthy controls. The specific primers to examine HLA-DQA1*05, HLA-DQB1*02 and HLA-DQB1*0302 alleles were used and results were compared with commercially available kits.

Results: Using this method, the presence of HLA-DQ2 and HLA-DQ8 alleles were determined with sensitivity and specificity 80% and 100% respectively and compared to low resolution commercially available kits, the results of this method were more efficient. The frequency of DQ2 and DQ8 in patients was 76% and 29%, respectively and overall 96% of patients were carries DQ2 and/or DQ8 alleles.

Conclusion: The result of this study showed that Real-time PCR using SYBR Green method with melting curve analysis has good efficiency to identify the HLA-DQ2/8 risk alleles.

Keywords: Celiac disease, Real-time PCR, Melting curve analysis, HLA-DQ2/8 alleles, HLA typing.

(Please cite as: Mashayekhi K, Rostami-Nejad M, Amani D, Rezaei-Tavirani M, Mohaghegh-Shalmani H, Zali MR. A rapid and sensitive assay to identify HLA-DQ2/8 risk alleles for celiac disease using real-time PCR method. Gastroenterol Hepatol Bed Bench 2018;11(3):250-258).


Celiac disease; Real-time PCR; Melting curve analysis; HLA-DQ2/8 alleles; HLA typing.


Meresse, B., G. Malamut, and N. Cerf-Bensussan, Celiac disease: an immunological jigsaw. Immunity, 2012. 36(6): p. 907-19.

Sollid, L.M., Coeliac disease: dissecting a complex inflammatory disorder. Nat Rev Immunol, 2002. 2(9): p. 647-55.

Dube, C., et al., The prevalence of celiac disease in average-risk and at-risk Western European populations: a systematic review. Gastroenterology, 2005. 128(4 Suppl 1): p. S57-67.

Fasano, A., et al., Prevalence of celiac disease in at-risk and not-at-risk groups in the United States: a large multicenter study. Arch Intern Med, 2003. 163(3): p. 286-92.

Maki, M., et al., Prevalence of Celiac disease among children in Finland. N Engl J Med, 2003. 348(25): p. 2517-24.

Rostami-Nejad, M., et al., Allele and haplotype frequencies for HLA-DQ in Iranian celiac disease patients. World J Gastroenterol, 2014. 20(20): p. 6302-8.

Mashayekhi, K., K. Koushki, and M. Rostami-Nejad, The correlation between HLA-DQ2 and DQ8 haplotypes and abnormal histology in patients with celiac disease. IJAPBS, 2015. 4(2): p. 152-156.

Zamani, M., et al., The involvement of the HLA-DQB1 alleles in the risk and the severity of Iranian coeliac disease patients. Int J Immunogenet, 2014. 41(4): p. 312-7.

Sollid, L.M., et al., Evidence for a primary association of celiac disease to a particular HLA-DQ alpha/beta heterodimer. J Exp Med, 1989. 169(1): p. 345-50.

Aggarwal, S., B. Lebwohl, and P.H. Green, Screening for celiac disease in average-risk and high-risk populations. Therap Adv Gastroenterol, 2012. 5(1): p. 37-47.

Clouzeau-Girard, H., et al., HLA-DQ genotyping combined with serological markers for the diagnosis of celiac disease: is intestinal biopsy still mandatory? J Pediatr Gastroenterol Nutr, 2011. 52(6): p. 729-33.

Hadithi, M., et al., Accuracy of serologic tests and HLA-DQ typing for diagnosing celiac disease. Ann Intern Med, 2007. 147(5): p. 294-302.

Megiorni, F., et al., A rapid and sensitive method to detect specific human lymphocyte antigen (HLA) class II alleles associated with celiac disease. Clin Chem Lab Med, 2008. 46(2): p. 193-6.

Murch, S., et al., Joint BSPGHAN and Coeliac UK guidelines for the diagnosis and management of coeliac disease in children. Arch Dis Child, 2013. 98(10): p. 806-11.

Zamani, F., et al., Prevalence of celiac disease among patients with Behcet's disease in Iran. Dig Dis Sci, 2009. 54(8): p. 1736-9.

Marsh, M.N., M.W. Johnson, and K. Rostami, Mucosal histopathology in celiac disease: a rebuttal of Oberhuber's sub-division of Marsh III. Gastroenterol Hepatol Bed Bench, 2015. 8(2): p. 99-109.

Husby, S., et al., European Society for Pediatric Gastroenterology, Hepatology, and Nutrition guidelines for the diagnosis of coeliac disease. J Pediatr Gastroenterol Nutr, 2012. 54(1): p. 136-60.

Sollid, L.M. and B.A. Lie, Celiac disease genetics: current concepts and practical applications. Clin Gastroenterol Hepatol, 2005. 3(9): p. 843-51.

Otten, H.G., et al., Serology versus PCR-SSP in typing for HLA-DR and HLA-DQ: a practical evaluation. Tissue Antigens, 1995. 45(1): p. 36-40.

Michalski, J.P., et al., HLA-DR, DQ genotypes of celiac disease patients and healthy subjects from the West of Ireland. Tissue Antigens, 1996. 47(2): p. 127-33.

Olerup, O., A. Aldener, and A. Fogdell, HLA-DQB1 and -DQA1 typing by PCR amplification with sequence-specific primers (PCR-SSP) in 2 hours. Tissue Antigens, 1993. 41(3): p. 119-34.

Monsuur, A.J., et al., Effective detection of human leukocyte antigen risk alleles in celiac disease using tag single nucleotide polymorphisms. PLoS One, 2008. 3(5): p. e2270.

Voorter, C.E., M.C. Kik, and E.M. van den Berg-Loonen, High-resolution HLA typing for the DQB1 gene by sequence-based typing. Tissue Antigens, 1998. 51(1): p. 80-7.

Saiki, R.K., et al., Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature, 1986. 324(6093): p. 163-6.

Arguello, J.R., et al., Mutation detection and typing of polymorphic loci through double-strand conformation analysis. Nat Genet, 1998. 18(2): p. 192-4.

Bunce, M., et al., 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(5): p. 355-67.

Profaizer, T., D. Eckels, and J.C. Delgado, Celiac disease and HLA typing using real-time PCR with melting curve analysis. Tissue Antigens, 2011. 78(1): p. 31-7.

Amirzargar, A., et al., Human leukocyte antigen class II allele frequencies and haplotype association in Iranian normal population. Hum Immunol, 2001. 62(11): p. 1234-8.

Bahari, A., et al., HLA-DQ2 and HLA-DQ8 Genotyping in a Sample of Iranian Celiac Patients and Their First-Degree Relatives. Govaresh, 2014. 18(4): p. 242-245.

Farjadian, S. and A. Ghaderi, HLA class II similarities in Iranian Kurds and Azeris. Int J Immunogenet, 2007. 34(6): p. 457-63.

Farjadian, S., F.A. Moqadam, and A. Ghaderi, HLA class II gene polymorphism in Parsees and Zoroastrians of Iran. Int J Immunogenet, 2006. 33(3): p. 185-91.

Farjadian, S., et al., Molecular analysis of HLA allele frequencies and haplotypes in Baloch of Iran compared with related populations of Pakistan. Tissue Antigens, 2004. 64(5): p. 581-7.

DOI: https://doi.org/10.22037/ghfbb.v0i0.1307