title: Simplifying celiac disease predisposing HLA-DQ alleles determination by the real time PCR method.
abstract: BACKGROUND
  Celiac disease is an autoimmune enteropathy triggered by the ingestion of gluten in genetically susceptible individuals. Genetic susceptibility is associated with two sets of alleles, DQA1*05 - DQB1*02 and DQA1*03 - DQB1*03:02, which code for class II MHC DQ2 and DQ8 molecules, respectively. Approximately 90%-95% of celiac patients are HLA-DQ2 positive, and half of the remaining patients are HLA-DQ8 positive. In fact, during a celiac disease diagnostic workup, the absence of these specific DQA and DQB alleles has a near perfect negative predictive value.
  
  
  OBJECTIVE
  Improve the detection of celiac disease predisposing alleles by combining the simplicity and sensitivity of real-time PCR (qPCR) and melting curve analysis with the specificity of sequence-specific primers (SSP).
  
  
  METHODS
  Amplifications of sequence-specific primers for DQA1*05 (DQ2), DQB1*02 (DQ2), and DQA1*03 (DQ8) were performed by the real time PCR method to determine the presence of each allele in independent reactions. Primers for Human Growth Hormone were used as an internal control. A parallel PCR-SSP protocol was used as a reference method to validate our results.
  
  
  RESULTS
  Both techniques yielded equal results. From a total of 329 samples the presence of HLA predisposing alleles was determined in 187 (56.8%). One hundred fourteen samples (61%) were positive for a single allele, 68 (36.3%) for two alleles, and only 5 (2.7%) for three alleles.
  
  
  CONCLUSIONS
  Results obtained by qPCR technique were highly reliable with no discordant results when compared with those obtained using PCR-SSP.
authors.affiliation: Graduate Program in Health Sciences, School of Health Sciences, Brasília, DF, Brazil.
authors.forename: Nicole
authors.initials: N
authors.lastname: Selleski
authors.affiliation: Graduate Program in Medical Sciences, School of Medicine, Brasília, DF, Brazil.
authors.forename: Lucas Malta
authors.initials: LM
authors.lastname: Almeida
authors.affiliation: Graduate Program in Medical Sciences, School of Medicine, Brasília, DF, Brazil.
authors.forename: Fernanda Coutinho de
authors.initials: FC
authors.lastname: Almeida
authors.affiliation: Graduate Program in Health Sciences, School of Health Sciences, Brasília, DF, Brazil.
authors.forename: Lenora
authors.initials: L
authors.lastname: Gandolfi
authors.affiliation: Graduate Program in Health Sciences, School of Health Sciences, Brasília, DF, Brazil.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Pratesi
authors.affiliation: Graduate Program in Medical Sciences, School of Medicine, Brasília, DF, Brazil.
authors.forename: Yanna Karla de Medeiros
authors.initials: YK
authors.lastname: Nóbrega
mesh_terms: D000483:Alleles; D002446:Celiac Disease / Q000175:diagnosis / Q000235:genetics*; D020022:Genetic Predisposition to Disease / Q000235:genetics*; D005838:Genotype; D006683:HLA-DQ Antigens / Q000235:genetics*; D006801:Humans; D011237:Predictive Value of Tests; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity
chemical_list: D006683:HLA-DQ Antigens