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

Aplicação da metodologia de PCR-SSP na identificação de HLA-B*27 como auxílio ao diagnóstico de espondilite anquilosante

Gisele Cristina A. Oliveira Eliane P. Ambrosio-Albuquerque Jeane Eliete L. Visentainer About the authors

ABSTRACT

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 primer-polymerase 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.

Key words:
HLA-B*27 antigen; ankylosing spondylitis; molecular biology

RESUMO

INTRODUÇÃO:

Os antígenos leucocitários humanos (HLA) são moléculas que apresentam antígenos ao sistema imune; a presença ou a ausência deles é descrita como fator influente em algumas doenças. O HLA-B*27 é um polimorfismo do HLA que tem sido associado à maior predisposição à espondilite anquilosante (EA) e a outras espondiloartropatias. A detecção do HLA-B*27 é utilizada como ferramenta diagnóstica e prognóstica nesses casos, assim como no diagnóstico diferencial de outras doenças.

OBJETIVO:

Padronizar a metodologia de reação em cadeia da polimerase-iniciador específico (PCR-SSP) para utilização no Laboratório de Imunogenética da Universidade Estadual de Maringá (UEM), considerando sua especificidade e seu custo-benefício.

MATERIAL E MÉTODOS:

Foram utilizados 30 indivíduos comprovadamente sem EA positivos para o alelo HLA-B*27 e 10 indivíduos negativos, testados previamente por PCR-oligonucleotídeo específico (PCR-SSO) e, neste estudo, por PCR-SSP.

RESULTADOS:

Cem por cento dos pacientes testados tiveram seus resultados confirmados, mesmo com diferentes subtipos.

CONCLUSÃO:

Tendo em vista a alta reprodutibilidade e o amplo espectro de subtipos que abrange, concluiu-se que a técnica de identificação de PCR-SSP para o HLA-B*27 pode ser utilizada como auxílio diagnóstico de rotina para espondiloartropatias.

Unitermos:
antígeno HLA-B*27; espondilite anquilosante; biologia molecular

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)(11 Martinez-Borra J, Gonzalez S, Lopez-Larrea C. Genetic factors predisposing to spondylarthropathies. Arthritis Rheum. 2000;43(3):485- 92.). HLA molecules are divided into classes: in class I region are loci A, B and C(22 Rudwaleit M. New approaches to diagnosis and classification of axial and peripheral spondyloarthritis. Curr Opin Rheumatol. 2010;22(4):375-80.), that generates products with high degree of allelic variation at each locus, resulting in a large variability in individuals(33 Castro-Santos P, Gutierrez MA, Diaz-Pena R. Genetics of ankylosing spondylitis. Rev Med Chil. 2014;142(9):1165-73.). The presence or absence of some of these antigens has been related to some diseases(11 Martinez-Borra J, Gonzalez S, Lopez-Larrea C. Genetic factors predisposing to spondylarthropathies. Arthritis Rheum. 2000;43(3):485- 92.). By August 2015, 3,977 HLA-B alleles were identified in different populations(44 International Immunogenetics databases [Internet]. Available at: http://www.ebi.ac.uk/ipd/imgt/hla/stats.html
http://www.ebi.ac.uk/ipd/imgt/hla/stats....
).

The HLA-B*27 is a polymorphism of the HLA-B, described in 1969(55 Thorsby E. HL-A antigens and genes. I. A study of unrelated Norwegians. Vox Sang. 1969;17(2):81-92.). The presence of this allele was associated with ankylosing spondylitis (AS) and other spondyloarthropathies(66 Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet. 1973;1(7809):904-7.

7 Brewerton DA. Discovery: HLA and disease. Curr Opin Rheumatol. 2003;15(4):369-73.
-88 Schlosstein L, Terasaki PI, Bluestone R, Pearson CM. High association of an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med. 1973;288(14):704-6.), a group of chronic inflammatory diseases with clinical and radiological manifestations in common(99 Zeidler H, Mau W, Khan MA. Undifferentiated spondyloarthropathies. Rheum Dis Clin North Am. 1992;18(1):187-202.). About 184 subtypes of this HLA (1010 Allele Search Tool. IMGT/HLA-IPD-EMBL-EBI. [Internet]. Available at: http://www.ebi.ac.uk/ipd/imgt/hla/allele.html
http://www.ebi.ac.uk/ipd/imgt/hla/allele...
) are known, and HLA-B*27:05 is the most widely distributed and probably is the allele from which others have developed(1111 Khan MA, Ball EJ. Genetic aspects of ankylosing spondylitis. Best Pract Res Clin Rheumatol. 2002;16(4):675-90.

12 Khan MA. HLA in spondyloarthropathies. In: Mehra NK, editor. The HLA complex in biology and medicine. A resource book. New Delhi: India Jaypee Brother Med Publ Ltd. 2010; p. 259-75.

13 Ankylosing spondylitis. Ankylosing spondylitis. [Internet]. Available at: http://www.hlab27.com/
http://www.hlab27.com/...
-1414 Van Gaalen FA. Does HLA-B*2706 protect against ankylosing spondylitis? A meta-analysis. Int J Rheum Dis. 2012;15(1):8-12.).

The HLA-B*27 connection to the AS is well established; 90%-95% of patients with the disease have this allele(1515 Oostveen J, Prevo R, den Boer J, van de Laar M. Early detection of sacroiliitis on magnetic resonance imaging and subsequent development of sacroiliitis on plain radiography. A prospective, longitudinal study. J Rheumatol. 1999;26(9):1953-8.

16 Svejgaard A. PP and LPR. HLA and disease suceptibility: clinical implications. Clin Immunol Allergy 4. 1984;567-80.

17 Breur-Vriesendorp BS, Dekker-Saeys AJ, Ivanyi P. Distribution of HLA-B27 subtypes in patients with ankylosing spondylitis: the disease is associated with a common determinant of the various B27 molecules. Ann Rheum Dis. 1987;46(5):353-6.
-1818 Khan MA, Kellner H. Immunogenetics of spondyloarthropathies. Rheum Dis Clin North Am. 1992;18(4):837-64.), suggesting a tendency to family association(1919 Khan MA. Ankylosing spondylitis: introductory comments on its diagnosis and treatment. Ann Rheum Dis. 2002;61 Suppl 3:iii3-7.

20 Sieper J, Braun J, Rudwaleit M, Boonen A, Zink A. Ankylosing spondylitis: an overview. Ann Rheum Dis. England. 2002;61 Suppl 3:iii8-18.
-2121 Zochling J, van der Heijde D, Burgos-Vargas R, et al. ASAS/EULAR recommendations for the management of ankylosing spondylitis. Ann Rheum Dis. 2006;65(4):442-52.). The prevalence of AS accompanies the frequency of HLA-B*27 allele in the population(2222 Van Gaalen FA. Does HLA-B*2706 protect against ankylosing spondylitis? A meta-analysis. Int J Rheum Dis. 2012;15(1):8-12.), however studies indicate that only 2% of the individuals HLA-B*27 positive develop the disease(2323 Braun J, Bollow M, Remlinger G, et al. Prevalence of spondylarthropathies in HLA-B27 positive and negative blood donors. Arthritis Rheum. 1998;41(1):58-67.), indicating the involvement of other genetic and/or environmental factors in the onset of it(2424 Brown MA, Kennedy LG, MacGregor AJ, et al. Susceptibility to ankylosing spondylitis in twins: the role of genes, HLA, and the environment. Arthritis Rheum. 1997;40(10):1823-8.,2525 Laval SH, Timms A, Edwards S, et al. Whole-genome screening in ankylosing spondylitis: evidence of non-MHC genetic-susceptibility loci. Am J Hum Genet. 2001;68(4):918-26.). The exact trigger mechanism has not been identified(2626 Allen RL, Bowness P, McMichael A. The role of HLA-B27 in spondyloarthritis. Immunogenetics. 1999;50(3-4):220-7.).

Moreover, some studies indicate that certain subtypes have a greater association with AS than others, suggesting clinical specificity(2727 Liu X, Hu LH, Li YR, Chen FH, Ning Y, Yao QF. The association of HLA-B*27 subtypes with ankylosing spondylitis in Wuhan population of China. Rheumatol Int. 2010;30(5):587-90.

28 Liu Y, Jiang L, Cai Q, et al. Predominant association of HLA-B*2704 with ankylosing spondylitis in Chinese Han patients. Tissue Antigens. 2010;75(1):61-4.

29 Garcia-Fernandez S, Gonzalez S, Mina Blanco A, et al. New insights regarding HLA-B27 diversity in the Asian population. Tissue Antigens. 2001;58(4):259-62.

30 Gonzalez S, Garcia-Fernandez S, Martinez-Borra J, et al. High variability of HLA-B27 alleles in ankylosing spondylitis and related spondyloarthropathies in the population of northern Spain. Hum Immunol. 2002;63(8):673-6.

31 Mou Y, Wu Z, Gu J, et al. HLA-B27 polymorphism in patients with juvenile and adult-onset ankylosing spondylitis in Southern China. Tissue Antigens. 2010;75(1):56-60.
-3232 Meryem A, Noureddine B, Ouafaa MS, et al. HLA-B27 subtypes distribution among moroccan patients with ankylosing spondylitis. Am J Med Biol Res. 2013;1(1):28-32.). The HLA-B*27:05 and the HLA-B*27:02 are the most common subtypes, 90% and 5%-10%, respectively, in Caucasians(3333 Ball EJ, Khan MA. HLA-B27 polymorphism. Joint Bone Spine. 2001;68(5):378-82.); the HLA-B*27:04 is the predominant subtype among Chinese and Japanese(3434 Lopez-Larrea C, Sujirachato K, Mehra NK, et al. HLA-B27 subtypes in Asian patients with ankylosing spondylitis. Evidence for new associations. Tissue Antigens. 1995;45(3):169-76.) and the HLA-B*27:04, the HLA-B*27:06 and the HLA-B*27:07 are found only in Asia(3535 Choo SY, Antonelli P, Nisperos B, Nepom GT, Hansen JA. Six variants of HLA-B27 identified by isoelectric focusing. Immunogenetics. 1986;23(1):24-9.,3636 Choo SY, Fan LA, Hansen JA. A novel HLA-B27 allele maps B27 allospecificity to the region around position 70 in the alpha 1 domain. J Immunol. 1991;147(1):174-80.). 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(3737 Sampaio-Barros PD, Bertolo MB, Kraemer MH, Neto JF, Samara AM. Primary ankylosing spondylitis: patterns of disease in a Brazilian population of 147 patients. J Rheumatol (Canada). 2001;28(3):560-5.).

The HLA-B*27 has been used as a diagnostic marker for AS and other spondyloarthropathies(3838 Reveille JD, Ball EJ, Khan MA. HLA-B27 and genetic predisposing factors in spondyloarthropathies. Curr Opin Rheumatol. 2001;13(4):265-72.,3939 Koh WH, Boey ML. Ankylosing spondylitis in Singapore: a study of 150 patients and a local update. Ann Acad Med Singapore. 1998;27(1):3-6.). 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(22 Rudwaleit M. New approaches to diagnosis and classification of axial and peripheral spondyloarthritis. Curr Opin Rheumatol. 2010;22(4):375-80.). 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(4040 Londono J, Romero-Sanchez MC, Torres VG, et al. Associação entre os níveis séricos de potenciais biomarcadores com a presença de fatores relacionados à atividade clínica e ao mau prognóstico em espondiloartrites. Rev Bras Reumatol. [Internet]. 2012;52(4):529-44. Available at: http://www.scielo.br/pdf/rbr/v52n4/v52n4a06.pdf
http://www.scielo.br/pdf/rbr/v52n4/v52n4...
).

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(4141 Allen RL, Bowness P, McMichael A. The role of HLA-B27 in spondyloarthritis. Immunogenetics. 1999;50(3-4):220-7.). The advantage is the differentiation of several alleles(4242 Conjunto de baixa resolução AllSet+TM Gold B27. Instruções de utilização para identificar a presença ou a ausência de alelos HLA B27. [Internet]. Available at: http://www.onelambda.com/content/dam/onelambda/en/TDX/Documents/securedocs/docs/Product_Insert/MAN0002577_AllSet+Gold HLA B27 SSP Kit Instructions for Use - Portuguese Translation_rev07.pdf.
http://www.onelambda.com/content/dam/one...
), 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(4343 McGinnis MD, Conrad MP, Bouwens AG, Tilanus MG, Kronick MN. Automated, solid-phase sequencing of DRB region genes using T7 sequencing chemistry and dye-labeled primers. Tissue Antigens. 1995;46(3):173-9.). 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(4444 Testes de tipagem LABType® SSO. [Internet]. Available at: http://www.onelambda.com/content/dam/onelambda/en/TDX/Documents/securedocs/docs/Product_Insert/RSSO-LTYP-PI-PT-00.pdf
http://www.onelambda.com/content/dam/one...
).

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.

MATERIAL AND METHODS

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). Table 1 describes the HLA-B*27 subtypes tested and confirmed in this study.

TABLE 1
Positive results for HLA-B*27 by PCR-SSO and PCR-SSP methods

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.(4545 Kulkarni B, Mohanty D, Ghosh K. Frequency distribution of human platelet antigens in the Indian population. Transfus Med. England; 2005 Apr;15(2):119-24.); 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(4646 Nathalang O, Tantimavanich S, Nillakupt K, Arnutti P, Jaruchaimontree C. HLA-B27 testing in Thai patients using the PCR-SSP technique. Tissue Antigens. 2006;67(3):233-6.). The HgH gene was used as an internal control of the reaction.

TABLE 2
Specific primers for HLA-B*27

The conditions of the reaction used were described by Parasannanavar et al. (2013)(4747 Parasannanavar DJ, Rajadhyaksha A, Ghosh K. Application of a simple in-house PCR-SSP technique for HLA-B* 27 typing in spondyloarthritis patients. Arthritis. [Internet]. 2013. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24490069PubMed PMID: 24490069.
http://www.ncbi.nlm.nih.gov/pubmed/24490...
) with modifications. The tests were initiated at the following conditions: 1 µl buffer, 0.5 µl magnesium chloride (MgCl2), 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.

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 MgCl2, 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.

FIGURE
Band pattern of positive and negative samples for HLA-B*27 by PCR-SSP in agarose gel with standardized mix

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.

TABLE 3
Positive and negative results for PCR-SSO and PCR-SSP

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(4848 Sérgio Fraco. Bioinforme. HLA-B27. [Internet]. Available at: http://www.sergiofranco.com.br/bioinforme/index.asp?cs=Hematologia&ps=hlab27
http://www.sergiofranco.com.br/bioinform...
).

According to Parasannanavar et al. (2013)(4747 Parasannanavar DJ, Rajadhyaksha A, Ghosh K. Application of a simple in-house PCR-SSP technique for HLA-B* 27 typing in spondyloarthritis patients. Arthritis. [Internet]. 2013. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24490069PubMed PMID: 24490069.
http://www.ncbi.nlm.nih.gov/pubmed/24490...
), 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)(4747 Parasannanavar DJ, Rajadhyaksha A, Ghosh K. Application of a simple in-house PCR-SSP technique for HLA-B* 27 typing in spondyloarthritis patients. Arthritis. [Internet]. 2013. Available at: http://www.ncbi.nlm.nih.gov/pubmed/24490069PubMed PMID: 24490069.
http://www.ncbi.nlm.nih.gov/pubmed/24490...
) 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(4949 John SW, Weitzner G, Rozen R, Scriver CR. A rapid procedure for extracting genomic DNA from leukocytes. Nucleic Acids Res. 1991;19(2):408.), 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)(5050 Frankenberger BL, Breitkopf S, Albert E, et al. Routine molecular genotyping of HLA-B27 in spondyloarthropathies overcomes the obstacles of serological typing and reveals an increased B *2702 frequency in ankylosing spondylitis. J Rheumatol. 1997;24(5):899-903.), 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.

REFERENCES

  • 1
    Martinez-Borra J, Gonzalez S, Lopez-Larrea C. Genetic factors predisposing to spondylarthropathies. Arthritis Rheum. 2000;43(3):485- 92.
  • 2
    Rudwaleit M. New approaches to diagnosis and classification of axial and peripheral spondyloarthritis. Curr Opin Rheumatol. 2010;22(4):375-80.
  • 3
    Castro-Santos P, Gutierrez MA, Diaz-Pena R. Genetics of ankylosing spondylitis. Rev Med Chil. 2014;142(9):1165-73.
  • 4
    International Immunogenetics databases [Internet]. Available at: http://www.ebi.ac.uk/ipd/imgt/hla/stats.html
    » http://www.ebi.ac.uk/ipd/imgt/hla/stats.html
  • 5
    Thorsby E. HL-A antigens and genes. I. A study of unrelated Norwegians. Vox Sang. 1969;17(2):81-92.
  • 6
    Brewerton DA, Hart FD, Nicholls A, Caffrey M, James DC, Sturrock RD. Ankylosing spondylitis and HL-A 27. Lancet. 1973;1(7809):904-7.
  • 7
    Brewerton DA. Discovery: HLA and disease. Curr Opin Rheumatol. 2003;15(4):369-73.
  • 8
    Schlosstein L, Terasaki PI, Bluestone R, Pearson CM. High association of an HL-A antigen, W27, with ankylosing spondylitis. N Engl J Med. 1973;288(14):704-6.
  • 9
    Zeidler H, Mau W, Khan MA. Undifferentiated spondyloarthropathies. Rheum Dis Clin North Am. 1992;18(1):187-202.
  • 10
    Allele Search Tool. IMGT/HLA-IPD-EMBL-EBI. [Internet]. Available at: http://www.ebi.ac.uk/ipd/imgt/hla/allele.html
    » http://www.ebi.ac.uk/ipd/imgt/hla/allele.html
  • 11
    Khan MA, Ball EJ. Genetic aspects of ankylosing spondylitis. Best Pract Res Clin Rheumatol. 2002;16(4):675-90.
  • 12
    Khan MA. HLA in spondyloarthropathies. In: Mehra NK, editor. The HLA complex in biology and medicine. A resource book. New Delhi: India Jaypee Brother Med Publ Ltd. 2010; p. 259-75.
  • 13
    Ankylosing spondylitis. Ankylosing spondylitis. [Internet]. Available at: http://www.hlab27.com/
    » http://www.hlab27.com/
  • 14
    Van Gaalen FA. Does HLA-B*2706 protect against ankylosing spondylitis? A meta-analysis. Int J Rheum Dis. 2012;15(1):8-12.
  • 15
    Oostveen J, Prevo R, den Boer J, van de Laar M. Early detection of sacroiliitis on magnetic resonance imaging and subsequent development of sacroiliitis on plain radiography. A prospective, longitudinal study. J Rheumatol. 1999;26(9):1953-8.
  • 16
    Svejgaard A. PP and LPR. HLA and disease suceptibility: clinical implications. Clin Immunol Allergy 4. 1984;567-80.
  • 17
    Breur-Vriesendorp BS, Dekker-Saeys AJ, Ivanyi P. Distribution of HLA-B27 subtypes in patients with ankylosing spondylitis: the disease is associated with a common determinant of the various B27 molecules. Ann Rheum Dis. 1987;46(5):353-6.
  • 18
    Khan MA, Kellner H. Immunogenetics of spondyloarthropathies. Rheum Dis Clin North Am. 1992;18(4):837-64.
  • 19
    Khan MA. Ankylosing spondylitis: introductory comments on its diagnosis and treatment. Ann Rheum Dis. 2002;61 Suppl 3:iii3-7.
  • 20
    Sieper J, Braun J, Rudwaleit M, Boonen A, Zink A. Ankylosing spondylitis: an overview. Ann Rheum Dis. England. 2002;61 Suppl 3:iii8-18.
  • 21
    Zochling J, van der Heijde D, Burgos-Vargas R, et al. ASAS/EULAR recommendations for the management of ankylosing spondylitis. Ann Rheum Dis. 2006;65(4):442-52.
  • 22
    Van Gaalen FA. Does HLA-B*2706 protect against ankylosing spondylitis? A meta-analysis. Int J Rheum Dis. 2012;15(1):8-12.
  • 23
    Braun J, Bollow M, Remlinger G, et al. Prevalence of spondylarthropathies in HLA-B27 positive and negative blood donors. Arthritis Rheum. 1998;41(1):58-67.
  • 24
    Brown MA, Kennedy LG, MacGregor AJ, et al. Susceptibility to ankylosing spondylitis in twins: the role of genes, HLA, and the environment. Arthritis Rheum. 1997;40(10):1823-8.
  • 25
    Laval SH, Timms A, Edwards S, et al. Whole-genome screening in ankylosing spondylitis: evidence of non-MHC genetic-susceptibility loci. Am J Hum Genet. 2001;68(4):918-26.
  • 26
    Allen RL, Bowness P, McMichael A. The role of HLA-B27 in spondyloarthritis. Immunogenetics. 1999;50(3-4):220-7.
  • 27
    Liu X, Hu LH, Li YR, Chen FH, Ning Y, Yao QF. The association of HLA-B*27 subtypes with ankylosing spondylitis in Wuhan population of China. Rheumatol Int. 2010;30(5):587-90.
  • 28
    Liu Y, Jiang L, Cai Q, et al. Predominant association of HLA-B*2704 with ankylosing spondylitis in Chinese Han patients. Tissue Antigens. 2010;75(1):61-4.
  • 29
    Garcia-Fernandez S, Gonzalez S, Mina Blanco A, et al. New insights regarding HLA-B27 diversity in the Asian population. Tissue Antigens. 2001;58(4):259-62.
  • 30
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Publication Dates

  • Publication in this collection
    Sept 2016

History

  • Received
    19 May 2016
  • Reviewed
    19 May 2016
  • Accepted
    28 June 2016
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