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-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.     

Predominant association of HLA-B*2704 with ankylosing spondylitis in Chinese Han patients

Abstract
The HLA-B27 subtypes have a varied racial and ethnic prevalence throughout the world. However, the association of B27-subtypes with ankylosing spondylitis (AS) in the mainland China is unknown. To determine the association of B27-subtypes with AS in the Mainland Chinese Han population, a total of unrelated 153 patients with AS were enrolled in a large case-control association study, and 1545 unrelated, healthy, ethnically matched blood donors were included as controls. The genotyping of B27 and its subtypes was performed using the polymerase chain reaction with sequence specific primers (PCR-SSP). A total of 130 (84.97%) AS patients and 61 (3.95%) healthy controls were B27 positive. Three B27-subtypes, B*2704, B*2705 and B*2710, were further identified, of which both B*2704 and B*2705 were strongly AS associated. B*2710 was only detected in one AS patient and two other healthy controls. Considering only B27-positive cases and controls, a statistically different frequency of B27-subtypes was observed, with an over-representation of B*2704 ( P = 0.018). B*2704 was clearly more strongly associated than B*2705 with AS [odds ratio (OR ) = 2.4, P = 0.011]. Furthermore, a combined analysis including three previous studies of B27-subtype distributions in Chinese AS cases confirmed the stronger association of B*2704 with AS than B*2705 (OR = 2.5, P = 0.00094).     

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 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.     

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.     

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。     

New insights regarding HLA-B27 diversity in the Asian population

A polymerase chain reaction-sequence-specific primer (PCR-SSP) method which distinguishes all B27 alleles described at present (B*2701-23) has been developed. The distribution of B27 alleles was characterised in six different Asian populations. HLA-B*2705, 02, 04, 07, 22 (formerly B*2706) subtypes found in Asian populations differ in their ethnic distribution, which may be the result of different genetic and geographic origins. Furthermore, two novel B27 alleles were found in this study. B*2714 was identified in two Siberians, one of whom was a patient with ankylosing spondylitis. B*2715 was found in two patients with ankylosing spondylitis in Thais. These associations have not previously been reported in either ethnic group.     

TAP1 and TAP2 polymorphism in Spanish patients with ankylosing spondylitis

OBJECTIVE: HLA-B27 is strongly associated with ankylosing spondylitis (AS); however, the association is not absolute and additional susceptibility factors in the MHC region could play a role. We studied the influence of polymorphism in the transporter associated with antigen processing (TAP) genes, including point mutations not previously analyzed. METHODS: HLA-B*27 typing and subtyping as well as TAP1 and TAP2 typing were performed by PCR-RFLP. Forty-four AS individuals were compared to 61 ethnically matched random individuals and 35 B*27-positive healthy unrelated individuals as controls. RESULTS: The frequency of the TAP1B allele was significantly greater in the patient group compared with the random controls (corrected p value (p(c)) = 0.035; odds ratio = 15.8, p = 0.01). A greater frequency was also evident when B*27-positive patients and B*27- positive healthy controls were compared, although it did not reach statistical significance. No differences were observed in TAP2 alleles between the groups studied. DISCUSSION: We did not find a primary association between TAP2 polymorphism and AS susceptibility. Formal confirmation of a linkage between the TAP and HLA-B loci would probably require family studies.     

HLA-B27 alone rather than B27-related class I haplotypes contributes to ankylosing spondylitis susceptibility

Characterization of non-B27 susceptibility genes will be required to know the pathogenesis of AS. The aim of this study was to examine whether ankylosing spondylitis (AS) susceptibility is controlled by B27 alone rather than B27 haplotypes and, whether other closely related class I loci, such as MICA and TNFA genes might play a role in AS. Three hundred eleven B27-positive samples from Caucasoid, Asian, and African populations were selected and genotypes were carried out by PCR/SSOP (HLA-B27 and HLA-C), PCR/SSP (MICA-TM polymorphism in the transmembrane region), PCR/SSCP (MICA alleles), and PCR-RFLP (TNF-alpha). Of these, 161 were AS patients, chosen in order to investigate the contribution of TNFA and MICA loci to AS in HLA-B27 positive individuals. Some findings can be concluded from the study: (a) No significant differences of TNF-alpha promoter alleles at position -308 and -238 (A/G) were found between AS patients and B27 matched alleles from healthy controls; (b) strong linkage disequilibrium was found between the B27 and the MICA alleles. The MICA-A4 was found to be in association with B*2705,02,03 and 08; MICA-A5 with B*2704 and B*2707 and MICA-A.5.1 with B*2706; (c) no significant differences of MICA alleles were found between AS and controls carrying the B27-associated alleles, and therefore no evidence is provided for an additional role of MICA gene in AS susceptibility; (d) there are a striking correlations between the structure of B27 extended haplotypes (from MICA region to HLA-C) and the ethnic distribution of these subtypes. The results of differential linkage disequilibrium with HLA-B27 subtypes suggest that B27 itself remains the primary gene for AS susceptibility, and TNFA and MICA are not involved in the pathogenesis of the disease.     

A single-nucleotide polymorphism marker within the FCRL5 gene and HLA-B27 positive Han Chinese ankylosing spondylitis patients

The aim of this study was to determine whether FCRL5 genes in concert with human leukocyte antigen-B27 (HLA-B27) genotypes are associated with susceptibility to ankylosing spondylitis (AS) in Chinese population. One hundred and sixty-nine HLA-B27-positive AS patients (107 males and 62 females) and 184 HLA-B27-positive matched controls (112 males and 72 females) were analyzed from Han Chinese populations by case–control design, and their samples were genotyped using a panel of two single-nucleotide polymorphism (SNP) markers (rs6427384, rs12036228) within the FCRL5 gene by ligase detection reactions (LDRs) and the HLA-B27 subtypes were determined by polymerase chain reaction (PCR) using sequence-specific primer (SSP) methods. Our results show that in addition to B27 , the SNPs rs6427384 and rs12036228 were associated with AS, and the C-T haplotype was higher in cases with AS than in the control population [74.8% vs 63.6%, Fisher's P = 0.003, odds ratio (OR) = 1.660,95% confidence interval (CI) = 1.184−− 2.326]. Our results suggest that, in addition to HLA-B27 , a novel polymorphism within the FCRL5 gene confers susceptibility to AS in Han Chinese population.     

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.     

High variability of HLA-B27 alleles in ankylosing spondylitis and related spondyloarthropathies in the population of northern Spain

The distribution of B27 alleles (B*2701-23) was characterized by PCR-SSP in ankylosing spondylitis and related spondyloarthropathies (SpA) in a sample of B27 positive patients from northern Spain. Six B27 alleles were identified: B*2705,02,03,07,08 and B*2713. B*2705 and 02 were the most common alleles in the SpA studied: ankylosing spondylitis (AS) (n = 89), reactive arthritis (ReA) (n = 11), psoriatic arthritis (PsA) (n = 29), and inflammatory bowel disease (IBD) (n = 21). B*2707 and B*2708 were found in PsA patients and B*2703 in one patient with IBD. B*2713 was identified in a healthy control family. B*2713 has not been reported to be represented in either ethnic group. Thus, this population shows higher levels of B27 diversity than other Caucasian groups.     

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.     

Discrimination of HLA-B27 alleles by group-specific amplification followed by solid-phase sequencing

HLA-B27 is known to be highly associated with ankylosing spondylitis. Until now, nine B27 subtypes have been sequenced and may contribute in different fashions to ankylosing spondylitis. Additionally, the divergent subtypes may be of clinical importance in bone marrow transplantation with alternative donors. The purpose of this study was to determine the different subtypes of HLA-B27 by a direct sequencing approach. The typing strategy is based on a group-specific amplification of the second and third exon followed by automated fluorescence sequencing of the polymorphic regions. The extensive sharing of sequence motifs between the different B alleles made it impossible to specifically amplify the B27 group under the precondition of including all sequence variations necessary for a postamplification specificity step. Therefore, for setting up a direct sequencing approach of B27, co-amplified B alleles had to be taken into account. In order to get unambiguous sequencing chromatograms without any heterozygous positions, nested sequencing primers were used which selectively matched sequence motifs only present in the second and third exon of the amplified B27 alleles. This strategy allowed in all cases investigated a clear separation of the haplotypes, revealing unequivocal sequencing results. Using this method, we have investigated 93 B27-positive individuals. Sequencing identified the alleles B*2702, 2703, 2704, 2705, and 2707. B*2701, 2706, 2708, and 2709 were not represented in the population studied.     

B*27 in molecular diagnostics: impact of new alleles and polymorphism outside exons 2 and 3

HLA-B*27 is known to be associated with ankylosing spondylitis and several methods have been applied to determine its presence or absence. In this report two molecular methods were used for detection of B*27. The polymerase chain reaction sequence-specific primer (PCR-SSP) method was performed to detect the presence or absence of B*27, whereas the sequence-based typing method (SBT) was used to identify the B*27 subtype. The PCR-SSP method used to detect B*27 was updated to enable the detection of all B*27 alleles. The typing results obtained by this method were compared with the serological typings of 262 individuals. Fifty of them were found to be B*27 positive by PCR-SSP and 46 also showed positive serological reactions with B27-specific sera. The four discrepancies were the result of the presence of B*2712 in three individuals and B*2715 in one individual; both alleles showed no serological reactions with B27-specific antisera. With SBT the sequences of exons 1 through 4 were determined to unequivocally assign the B*27 alleles. Eleven different subtypes were detected in 78 individuals, including three new B*27 alleles: B*27054, B*2715 and B*2717. The allele B*27054 showed an allelic drop out when exon 3 was amplified. Three differences with B*27052 were demonstrated; one in exon 1, one in intron 1 and one in intron 2, the latter being responsible for the allelic drop out. The B*2715 allele was serologically not detectable with several B27-specific sera, but showed Bw4-positive reactions. The sequence of B*2715 showed two mismatches with B*2704. The sequence of B*2717 showed one mismatch with B*27052 at position 248 (A-->T), which was considered to be a conserved position in all B alleles.     

Subtypes of HLA-B27 detected by cytotoxic T lymphocytes and their role in self-recognition

In the present study cytotoxic T lymphocytes were generated in MLC of lymphocytes from two unrelated HLA-A, B, C-identical, B27-positive, but D/DR-different, individuals. These CTL were shown to detect subtypes of HLA-B27. CTL specific for influenza virus lysed infected target cells matched for HLA-B27 only when they shared the same subtype. This indicates that the two subtypes of HLA-B27 detected by CTL function also as distinct elements in a self-restricted CTL response. Both subtypes were found among patients with ankylosing spondylitis.     

MICA gene and ankylosing spondylitis: linkage analysis via a transmembrane-encoded triplet repeat polymorphism

In order to address the possibility that the MICA gene located 47 kb upstream from HLA-B is involved in the pathogenesis of ankylosing spondylitis (AS), we have investigated microsatellite polymorphism in the transmembrane region of MICA in Caucasian patients with AS. The microsatellite allele consisting of 4 repetitions of GCT/AGC was present at significantly higher frequency in the patient group (Pc<0.0000001) than in the ethnically matched control group. However, the frequency of the (GCT/AGC)₄ allele was significantly low in the B27-positive patients than in the B27-positive healthy controls (Pc=0.0145). These observations suggest that B27 itself remains the primary genetic marker for AS, although the significantly dissimilar pheno-type frequency of the (GCT/AGC)₄ allele in B27-positive patients and healthy individuals may reflect the existence of other genetic factor(s) in the HLA-B27 haplotype involved in the development of AS.     

Prevalence of HLA-B*27 subtypes in the Tamil population of India with Ankylosing spondylitis and its correlation with clinical features

IntroductionHLA-B*27 is strongly associated with Ankylosing spondylitis (AS). Its subtypes show considerable geographic and ethnic difference. The main aim of this study was to assess the frequency of subtypes of HLA-B*27 in the Indian Tamil AS patients.Methods and materialsAdult AS patients positive for HLA-B*27 were considered for the study. The high-resolution typing to define HLA-B*27 subtypes were done using Invitrogen B kits from One Lambda (SeCore® Sequencing Kits, Thermo Fisher, United States).Results and conclusionPrevalence of subtypes identified were HLA-B*27:04 (52.2%), HLA-B*27:05 (41.6%), HLA-B*27:07 (3.5%) and HLA-B*27:02 (2.7%). All subtypes showed disease predisposition for males. The most common extra articular manifestation seen was enthesitis in HLA-B*27:04 and HLA-B*27:05. Uveitis was mainly associated with HLA-B*27:05 and dactylitis with HLA-B*27:04. A significant peripheral joints involvement for female and axial joint involvement for males was seen in HLA-B*27:04. Our study establishes the prevalence of HLA-B*27 subtypes and the associated clinical phenotypes among the Indian Tamil population. Considering the variability of presentation, organ involvement, and disease course in different subtypes and across ethnicities it is critical to define these associations in the ethnic populations we treat for their appropriate care considering the significant negative health and socioeconomic effects of AS.