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.     

Unexpected Bw4 and Bw6 reactivity patterns in new alleles

The Bw4 and Bw6 epitopes were the first HLA-B differences to be recognized by serological methods. Since then 44 serological groups have been identified and more than 250 alleles assigned by molecular typing methods. In general each serological HLA-B group is associated with the presence of either the Bw4 or the Bw6 epitope. There are several exceptions to this rule. Four alleles, B*4601, *7301, *5503 and *1806, show no serological reactivity with either Bw4 or Bw6. Although the Bw6 motif at residues 77-83 is present in these alleles the Bw6 epitope is modified by a valine at residue 76. One or more alleles from the B8, B40 and B62 groups are identified as Bw4 positive, whereas all others are Bw6 positive. In the groups B27, B44 and B47 several alleles are found to be Bw6 positive, while the majority is Bw4 positive. Histocompatibility testing of dialysis patients and their families revealed the serological presence of an unexpected Bw4 epitope associated with B18 in one patient and B56 in another. Allele-specific amplification and sequencing of exons 2 and 3 of these HLA-B alleles revealed the presence of the Bw4 sequence motif for both. The new alleles were assigned B*1809 and B*5607, respectively. In 2 other patients the presence of a new B*07 allele was determined by sequence based typing. Although the new allele, B*0715, showed the Bw6 sequence motif at positions 77 to 83, a substitution of amino acid 76 from glutamic acid to valine was identified. This change resulted in an aberrant Bw6 serological reaction pattern.     

HLA B27 polymorphism in Western India

We have characterized HLA B27 alleles in a sample population of Maharastra, Western Indians (n = 51), with the aim to investigate the different subtypes present among this population. The study was carried out using polymerase chain reaction with sequence-specific primers (PCR-SSP) and reverse line strip (RLS) techniques. Significant new findings have arisen from this study: B*2704, B*2705, B*2707, B*2708 and B*2714 alleles were found to be present, and two novel B27 alleles, B*2708 and B*2714, were found in this Indian population. In addition, B*2714 was observed in a patient with ankylosing spondylitis. This association has not been previously reported in ethnic groups from India.     

Characterization of a new HLA-B18 allele, B*1806, which lacks expression of the Bw6 epitope

Abstract: HLA-B18 is a well defined Bw6-associated serologic specificity. Up to now, four different sequences have been characterised in Caucasian populations (B*1801,3,4,5), and one in Orientals (B*1802). We report a new HLA-B18 subtype (B*1806) which was serologically detected in a Spanish Caucasian individual as a B18 Bw4-associated antigen. Complete coding region sequencing showed that B*1806 differs from B*1801 in a unique nu-cleotide at position 299 (A to T), giving rise to an amino acid replacement in residue 76 (glutamic acid to valine) placed at the α1 domain. Therefore, in contrast to the serologic results, B*1806 possesses the canonical Bw6 motif at position 77–83. Subsequent flow cytometric assays proved that B*1806 evidences neither Bw4 nor Bw6 epitopes. Only three additional HLA-B alleles encode valine at codon 76, B*4601, B*7301 and B*5503, and like B*1806, all of them would include a Bw6 motif associated to the negative recognition by Bw6 antibodies. These findings support that valine at position 76 will modify the Bw6 epitope drastically, and suggest that this group of HLA-B alleles would define a third, Bw4 and Bw6-negative, lineage of molecules. Furthermore, valine 76 will also prevent the binding of Bw6 antibodies to those HLA-C antigens with the canonical Bw6 epitope (Cw*1,3,7,8,12,13,14,16).     

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.     

Characterization a novel HLA-B40 allele with serological Bw4 motif, HLA-B*4047, in the Finnish population and confirmation of B*270503 allele

We describe a novel HLA-B*40 allele assigned as B*4047*. The B*4047 allele was detected in a Finnish patient awaiting kidney transplantation. The patient had a "short" B60-like serological specificity with Bw4 association. After sequencing, the B*4047 allele was found to be identical to B*4001, except having five amino acid changes in exon 2, including the entire motif corresponding to Bw4 and w6 specificity. As a result of recombination or gene conversion, B*4047 has the Bw4 motif instead of expected Bw6. Screening of B40 alleles in the Finnish population revealed no other cases with this pattern, suggesting that this allele is rare. The sequence of B*270503 presented here provides the complete sequence for exons 2 and 3 for this allele. B*270503 allele differs from B*270502 by a single synonymous nucleotide substitution at non-variable position 489 in exon 3.     

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.     

Functional characterization and exon 2 – intron 2 – exon 3 gene sequence of HLA-B*2712 as found in a British family

The HLA-B*27 group of alleles has been extensively studied due to the association of particular B*27 alleles with ankylosing spondylitis (AS). We describe here an HLA-B*27 allele (B*2712) encoding an antigen that lacks reactivity with B27 monoclonal antibodies (moabs) and alloantisera but reacts with some B40/B60 moabs and alloantisera and expresses the Bw6 public epitope. This allele was discovered by the segregation of an HLA-B allele undetectable by PCR–SSP within a Caucasian family from the British population referred for routine bone marrow transplant HLA typing and found in the haplotype A*29; B*2712 ; Cw*1203; DRB1*13; DQB1*0603. Serological typing showed a lack of reactivity with four B27 moabs and four alloantisera but positive reactivity with moabs and alloantisera specific for B40/B60 and Bw6 public epitopes. Subsequent sequencing showed the closest homology was with B*2708 with three mismatches in exon 2 at positions 204, 209 and 210. The intron 2 sequence was identical with other B*27 lineage alleles including a 2 base pair deletion at positions 95 and 96. The relationship between HLA-B*2712 and reported B60 associations with susceptibility to AS remains to be determined.     

HLA-B27 polymorphism and worldwide susceptibility to ankylosing spondylitis

HLA-B27 is strongly associated to ankylosing spondylitis (AS) and represents a family of eleven B27 alleles (B*2701–11). Our aim was to analyze the distribution of B27 subtypes by PCR/SSOP and genomic sequencing in a large group of populations ( n =17). 711 B27-positive samples from Caucasoid, Asian, African, Amerindian and Polynesian populations were selected to ascertain transracial gene mapping of the B27 subtypes. 476 of these were AS patients, chosen to investigate the contribution of B27 alleles to AS susceptibility. Some significant new findings have arisen from this study: 1) B*2705 was the predominant subtype in circumpolar and subarctic areas. B*2702 was found to be practically restricted to Caucasian populations, showing a higher frequency in Middle-East (Jews) and North Africa (Arabs/Berbers) groups. 2) B*2703 appears associated with AS in Western Africans. This is of remarkable interest since it was suggested that B*2703 would be negatively disease-associated. 3) Although B*2706 appears negatively associated with AS in Thais, we identified two patients from northern China carrying it. This may be a reflection of a disease heterogeneity and could indicate that more than one pathogenic agent can be involved in AS. B*2709 has been recently described as negatively associated with AS in Sardinians. The molecular changes His 114Asp (B*2706) and Asp 116His (B*2709) could modify the genetic susceptibility to AS.     

Identification of a new HLA-B allele,B*3705 containing a Bw6 sequence motif

HLA-B37, which is Bw4 type antigen frequently found in linkage disequilibrium with A1, Cw6 and DR10 in all ethnic groups, generally has a very low frequency all over the world. We report a new HLA-B*37 allele, B*3705, identified in two potential bone marrow donors in the Korean population. B*3705, which has the Bw6 nucleotide segment, differs from B*3701 in three nucleotide positions: 311, 317 and 319 in exon2. The serological profile of B*3705 did not exhibit the B37 specificity. The putative haplotype associated with B*3705 in the Korean population could be A*02-Cw*0602-DRB1*1001-DQA1*0101-DQB1*0501-DPB1*02011.     

Expression of an unusual Bw4 epitope by a subtype of HLA-B8 [B*0802]

Abstract: The primary structure of a variant HLA-B8 antigen has been determined by cDNA cloning and sequencing. The variant, B*0802 differs, from the common B*0801 subtype at positions 77–83 of the α₁ helix that determine the Bw4 and Bw6 public epitopes. Whereas B*080l has the common Bw6 motif, B*0802 has the Bw4 motif found in B*13 and B*44 allotypes. Serological analysis of B cell lines expressing B*0802 and of a B*0802 transfectant made with the HLA-A, B negative cell line 721.221 shows that B*0802 reacts with Bw4-specific antibodies, but at a level much lower than expected for Bw4 positive HLA-B allotypes.     

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-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.     

A new HLA-B27 allele found in a healthy Japanese

We have found a rare variant antigen (tentatively named B27KH) which reacts with B27-monospecific antisera and some B40 group specific and Bw6-specific antisera. The reaction pattern of B27KH was clearly distinguished from those of the B27 antigens encoded by B*2702, 2704, 2705 and 2706. On the other hand, B27KH and B*2707 antigen (originating from non-Japanese) showed very similar serological reaction patterns. Nevertheless, the B27KH allele was discriminated from B*2707 by PCR-SSOP typing, suggesting that the allele is a new member of the B27-group alleles. In the nucleotide sequence analysis, the allele encoding B27KH was found to differ from B*2707 by two base substitutions which resulted in an amino acid change in the 1 domain.     

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.     

Characterization of a new HLA-B allele (B3702) generated by an intronic recombination event

Routine serological HLA typing of a Syrian family revealed a Bw4-associated HLA-B blank antigen showing Mendelian segregation together with the haplotype A1, Cw2, DR11, DQ7. A more extensive serological analysis showed no conclusive typing reactions with sera towards HLA-B27 and HLA-B37 antigens. Full length cDNA was sequenced in both senses for two individuals. Comparison of the consensus sequence with all other HLA-B published sequences evidenced a new HLA-B allele, confirming, as serologically predicted, the greatest relationship to HLA-B37 and HLA-B27 genes. Based on the exon 2 sequence identity to B^*3701, this new allele has been designated B^*3702. Proteins derived from B^*3702 and B^*3701 differ in 9 amino acids at alpha 2 (7 residues), and in transmembrane and cytoplasmic domains. Exons 3 to 8 are identical between B^*3702 and a number of HLA-B27 subtypes (all except Oriental HLA-B27 subtypes B^*2704, B^*2706 and B^*2707). B^*3702 alpha 1 domain differs from those of B^*2701, B^*2702, B^*2703, B^*2705, and B^*2708 in 9, 11, 9, 7, and 11 residues, respectively. The mosaicism found in B^*3702 strongly suggests that this new allele could be derived by homologous recombination at the intron 2 region between the B^*3701 gene and some of the 5 non-oriental B27 subtypes.     

Complete coding sequence of HLA-B*2712: a serologic B27-negative antigen associated to Bw6

Abstract: We report the complete coding sequence of a new HLA-B27 subtype, B*2712, which was found in a Caucasian Spanish family within the chromosome A2-Cw2-B*2712-DR15-DQ6. B*2712 was first detected as a segregating B blank Bw6-associated antigen. Extensive serologic analysis demonstrated that this new B27 subtype was not recognised by any of the B27-monospecific antibodies, giving positive reactions only with some monoclonal reagents against B40 or B27,40. Sequencing analysis showed a high similarity with B*2708, only differing in three clustered amino acid residues at positions 69 to 71 located in the α helix of the α1 domain. Residues 69 and 71 point towards the T-cell receptor, while amino acid 70 points to the antigen binding site. Loss of the conserved structure of pocket B as well as the differentiated pocket F configuration suggests that B*2712 does not confer ankylosing spondylitis susceptibility, Misleading serologic definition supports the usefulness of DNA-typing methods to complement HLA class I typing.     

HLA-B27 allele diversity in Indians: impact of ethnic origin and the caste system

HLA-B27 is a serological specificity which encompasses an increasing number of subtypes that show varied racial/ethnic prevalence in the world. Here, data from 5129 Indians (4500 population and caste; 629 tribal) is compiled from the literature. In addition, HLA-B27 subtyping of 58 positive individuals from Maharastra is presented. Analysis revealed an increased B27 antigen frequency among the north Indian groups (>5%) compared to the south Indian groups (<5%). HLA-B27 subtyping identified B*2704 (34.48%), B*2705 (36.2%), B*2707 (15.51%), B*2708 (10.34%) and B*2714 (3.44%) alleles in the population groups from Maharastra, but these differed in their distribution among the caste and tribal groups studied. The study showed that more 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.     

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.     

Routine HLA-B27 typing by flow cytometry: differentiation of the products of HLA-B*2702, B*2705 and B*2708

Patient HLA-B27 typing is widely performed as an aid to the diagnosis of several diseases, particularly ankylosing spondylitis. Typing by flow cytometry, using monoclonal antibodies, has been shown to be a potentially useful alternative to classical serology on account of its speed, simplicity and economy. However, we required a flow cytometry typing procedure that would accurately differentiate HLA-B27 (Bw4) from B2708 (Bw6) and not be confounded by other HLA-B7/B27 cross-reactive group antigens. Accordingly, we evaluated the simultaneous use of two monoclonal antibody preparations, ABC-m3-FITC (anti-B27 + weak B7)/BB7.1-PE (anti-B7) and FD705-FITC (anti-B27), by testing a highly selected panel of 62 reference lymphocytes containing examples of all HLA-B7/B27 cross-reactive group antigens, including: HLA-B42, B47, B48, B73, B703, B2702, B2705 and B2708. In addition, 268 whole blood samples from routine patient requests for B27-associated disease typing were tested in parallel with HLA-B typing using the standard complement-dependent microlymphocytoxicity test. The detailed specificity of the three monoclonal antibodies was established and the products of HLA-B*2702, B*2705 and B*2708 were found to be readily differentiated from each other and all other HLA-B7/B27 cross-reactive HLA-B antigens.