HLA-B27 and ankylosing spondylitis: tales from China

Abstract
The two most frequent HLA-B27 subtypes worldwide are B*2704 and B*2705. In the Han population of China B*2704 and, to a lower extent, B*2705 are found with significant frequency, and both are associated to ankylosing spondylitis (AS). Two articles in this issue report that the association to AS in this ethnic group is stronger for B*2704 than for B*2705. Thus, at least among the Han, B*2704 would be the strongest known susceptibility factor for AS.     

Diversity of human leukocyte antigen-B27 alleles in Han population of Hunan province, southern China

This study was to investigate the frequency of HLA-B27 and its subtypes in the Han population of Hunan province, southern China. One hundred and sixty-nine healthy unrelated donors were tested for HLA-B27 by polymerase chain reaction-sequence-specific primer (PCR-SSP). One hundred and twenty-eight B27-positive spondyloarthropathy patients and 18 B27-positive healthy controls were subtyped using the high-resolution PCR-SSP. The phenotype frequency of human leukocyte antigen (HLA)-B27 was found to be 2.36% in healthy population. Five B27 alleles were identified: B*2704, B*2705, B*2706, B*2707, and B*2724. No significant difference was found in the distribution of HLA-B27 subtypes between the patients and controls studied. Notably, B*2724 was observed in a juvenile patient with ankylosing spondylitis. This subtype has not been previously reported in Chinese ankylosing spondylitis (AS) patients and other ethnic groups.     

HLA-B*27 subtypes in Northern and Northeastern Thais, Karens, and Bamars determined by a high-resolution PCR-SSP technique

Human leukocyte antigens (HLA), class I, are a group of antigens expressed on most nucleated cell surfaces. They transport endogenous peptides to the cell surface for recognition by T-cell receptors. Their functions are involved in immune responses. Many diseases are associated with HLA alleles, especially HLA-B*27 that is strongly associated with ankylosing spondylitis (AS). HLA-B*27 consists of 42 subtypes. Different subtypes of HLA-B*27 were reported in different ethnic groups of AS patients. In this study, a high-resolution polymerase chain reaction–sequence-specific primer technique has been developed to define all the HLA-B*27 subtypes with a total of 29 primer mixtures. Two of the primer mixes were used to detect the HLA-B*27 -specific group, and 27 primer mixes were used to identify 42 subtypes ( B*2701–B*2721 and B*2723–B*27 43). The HLA-B*27 -group-specific primers have been tested in unrelated healthy subjects; 846 Northeastern Thais (NET), 334 Northern Thais (NT), 264 Karens, and 310 Bamars. Sixty-three NET (phenotype frequency, PF = 7.4%), 24 NT (PF = 7.1%), 5 Karens (PF = 1.8%), and 12 Bamars (PF = 3.9%) were positive for HLA-B*27 . Only B*2704 was found in Karens, whereas B*2704 , B*2705/37/39 , B*2706 , and B*2707 were found in NET and NT. In Bamars, B*2704 , B*2705/37/39 , B*2706 , and B*2725 were found. The distribution of HLA-B*27 subtypes was compared with other studies in Asian and Caucasian populations. Significant differences of the distribution of HLA-B*27 subtypes were found in most of the populations. This study established a simple technology for HLA-B*27 subtyping and provided basic information for anthropology and further studies in disease associations.     

HLA-B27 subtypes determination in patients with ankylosing spondylitis from Zulia,Venezuela

To perform an investigation regarding the distribution of the human leukocyte antigen (HLA)-B27 subtypes in the Zulian population with ankylosing spondylitis (AS), 48 unrelated Mestizos, HLA-B27 positive by serology, were studied using the polymerase chain reaction-specific sequence oligonucleotides probe (PCR-SSOP) and specific sequence primers (SSP) to analyze the polymorphism in exons 2 and 3 of the HLA-B27 gene. Only two of eight HLA-B27 subtypes studied (B*2701-B*2708) were found. The distribution of these alleles in the population of patients was: B*2705, 68.8%, and B*2702, 31.2%. B*2705 subtype showed significant association with patients being male. In the healthy controls, the most common subtype was B*2708. These results were compared with frequencies reported in other Mestizo and Spanish populations and showed significant differences, such as a high frequency of B*2702. Such results show that HLA*B2705 and HLA*B2702 are the subtypes most frequently associated with AS in our Mestizo population and suggest a possible protector role for HLA*B2708, which was found only in the healthy population.     

HLA-B27 subtypes in chinese patients with ankylosing spondylitis

The HLA-B27 allele has been extensively studied due to its strong association with ankylosing spondylitis (AS). In order to identify B27 alleles in Chinese patients with AS from the Shanghai area, we joined the AHS#5 of the 12 IHW and total of 68 B27 positive patients and 7 B27 positive normal persons have been investigated using polymerase chain reaction and Dig-ddTUP labeled oligonucleotides. Three primer pairs, E403 and E90as, E91As and E136as, E91Bs and E18as, were used to amplify codons 40-90 of HLA-B related alleles, codon 91 to 136 of HLA-B^*2701-B^*2706 and B^*2708 and codons 91-180 of B^*2707. A total of 11 probes were used to distinguish 8 B^*27 alleles from B^*2701 to B^*2708. 68 AS patients contain 69 B27 alleles because one patient is heterozygous B^*2704/B^*2705. A total of 4 alleles of B^*27 were detected in the AS-patient group. B^*2704 is the most common B^*27 allele in both AS patients and controls with similar frequency, 76.8% and 71.4%, respectively. We found a high proportion of B^*2705 in both AS patient (20.3%) and control (28.6%) groups. Although the control group is quite small we are still able to deduce that B^*2704 and probably also B^*2705 seem to be associated with AS in Shanghai area patients We also found one AS allele typed as B^*2707. Interestingly, for the first time we detected B^*2706 in an AS patient, which would argue against a protective effect of B^*2706 on AS susceptibility in Shanghai Chinese. The conclusion from this study is that the distribution of B^*27 alleles is not significantly different between AS patients and controls. Expanded numbers of AS patients and especially of healthy controls in different ethnic groups will be necessary to assess the contribution of different B27 subtypes to AS susceptibility.     

少儿强直性脊柱炎与幼儿类风湿性关节炎HLA-B27亚型的鉴别诊断

目的：探讨HLA－B27等位基因亚型与少年强直性脊柱炎和幼年类风湿性关节炎的关联。方法：用PCR－SSP方法对74人HLA－B27等位基因亚型进行研究，其中少年强直性脊柱炎32例，幼年类风湿性关节炎28例，5个家系中患者的父亲或母亲5例，正常对照组9例，并进行关联分析。结果：本组人群的HLA－B27等位基因由HLA－B*2704、*2705、*2702、*2707 4种亚型组成，其中少年强直性脊柱炎患者HLA－B27等位基因亚型频率为B*2704 56.25%、B*2705 40.63%、B*2702 3.13%；幼年类风湿性关节炎HLA－B27等位基因亚型频率为B*2705 60．7％、B*2704 28．57％、B*2702 3．57％及B*2707为7．14％；少年强直性脊柱炎与幼年类风湿性关节炎结果比较，HLA－B*2704基因频率在少年强直性脊柱炎组高于幼年类风湿性关节炎组（RR＝3．21，P＜0．05）。结论：少年强直性脊柱炎与HLA－B*2704等位基因亚型关联。对HLA－B27等位基因亚型的检测可成为少年强直性脊柱炎和幼年类风湿性关节炎鉴别诊断中一个有价值的实验指标。     

HLA-B*27 subtyping by PCR-RFLP in Spanish patients with ankylosing spondyiitis

Abstract: We describe the use of restriction analysis on PCR-amplified DNA for detecting all B*27 subtypes except B*2710 and B*2711 (i.e. from B*2701 to B*2709). After detecting B*27 by Sty I, double digestions consisting of Sty I plus another informative enzyme led to subtype assignment. We used mismatched primers to create restriction sites when necessary. The method avoids group-specific amplifications and other laborious optimization procedures. It was successfully tested on a panel of well characterized cell lines covering different B*27 subtypes. Then, we studied a group of 57 ankylosing spondyiitis patients and 746 controls from the south of Spain. B*27 showed a very strong association with the disease (OR=211.27, P=\0˜7). B*2702 and B*2705 distribution in controls (20% and 77.1%, respectively) differed from previously reported data in the Spanish population. We unexpectedly found the B*2707 allele in our population (one control).     

HLA-B27 polymorphism in patients with juvenile and adult-onset ankylosing spondylitis in Southern China

Abstract
Distribution of B27 subtypes in juvenile and adult-onset ankylosing spondylitis (JAS and AAS) in Southern China was studied. A total of 505 patients belonged to Han population were included (145 JAS and 360 AAS patients), and 1368 healthy individuals were included as controls. Human leukocyte antigen (HLA)-B27 typing was performed by Luminex liquid array combining polymerase chain reaction-sequence specific oligonucleotide probe (PCR-SSOP) and/or serological method. HLA-B27 subtyping was performed by polymerase chain reaction-sequence specific primer (PCR-SSP). The sequence-based typing was performed for the B*2715 samples to verify the PCR-SSP results. HLA-B27 was presented in 453 of 505 patients (89.7%), compared with 74 of 1368 controls (5.41%). B*2704 subtype in AS group was significantly higher than controls and B*2705 subtype significantly lower. B*2715 and B*2702 were found in 1.32% and 0.66% of the B27-positive patients but none in controls, and there was no significant difference between either of them and controls. B27-positive patients were 134 (92.4%) in JAS group and 319 (88.6%) in AAS group. There was no significant difference for B27 subtypes distribution between JAS (B*2704, 05, 15) and AAS (B*2704, 05, 15, 02) groups. The frequency of B*2715 in two groups was 3 (2.24%) and 3 (0.94%), respectively. The onset age of three JAS patients carrying B*2715 was 5, 9 and 13 years old, respectively. Our results suggested that B*2704 was the predominant subtype in AS patients in Southern China. B*2715 was observed in AS group only and slightly more in JAS than in AAS, and the patients carrying this allele tended to have early onset, B*2715 may be disease-association subtype.     

HLA-B27 subtypes in Asian patients with ankylosing spondylitis Evidence for new associations

Abstract: The aim of this study was to investigate the contribution of the different B27 subtypes to ankylosing spondylitis (AS) susceptibility. The polymerase chain reaction (PCR) in combination with the sequence-specific oligonucloetide probes (SSOs) was used to analyse the polymorphism in exon 2 and 3 of HLA-B27 in two Asian groups with different genetic HLA structures: Indian (I) and Thai (T) populations. The same number of AS patients (45) and healthy B27 positive donors (n=17) from both populations were analysed in order to ascertain the B27 subtypes. Three different findings can be concluded from this study: 1) B*2707 has been found to be associated with AS in both populations. This association has not been previously reported in either ethnic group. 2) B*2704 is strongly associated with AS in the Thai patients (91% in AS vs. 47% in C; RR=11.5; EF=0.83). In contrast, B*2704 was found with similar frequency in Asian Indians AS patients and controls (41% in AS vs. 41% in G). 3) B*2706 was found overrepresented in control populations and absent in AS patients (0% in AS vs. 47% in C; p_c<10^-6) showing the maximum value of protective fraction (PF=1). The B*2706 negative association with AS has not been previously described in other ethnic groups and could indicate a protective effect of this subtype on AS susceptibility. The B*2706 allele has two changes relative to B*2704 at residue 114 (His to Asp) and 116 (Asp to Tyr) in the pockets D/E. The importance that these differences can play in the pathogenesis of AS are discussed.     

HLA-A*9, a probable secondary susceptibility marker to ankylosing spondylitis in Basque patients

HLA-B27 is strongly associated to ankylosing spondylitis (AS). The objective of our study was to analyze HLA-B27 association, B27 subtype distribution and frequency of other HLA class I and DR antigens in a group of Basque AS patients. HLA class I antigens were typed serologically and HLA-B27 and A9 subtypes were determined by DNA typing in samples from 46 patients with AS, 54 B27-positive spondyloarthropathies, 82 healthy subjects and 20 B27-positive controls. A class I HLA 9.2 kb PvuII restriction fragment length polymorphism (RFLP), previously associated with AS, was analyzed in a representative group of patients and controls. We found that HLA-B*2705 conferred a relative risk of 126 for AS in this group. HLA-A9 (A*2402) allele was significantly increased in AS patients compared with healthy controls and B27-positive control group (Pcorr<0.0001) and also increased in patients affected with peripheral arthritis. No association between class I HLA 9.2 Kb RFLP and AS was found. These results suggest that HLA-A*9 allele itself or another linked gene could act as a secondary and independent susceptibility allele to AS.     

HLA B-27 subtypes in Turkish patients with spondyloarthropathy and healthy controls

The frequency and the distribution of HLA-B27 subtypes in spondylarthropathy (SpA) patients and controls were investigated in a sample Turkish population. B27 subtyping was performed by PCR-SSP method in two groups: 49 unrelated HLA-B27 positive Turkish patients with the diagnosis of SpA according to the European Spondyloarthropathy Study Group Criteria, and 55 HLA-B27 positive healthy controls. The frequency of HLA-B*27 was 2.6% in the Turkish population, and B*2705 was the predominant allele among patients with SpA. The difference was mainly between male patients and male controls The proportion of B*2705 among B27-positive patients and controls was significantly different (P=0.02). Our study supports other reports from different populations which showed that B*2705 and B*2702 were more frequent in Caucasian patients with SpA.     

Lack of carboxyl–terminal tyrosine distinguishes the B2706–bound peptide repertoire from those of B2704 and other HLA–B27 subtypes associated with ankylosing spondylitis

B*2704 and B*2706 are two closely related HLA-B27 subtypes, which differ from the common B*2705 by the Asp>Ser77, Val>Glu152, and Ala>Gly211 amino acid changes. In addition, B*2706 differs from B*2704 by the His>Asp114 and Asp>Tyrl 16 changes. In spite of their similarity B*2704, but not B*2706, was associated to ankylosing spondylitis in a same population. We have carried out pool sequence analyses of the peptides naturally bound to each of these subtypes, and of several individual peptide ligands. B*2704 and B*2706 shared with B*2705, among other features, their selectivity for Arg² and their allowance for some aliphatic and aromatic C-terminal residues in their bound peptides. The main features that distinguished both subtypes from B*2705 were: 1) their failure to present peptides with C-terminal basic residues, and 2) their allowance for both polar and nonpolar residues at peptide position 3. A major difference between B*2704 and B*2706 was that C-terminal Tyr was prominent among the peptides bound to B*2704, but was not detected among those from B*2706. The use of Tyr as a C-terminal anchor motif is the only functional feature shared by the disease-associated B*2705, B*2702, and B*2704 subtypes that is absent in B*2706. This suggests that the ability of HLA-B27 to present peptides with C-terminal Tyr might be critical for its association to spondyloarthropathy,     

Modulation on immunogenicity by HLA-B27 subtype polymorphism

Cells from the same HLA-B27- individual, PA, were stimulated in vitro in primary mixed lymphocyte culture, with either B*2705+ or B*2704+ lymphoblastoid cell lines, in independent experiments. Cytolytic T lymphocytes (CTL) were cloned at limiting dilution and the clones obtained were screened for anti-B27 alloreactivity. Most of the CTL clones generated against the B*2705+ stimulator cells were directed against the B*2705 antigen. In contrast, no anti-B27 CTL clones were found among those derived against the B*2704+ stimulator cells. This was not due to a poor cytotoxic response against these cells because a large proportion of the T cell clones derived from this stimulation were cytotoxic. B2704 differs from B*2705 by only two amino acid changes at positions 77 and 152. Previous studies (Aparacio, P. et al., Eur. J. Immunol. 1988.18: 203) have shown that none of the anti-B*2705 CTL clones derived from donor PA and amenable to detailed characterization cross-reacted with B*2704, suggesting that most of this cytotoxic response was directed against an immunodominant determinant contributed for by residues 77 and/or 152 from B*2705. The present results further suggest that the changes at these positions in B*2704 alter this determinant in such a way that B*2704 becomes less immunogenic for the particular individual PA. Furthermore, a similar poor anti-B*2704 CTL response was obtained from a second B27- responder individual, AE, stimulated with another B2704+ cell line. The single anti-B*2704 CTL clone, 64.8P, isolated from this second individual, displayed an unusual reaction pattern in that it cross-reacted with all B27 subtypes with changes only at or close to positions 77 and 152, including B*2705. Significantly, the only HLA-B27 subtype that was not recognized by CTL 64.8P was B*2703, which differs from B*2705 only at residue 59. This residue is located in the three-dimensional structure at the opposite end from residues 77 and 152 at the surface of the antigen-binding groove of the class I molecule. Thus, the area around residues 77 and 152 is not an essential part of the epitope recognized by CTL 64.8P.     

HLA-B27 in the Greek Cypriot population: distribution of subtypes in patients with ankylosing spondylitis and other HLA-B27-related diseases. The possible protective role of B*2707

The major purpose of the present study was to investigate the frequency of human leukocyte antigen (HLA)-B27 alleles in healthy controls and in patients with ankylosing spondylitis (AS) and other HLA-B27–related diseases in the Greek Cypriot population. We selected 102 HLA-B27–positive individuals (60 controls and 42 patients). Typing of the HLA-B27 alleles was performed by polymerase chain reaction amplification with sequence-specific primers. Only two alleles were detected in the patient group: B*2702 (n = 31, 73.8%) and B*2705 (n = 11, 26.2%). The HLA-B*2707 allele was detected (n = 10, 16.7%) only in the healthy controls in addition to the B*2702 (n = 31, 51.7%) and B*2705 (n = 19, 31.7%) alleles. Our results show a restricted number of HLA-B27 subtypes associated with AS and other B27-related diseases and an elevated frequency of the B*2702 allele in the AS patients. The allele B*2707 seems to have a protective role in the population studied because it was found only in the healthy controls.     

An HLA-B27 polymorphism (B*2710) that is critical for T-cell recognition has limited effects on peptide specificity

Abstract: The B*2710 subtype differs from the HLA-B27 prototype (B*2705) only by having Glu instead of Val at position 152, in the α2 helix of the peptide-binding site. In spite of its structural similarity most allore-active CTL raised against B*2705 fail to cross-react with B*2710. Indeed, of the residues that are polymorphic among HLA-B27 subtypes, the Val> Glu152 change has the greatest influence on HLA-B27 T-cell antigenicity. The molecular basis for this antigenic disparity was analyzed in this study. Sequence analysis indicated that B*2710-bound peptides have very similar motifs to B*2705-bound ones both at the main and auxiliary anchor positions. In addition, most of the individual ligands sequenced from B*2710 were previously found in B*2705. Together these results indicate that both subtypes have largely overlapping peptide repertoires. Molecular dynamics simulations of a common ligand in complex with either B*2710 or B*2705 failed to detect significant conformational changes in the peptidic main chain or in solvent accessibility of the side chains. In addition, modeling of the Val>Glu152 change into the MHC-peptide-TCR structure suggested a direct role of residue 152 in interaction with the TCR. Thus, the large differences in T-cell recognition between B*2710 and B*2705 are not explained by an effect of the Glu152 change on peptide specificity or conformation, but by different direct interactions with the TCR.     

HLA-B27等位基因和亚型与强直性脊柱炎关系研究进展

大量的研究证明,强直性脊柱炎(AS)是与人类白细胞抗原(HLA)相关性最强的疾病。AS的发病与HLA-B27阳性密切相关,并与B7、B13、B40等几个等位基因有一定关系。HLA-B位点有42个等位基因,其中HLA-B27具有高度多态性,含有22个以上的亚型,不同亚型的碱基序列间只有个别差异。B27亚型在AS患者中的分布因地区和种族上的差别而不同,在中国主要以B2704和B2705为主,但以B2705分布最广。这几年大量的人B27转基因鼠实验证明AS与B27的关联性。     

Frequency of HLA-B27 subtypes in a Danish population and in Danish patients with ankylosing spondylitis

Polymerase chain reaction in combination with sequence-specific oligonucleotide probes were used to analyze nine HLA-B27 subtypes among 51 healthy I HLA-B27 positive Danish blood donors and 30 Danish HLA-B27 positive patients with ankylosing spondylitis (AS). In the group of healthy Danes we found two subtypes, B*2705 (90.2%) and B*2702 (9.8%), however, among the AS patients only the B*2705 subtype was detected. We did not find a significant evidence for associations between AS and a particular HLA-B27 subtype in a Danish population.     

Association of an extended haplotype of HLA class I alleles and their flanking microsatellites with spondyloarthropathies in South Indian patients

Spondyloarthropathy (SpA) is a complex autoimmune disease known to have an association with the HLA system. The aims of the present study were to compare the suballelic association of HLA-B27 and other HLA class I genes with microsatellite markers spanning the HLA class I region in the South Indian population of Kerala. The five microsatellites were C1_2_A (D6S2793), C1_2_5 (D6S2811), C1_4_1 (D6S2927), MIB (D6S2810), and STR-MICA. HLA typing was performed in 67 SpA patients and 77 ethnically matched healthy controls by polymerase chain reaction using sequence-specific primers, whereas fluorescence-labeled microsatellites were analyzed using GeneScan analysis. There was a significant association of HLA-B27 and Cw*02 with SpA, whereas B*44 had a negative association with the disease. Only two HLA-B27 subtypes, B*2704 and B*2705, were observed in the South Indian population. We were able to successfully predict the major B27 subtype B*2705 based on the C1_2_5 microsatellite. A significant association of different alleles of all the microsatellite markers with SpA was observed. An extended six-locus haplotype, B*2705-Cw*02-STR-MICA(A4)-C1_4_1 (213 bp)-C1_2_5 (178 bp)-MIB (340 bp), was significantly associated with SpA.     

Binding of peptides naturally presented by HLA–B27 to the differentially disease–associated B*2704 and B*2706 subtypes, and to mutants mimicking their polymorphism

B*2704 and B*2706 are closely related HLA–B27 subtypes of which the former but not the latter is associated to ankylosing spondylitis. Their peptide specificity relative to other disease–associated subtypes was analyzed by testing binding of self–peptides naturally presented by B*2705 or B*2702, and synthetic analogs, to B*2704, B*2706, and site–specific mutants mimicking their changes. Peptides with basic, aliphatic or aromatic C–terminal residues bound to B*2705 with similar affinity. In B*2704 C–terminal aliphatic/aromatic residues were preferred. B*2706 discriminated drastically between polar and nonpolar C–terminal residues, showing strong preference for Leu and Phe, and less than B*2704 for basic and Tyr residues. Loss of single acidic charges (D>S77, D>Y116) increased preference for C–terminal Leu and Phe, but allowed efficient binding of peptides with basic residues or Tyr. Their gain (V>E152, H>D114) maintained wide C–terminal specificity, but severely impaired binding, presumably by disrupting interactions with internal peptide residues. This was compensated by Y116 in the double Dl 14Y116 mutant. The specificity of B*2704 and B*2706 was explained only partially by the separate effects of single mutations, indicating that novel properties arise from concomitant changes at various positions. For instance, specificity of B*2706 for nonpolar C-terminal residues required simultaneous removal of Asp⁷⁷ and Asp116. B*2706 differed from B*2705, B*2702, and B*2704 in its lower suitability for C-terminal Tyr, suggesting that this feature might be relevant for HLA–B27 association to spondyloarthropathy.     

HLA-B27 polymorphism in Mumbai, Western India

Human leucocyte antigen (HLA)-B27 encompasses an increasing number of subtypes that show diverse racial/ethnic prevalence in the world. One thousand-one-hundred and seventy unrelated individuals from Mumbai, Maharashtra, Western India were typed for HLA-B27 antigen by serological methods. HLA-B27 positivity was confirmed by polymerase chain reaction using sequence specific primers. High-resolution typing using sequence specific primers for HLA-B27 alleles (B*2701 - B*2721) was carried out in 70 HLA-B27-positive individuals. The frequency of B27 ranged between 1.48 and 9.6% among the caste groups studied. HLA-B27 subtyping identified B*2702 (1.43%), B*2704 (14.29%), B*2705 (70%), B*2707 (12.86%) and B*2718 (1.43%), respectively. The findings illustrate substantial genetic variation and heterogeneity within population groups from India. Extensive subtyping in other Indian caste groups will be necessary to resolve the evolutionary implications of HLA-B27 subtypes and their relationship to disease association in the Indian context.