Abstract
OBJECTIVES:HLA-B27 is strongly associated with ankylosing spondylitis (AS) and its presence helps to confirm AS diagnosis. Due to the high HLA polymorphism and the differentiated contribution of alleles and molecules encoded by them, HLA-B*27 allele identification is relevant in the clinical follow-up, diagnosis, and treatment of this spondyloarthropathy. Inexpensive genotyping techniques with high specificity and sensitivity are of great interest in histocompatibility laboratories. This work aimed to optimize HLA-B*27 genotyping by Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP), which is an accessible and inexpensive technique.METHODS:The PCR-SSP was standardized using 26 HLA-B*27 positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) (medium resolution, One Lambda®) and primers described by Duangchanchot et al. (2009). For validating the technique, 397 samples were genotyped using PCR-SSP as well as PCR-SSOP.RESULTS:The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%). There was 100% agreement between the two techniques for heterozygous samples; however, two homozygous samples could not be detected by PCR-SSP.CONCLUSION:The HLA-B*27 genotyping using PCR-SSP, an easy-to-use, specific, and affordable technique, was optimized for heterozygous samples. This technique may contribute to AS diagnosis.
Highlights
Leukocyte antigen system (HLA), a major histocompatibility complex (MHC), is located on the short arm of chromosome 6 in humans [1]
The Polymerase Chain Reaction Sequence-specific Primer (PCR-SSP) was standardized using 26 HLA-B*27 alleles (B*27) positive and 3 HLA-B*27 negative samples previously defined by Polymerase Chain Reaction Sequence-specific Oligonucleotide Probes (PCR-SSOP) and primers described by Duangchanchot et al (2009)
The PCR-SSP technique was standardized for identifying the alleles HLA-B*27:02, HLA-B*27:CAFRW (05/13/16/17/28/37/38/39/42), HLA-B*27:CAFRZ (08/26/40), HLA-B*27:09 and HLA-B*27:12, which were found in 90 positive samples (22.67%)
Summary
Leukocyte antigen system (HLA), a major histocompatibility complex (MHC), is located on the short arm of chromosome 6 in humans [1]. This system has an extensive polymorphism for class I and II genes, characterized by a high number of alleles. HLA-B is the most polymorphic locus of the HLA system, with 7,053 alleles identified [2]. Class I human leukocyte antigens are expressed on most nucleated cell surfaces. They carry endogenous peptides to the cell surface for recognition by T-cell receptors and their
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