Allele-specific quantification of HLA-DQB1 gene expression by real-time reverse transcriptase-polymerase chain reaction

• Published in: Genes and immunity Vol. 5; no. 5; pp. 405 - 416
• Main Authors: Ferstl, B, Zacher, T, Lauer, B, Blagitko-Dorfs, N, Carl, A, Wassmuth, R
• Format: Journal Article
• Language: English
• Published: England Nature Publishing Group 01.07.2004
• Subjects: Alleles; Base Sequence; Cell Differentiation - drug effects; Cell Line; Cytokines; Cytokines - pharmacology; Dendritic cells; Dendritic Cells - metabolism; DQB1 gene; Gene Expression; Gene Frequency - genetics; Gene polymorphism; Genetic aspects; Heterozygote; Histocompatibility antigen HLA; Histocompatibility antigens; HLA histocompatibility antigens; HLA-DQ Antigens - biosynthesis; HLA-DQ Antigens - genetics; HLA-DQ beta-Chains; Humans; Molecular Sequence Data; Monocytes; Monocytes - drug effects; Monocytes - metabolism; Physiological aspects; Polymerase chain reaction; Polymorphism; Polymorphism, Genetic; Promoter Regions, Genetic - genetics; Reporter gene; Reverse Transcriptase Polymerase Chain Reaction; RNA-directed DNA polymerase; Transfection; Germany
• ISSN: 1466-4879; 1476-5470
• DOI: 10.1038/sj.gene.6364108

In addition to coding region polymorphism, allele-specific variation in the upstream regulatory region of the HLA-DQB1 gene has been detected. Reporter gene assays and transfection studies have indicated that HLA-DQB1 promoter polymorphism may be of functional significance. The aim of this study was to utilize real-time reverse transcriptase-polymerase chain reaction (RT-PCR) for allele-specific quantification of HLA-DQB1 expression and to analyze cell-specific HLA-DQB1 expression in vivo. For the allele-specific quantification of DQB1 gene products, a real-time RT-PCR set of primer pairs (n=27) and probes (n=5) targeting exon 2 variability was established. The robustness and integrity of the assay system were confirmed by using recombinant DQB1 exon 2 plasmid clones as active exogenous controls. Sensitivity and reproducibility were assessed by serial dilution and allelic mixing analyses. In application to the study of allele-specific expression of DQB1 gene products during cytokine-driven maturation of monocyte-derived dendritic cells, differential patterns of allelic expression in heterozygous individuals were observed for DQB1*0301, compared to DQB1*0501 and DQB1*0602. At maximum, 1.9-fold (*0301/*0501) and 2.5-fold (*0301/*0602) higher induction was seen for DQB*0301. In conclusion, HLA-DQB1 expression can be analyzed by real-time RT-PCR suitable for cell- and allele-specific detection of HLA-DQB1 transcripts in homo- and heterozygous combinations.