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Abstract
Objectives: The HLA-B*27 genotype is strongly associated with ankylosing spondylitis (AS) and also other spondyloarthropathies (SpA) such as psoriatic SpA. Our objective was to develop and validate a real time PCR for HLA-B*27 with greater capacity than our current allele specific approach without loss of fidelity.
Methods: We describe a real-time PCR method for the detection of HLA-B*27 using allele specific primers combined with TaqMan fluorescent minor groove binder (MGB) probes. One probe is specific for human leucocyte antigen (HLA) B*27 conjugated with the fluorochrome FAM and the other specific for human growth hormone (HGH) conjugated with the fluorochrome VIC which acts as endogenous control.
Results: 441 consecutive samples were tested of which 139 tested HLA-B*27 positive and 302 tested negative. We tested a further 12 external quality control samples of which four were positive and eight negative for HLA-B*27.
Conclusions: RT-PCR is suitable for HLA-B*27 genotyping and has the advantages of lesser hands-on time, does not require any post amplification processing, and does not use the toxic DNA intercalating dye ethidium bromide.
Keywords: HLA-B*27, TaqMan, PCR, MGB, minor groove binder, NFQ, non-fluorescent quencher, real time PCR, ankylosing spondylitis.
N Z J Med Lab Sci 2014; 68: 04-08
Introduction
The human leucocyte antigen (FILA) B*27 is strongly associated with the spondyloarthropathies (SpA's). FILA-B*27 is found in 90-95% of ankylosing spondylitis (AS) patients and also lesser frequency in forms of psoriatic arthritis and reactive arthritis (1,2). HLA-B*27 may have a direct role in the pathogenesis of SpA which is supported by epidemiological studies and transgenic rat studies (3). Different hypotheses exist, such as the arthritogenic peptide hypothesis, the misfolding hypothesis or molecular mimicry (3-5). Less than 5% of those members of the general population that are FILA-B*27+ develop AS, and HLA-B*27 has been estimated to account for only 20-50% of the overall genetic susceptibility to AS (6). In addition to FILA-B*27, a recent genome wide association study has confirmed 25 loci that confer increased genetic risk for AS, including genes involved in peptide processing prior to MFIC class I presentation and alterations in the IL-23 pathway (7).
There are more than 50 subtypes of FILA-B27 identified which mostly differ by only a small number of nucleotides. (IMGT/FILA database: http:Wwww.ebi.ac.uk/imat/hla/). The subtypes FILA-B*27:02, B*27:04,...