Application of PCR-SSP method for HLA-B * 27 identification as an auxiliary tool for diagnosis of ankylosing spondylitis

Introduction: Human leukocyte antigens (HLA) are molecules that present antigen to the immune system; their presence or absence have been described as an influential factor in some diseases. HLA-B*27 is an HLA polymorphism that has been associated with increased susceptibility to ankylosing spondylitis (AS) and other spondyloarthropaty. The detection of HLA-B*27 has been used as diagnostic and prognostic tool in these cases, as well as in the differential diagnosis of other diseases. Objective: Standardize the single specific primerpolymerase chain reaction (PCR-SSP) methodology for use in the Immunogenetics Laboratory of the Universidade Estadual de Maringá (UEM), considering its specificity and cost-effectiveness. Material and methods: A total of 30 individuals without AS positive for HLA-B*27 allele and 10 AS negative individuals, were previously tested by PCR-sequence specific oligonucleotide (PCR-SSO) and, in this study, by PCR-SSP. Results: One hundred percent of the patients tested confirmed their results, even with different subtypes. Conclusion: Considering the high reproducibility and the broad spectrum of subtypes covering, it was concluded that the PCR-SSP identifying method for HLA-B*27 can be used as a routine diagnostic tool for spondyloarthropathies.


introDuCtion
The major histocompatibility complex is a set of mapped genes on the short arm of chromosome 6, which encodes molecules presenting antigens to the immune system, called human leukocyte antigen (HLA) (1) .HLA molecules are divided into classes: in class I region are loci A, B and C (2) , that generates products with high degree of allelic variation at each locus, resulting in a large variability in individuals (3) .The presence or absence of some of these antigens has been related to some diseases (1) .By August 2015, 3,977 HLA-B alleles were identified in different populations (4) .
The HLA-B*27 is a polymorphism of the HLA-B, described in 1969 (5) .The presence of this allele was associated with ankylosing spondylitis (AS) and other spondyloarthropathies (6)(7)(8) , a group of chronic inflammatory diseases with clinical and radiological manifestations in common (9) .About 184 subtypes of this HLA (10) are known, and HLA-B*27:05 is the most widely distributed and probably is the allele from which others have developed (11)(12)(13)(14) .
The HLA-B*27 connection to the AS is well established; 90%-95% of patients with the disease have this allele (15)(16)(17)(18) , suggesting a tendency to family association (19)(20)(21) .The prevalence of AS accompanies the frequency of HLA-B*27 allele in the population (22) , however studies indicate that only 2% of the individuals HLA-B*27 positive develop the disease (23) , indicating the involvement of other genetic and/or environmental factors in the onset of it (24,25) .The exact trigger mechanism has not been identified (26) .
Moreover, some studies indicate that certain subtypes have a greater association with AS than others, suggesting clinical Application of PCR-SSP method for HLA-B*27 identification as an auxiliary tool for diagnosis of ankylosing spondylitis specificity (27)(28)(29)(30)(31)(32) .The HLA-B*27:05 and the HLA-B*27:02 are the most common subtypes, 90% and 5%-10%, respectively, in Caucasians (33) ; the HLA-B*27:04 is the predominant subtype among Chinese and Japanese (34) and the HLA-B*27:04, the HLA-B*27:06 and the HLA-B*27:07 are found only in Asia (35,36) .This allele is rare in black African populations, as well as the frequency of AS in this ethnic group.In Brazil, the disease is found in mulattos, since they have the Caucasian genetic influence (37) .
The HLA-B*27 has been used as a diagnostic marker for AS and other spondyloarthropathies (38,39) .The presence of the allele is not linked to the development of AS, but its presence may be useful in the diagnosis and also to auxiliate in the differential diagnosis of other diseases (2) .Furthermore, it can be used as indicator of patient prognosis; individuals with AS and HLA-B*27 tend to have more prolonged and severe symptoms (40) .
The identification of HLA-B*27 and its subtypes is commonly performed by polymerase chain reaction-sequence specific oligonucleotide (PCR-SSO) and polymerase chain reaction-sequence specific primer (PCR-SSP) methodologies.This is a method considered fast, efficient and relatively low cost (41) .The advantage is the differentiation of several alleles (42) , this method allows to detect a single different base in the the deoxyribonucleic acid (DNA) sequence between two alleles, though it can not detect a new undefined allele, unless the change happen in the location detected by the primer (43) .The PCR-SSO, when compared to the PCR-SSP, may present more ambiguities because the probes used are able to bind to the DNA of the sample in a single region per test; the PCR-SSP can bind to the DNA of the sample in two regions per test (44) .
Due to the benefits of PCR-SSP methodology and clinical applicability of HLA-B*27, the standardization of this method for use in routine and research in the Immunogenetics Laboratory of the Universidade Estadual de Maringá (UEM) is justified.

Case series
We included 30 patients without AS previously tested by PCR-SSO (One-Lambda, low-medium resolution) and proven positive for HLA-B*27 allele from the National Register of Bone Marrow Donor (Registro Nacional de Doadores de Medula Óssea [REDOME]) of the city of Maringá and surrounding region.The control-group consisted of 10 individuals from the same database, but negative for HLA-B*27 allele, evidenced by the same methodology.
The selection of individuals in the database considered the heterogeneity of the HLA-B*27 alleles to identify the greatest number of subtypes.These have been identified, including crossreactions, by the National Marrow Donor Program (NMDP) code (http://www.marrow-donor.org/cgi-bin/DNA/dnatyp.pl).

DNA extraction
The DNA extraction was performed with the BIOPUR extraction kit (Kit de Extração Mini Spin Plus 250, Biometrix), using from 200 μl of whole blood collected with ethylenediaminetetraacetic acid (EDTA).After extraction, the DNA was quantified in Nanodrop (NanoDrop 2000 Spectrophotometer, Thermo Scientific-Uniscience).

PCR-SSP
The primers specific for HLA-B*27 were selected from sequences of the International ImMunoGeneTics Database (IMGT)/HLA (http://www.ebi.ac.uk/ipd/imgt/hla/probe.html) and described by Kulkarni et al. (45) ; the sequences are shown in Table 2.The Amplicon flanked by these primers was designed to identify the HLA-B*27 subtypes from 27:01 to 27:73 and expand the identification, an additional primer was included (46) .The HgH gene was used as an internal control of the reaction.

rESuLtS
To standardize the PCR-SSP, tests were performed with several amounts of each compound to improve the visibility of the bands in the gel testes.In the first mix tested, the reaction was not satisfactory, the control bands were visible, but the bands of the HLA-B*27 were poorly visible.Modifications and sequential test were conducted by modifying a test variable to reach the best reaction condition, which is composed of: 12.5 μl buffer, 4 μl MgCl 2 , 2.5 μl dNTP, 1.5 μl primers, 1 μl primers of HgH gene and 1.25 μl Taq.Moreover, cycling conditions were also changed to improve the visibility of the bands in the agarose gel, reaching the optimum file of: initial denaturation at 94ºC for 5 minutes, 30 cycles at 94ºC for 40 seconds, 64ºC for 1 minute, 72ºC for 1 minute and final extension at 72ºC for 10 minutes.The Figure shows the pattern of bands obtained using the final mix, standardized.
From the standardization, the HLA-B*27 alleles were tested and all subtypes were amplified by PCR-SSP.Table 3 shows the results.One hundred percent of sensitivity and specificity were obtained, there was no false positive or negative result.
The conditions of the reaction used were described by Parasannanavar et al. (2013) (47) with modifications.The tests were initiated at the following conditions: 1 μl buffer, 0.5 μl magnesium chloride (MgCl 2 ), 0.5 μl deoxyribonucleotide phosphates (dNTP), 2.5 μl primers and 0,5 μl Taq, this volume was used for five samples.The amplifications were performed in a thermocycler (Veriti 96 Well Thermal Cycler, Applied Biosystems) at settings: initial denaturation at 94ºC for 5 minutes, 30 cycles at 94ºC for 1 minute, at 65ºC for 2 minutes, at 72ºC for 1 minute and final extension at 72ºC for 10 minutes.The amplification products were subjected to electrophoresis (Electrophoresis Power Supply LPS -300 V, Loccus Biotecnologia) in 2% agarose gel with 0.5 µg/ml Syber safe and bromophenol blue in 0.5× buffer of tris-borate-EDTA (TBE) at 80 V for 30 minutes.The identification of bands was performed using a transilluminator (Transiluminador UVB LTB 20 × 20 STV, Loccus Biotecnologia); the band corresponding to the HgH gene has 434 pb and the positive band for HLA B*27, 149 pb.

DiSCuSSion
Due to the association of the presence of the HLA-B*27 allele with spondyloarthropathies, the molecular detection requires this allele, which is of utmost importance for both the study and the diagnosis of these diseases.Furthermore, this detection in asymptomatic patients and the research on the risk of patients relatives developing the disease is also essential, that's because epidemiological studies have shown that 10%-20% of first-degree relatives of patients with AS have higher risk of developing the disease (48) .
According to Parasannanavar et al. (2013) (47) , the methods used for identification of HLA-B*27 (microlymphocytotoxicity and flow cytometry) are difficult because of the lack of a specific antiserum and the possibility of false results due to the change on epitopes.Molecular techniques are more sensitive and specific.The authors tested 90 samples from healthy controls by PCR-SSP and PCR-SSO and found no discrepancy in results between them, data compatible with this study, in which the efficiency of PCR-SSP and the applicability of the primers used was proven, since all results were consistent with those obtained by PCR-SSO, reaching 100% of specificity and sensitivity.
The difficulty encountered was the standardization of the DNA concentration to be used, Parasannanavar et al. (2013) (47) suggest the use of 80-100 ng, and this value is not always obtained.
Alternative for DNA extraction methods may be used, such as salting out method (49) , which expects a higher performance, but requires more time and the use of larger amounts of blood or buffy-coat.
Other authors, such as Frankenberger et al. (1997) (50) , also found that identification of HLA-B*27 by PCR is reliable and reproducible, and therefore recommended for the routine, since it overcomes the weaknesses of serology, such as cross-reactivity and ambiguous results.
It was concluded that the PCR-SSP identification method can be used as a routine diagnostic aid for spondyloarthropathies.It is a relatively simple, quick, low costly, high sensitivity and specificity technique.
FigurE − Band pattern of positive and negative samples for HLA-B*27 by PCR-SSP in agarose gel with standardized mix PCR-SSP: polymerase chain reaction-single specific primer; HLA: human leukocyte antigens; H: HLA-B*27 positive; C: HLA-B*27 negative; BNK: blank.Step ladder: 50 pb.
Table 1 describes the HLA-B*27 subtypes tested and confirmed in this study.