title: A multiplex assay to rapidly exclude HLA-DQ2.5 and HLA-DQ8 expression in patients at risk for celiac disease.
abstract: 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.
authors: Department of Clinical Chemistry, Hematology and Immunology, Diakonessenhuis Utrecht, Utrecht, The Netherlands. evbeek@diakhuis.nl
authors: Ellen M
authors: EM
authors: van Beek
authors: Elianne A
authors: EA
authors: Roelandse-Koop
authors: Raymon
authors: R
authors: Vijzelaar
authors: Rizkat
authors: R
authors: Yilmaz
authors: Ingrid M W
authors: IM
authors: van Hoogstraten
authors: Marco W J
authors: MW
authors: Schreurs
authors: Alice A M
authors: AA
authors: Verheul
authors: Arend Jan
authors: AJ
authors: van Houte
authors: Wouter
authors: W
authors: Kortlandt
mesh_terms: D002446:Celiac Disease / Q000175:diagnosis* / Q000235:genetics / Q000276:immunology*; D019075:Electrophoresis, Capillary / Q000379:methods; D020022:Genetic Predisposition to Disease; D006683:HLA-DQ Antigens / Q000032:analysis* / Q000096:biosynthesis* / Q000235:genetics; D006801:Humans; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods; D018807:Polymorphism, Single-Stranded Conformational; D012307:Risk Factors; D017422:Sequence Analysis, DNA
chemical_list: D006683:HLA-DQ Antigens; C053186:HLA-DQ2 antigen; C109756:HLA-DQ8 antigen