Clinical Chemistry and Laboratory Medicine (CCLM)
Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)
Editor-in-Chief: Plebani, Mario
Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.
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A multiplex assay to rapidly exclude HLA-DQ2.5 and HLA-DQ8 expression in patients at risk for celiac disease
1Department of Clinical Chemistry, Hematology and Immunology, Diakonessenhuis, Utrecht, The Netherlands
2MRC-Holland B.V., Amsterdam, The Netherlands
3Department of Pathology, VU University Medical Center, Amsterdam, The Netherlands
4Department of Immunology, Erasmus MC, University Medical Center, Rotterdam, The Netherland
5Department of Medical Microbiology and Immunology, Diakonessenhuis, Utrecht, The Netherlands
Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 51, Issue 6, Pages 1191–1198, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/cclm-2012-0774, December 2012
- Published Online:
Background: Celiac disease (CD) is an inflammatory disorder of the small intestine induced by gluten ingestion. CD has a strong genetic association with human leukocyte antigen (HLA)-DQ2.5 and HLA-DQ8. The absence of HLA-DQ2.5 and HLA-DQ8 has a strong negative predictive value for CD. Genetic screening of HLA-DQ2.5 and HLA-DQ8 in patients at risk is of great value.
Methods: We designed, developed, and validated a multiplex assay based on multiplex ligation-dependent probe amplification (MLPA) technology, allowing the simultaneous detection of DQA1*05-DQB1*02, encoding HLA-DQ2.5, and DQA1*03-DQB1*03:02, encoding HLA-DQ8. The amplified products were separated and identified using capillary electrophoresis.
Results: When compared with a polymerase chain reaction followed by single-strand conformation polymorphism/ heteroduplex analysis, one discrepancy was found. Sequencing analysis showed that the developed MLPA assay result was correct. Furthermore, we demonstrated that the MLPA method is able to distinguish between the heterozygote and homozygote expression of HLA-DQ2.5 or HLA-DQ8.
Conclusions: This study shows that it is possible to rapidly and accurately screen for the absence of HLA-DQ2.5 and HLA-DQ8 using MLPA, excluding patients at risk for CD for further serological or histological follow-up. In addition, MLPA might be an accurate tool to screen for other specific HLA types in the context of disease association in a diagnostic laboratory setting.
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