HLA-B27 is a risk marker for ankylosing spondylitis and other associated seronegative spondyloarthropathies. We compared three methods of HLA-B*27 typing in a New South Wales (NSW) population: flow cytometry, rs4349859 single nucleotide polymorphism (SNP) detection assay, and allele-specific real-time polymerase chain reaction (RT-PCR) analysis of exons 2 and 3. Over a 5-month period, 543 samples underwent flow cytometric testing and RT-PCR high-resolution melt analysis of rs4349859 SNP and of exon 2 (5' fragment) and exon 3. In the third method, positive samples were further analysed with fluorescent resonance emission transfer (FRET) RT-PCR of exon 2 fragments, 2a and 2b. HLA-B*27 and other genotypes were confirmed by Sanger sequencing of a 600 base pair fragment of exons 2 and 3. In our cohort, the rs4349859 SNP method had 78.6% sensitivity and 98.7% specificity. Screening with exon 2 (5' fragment) and exon 3 RT-PCR provided 100% sensitivity. Further testing with exon 2a and 2b FRET RT-PCR produced 100% specificity. This cascade approach with allele-specific RT-PCR assays was able to differentiate all samples into HLA-B*27 subtypes. HLA-B*27 genotyping with allele-specific RT-PCR assays, to screen for and confirm HLA-B27 positive samples, was more sensitive and specific than flow cytometry and rs4349859 SNP assays. It is a potentially cost-effective method for differentiating HLA-B27 subtypes. Our cascade genetic testing approach is suitable for replacing the current flow cytometric HLA-B27 assay for the heterogeneous NSW population.
Keywords: HLA-B27; ankylosing spondylitis; melting; real-time PCR.
Copyright © 2019 Royal College of Pathologists of Australasia. Published by Elsevier B.V. All rights reserved.
HLA-B27 typing: evaluation of an allele-specific PCR melting assay and two flow cytometric antigen assays.Cytometry B Clin Cytom. 2005 Jan;63(1):10-5. doi: 10.1002/cyto.b.20039. Cytometry B Clin Cytom. 2005. PMID: 15624199
HLA-B27 genotyping by fluorescent resonance emission transfer (FRET) probes in real-time PCR.Hum Immunol. 2004 Aug;65(8):826-38. doi: 10.1016/j.humimm.2004.05.009. Hum Immunol. 2004. PMID: 15336784
Do major histocompatibility complex tag single nucleotide polymorphisms accurately identify HLA-B27 in the Turkish population?Int J Rheum Dis. 2017 Dec;20(12):2035-2039. doi: 10.1111/1756-185X.12719. Epub 2015 Jul 22. Int J Rheum Dis. 2017. PMID: 26200952
Rapid screening for HLA-B27 by a TaqMan-PCR assay using sequence-specific primers and a minor groove binder probe, a novel type of TaqMan trade mark probe.J Immunol Methods. 2004 Apr;287(1-2):179-86. doi: 10.1016/j.jim.2004.01.027. J Immunol Methods. 2004. PMID: 15099766
Rapid HLA-B27 screening with real-time TaqMan PCR: a clinical validation in the Dutch population.Clin Chem Lab Med. 2011 Sep 6;49(12):1979-85. doi: 10.1515/CCLM.2011.252. Clin Chem Lab Med. 2011. PMID: 21892909