HLA Genotyping: Methods for the Identification of the HLA-DQ2,-DQ8 Heterodimers Implicated in Celiac Disease (CD) Susceptibility

Abstract

In this chapter we will present the principal technical methods to genotype the HLA-DQA1* and -DQB1* alleles associated with celiac disease (CD), corresponding to the serological heterodimers HLA-DQ2 and -DQ8. We will present the methods specific for the genotyping of these heterodimers, which represents a common request from consultant doctors. Because these alleles are also common in healthy subjects, their presence is not diagnostic for CD. Conversely, their absence is more important because it excludes the disease, since CD patients negative for these heterodimers are very rare. Accordingly, HLA typing has been included as a useful test to exclude celiac disease in the ESPGHAN guidelines for diagnosis of celiac disease.

The methods for HLA typing described in the present chapter are based on the following techniques:

PCR-SSP (Polymerase Chain Reaction-Sequence Specific Primers): PCR with primers specific for HLA alleles encoding the CD risk heterodimers, whose presence is revealed through the electrophoresis of PCR products.

Reverse PCR-SSOP (PCR-Sequence Specific Oligonucleotide Probes): PCR with primers specific for a single locus or a large group of alleles followed by hybridization with enzyme-conjugated probes specific for a single allele, immobilized on different supports (i.e., nitrocellulose strips), in which DNA-probes binding is revealed by the production of a colored precipitate derived from the enzymatic modification of a specific substrate.

Real-Time PCR (RT-PCR): PCR with locus or allelic specific primers whose amplification is revealed by particular probes (i.e., Taqman probes) hybridizing the DNA template within the two PCR primers and emitting fluorescent while the PCR reaction occurs.