HLA-B27 (B*2701) specificity for peptides lacking Arg2 is determined by polymorphism outside the B pocket

B*2701 differs from B*2705 by three amino acid changes: DY74, DN77, LA81, and from B*2702 only by two: DY74 and T180. Tyr74 is located in the C/F cavity of the peptide-binding site, and is unique to B*2701 among HLA-B27 subtypes. Binding of natural B*2705 and B*2702 ligands to B*2701, and to mutants mimicking subtype changes, was analyzed. In addition, sequencing of the peptides bound in vivo by B*2701 and the Y74 mutant was carried out. The main distinctive feature of B*2701 was its presentation of peptides with Gln2. Synthetic analogs bound in vitro similarly as the corresponding ligands with Arg2. Moreover, both Gln2 and Arg2 were dominant upon pool sequencing of B*2701- bound peptides, and 2 of 8 natural ligands contained Gln2. Suitability of Gln2 was largely determined by the Y74 change, as indicated by: 1) binding of Gln2 analogs to this mutant, and 2) detection of Gln2 by pool sequencing of Y74-bound peptides. B*2701 bound peptides with C-terminal aromatic or Leu residues, and interacted with these motifs more strongly than B*2702. The Y74 mutation alone was not responsible for poor binding of peptides with C-terminal basic residues to B*2701, since they bound efficiently and at least one was presented in vivo by this mutant. Most peptides bound to the A81 mutant worse than to B*2705, but frequently better than to B*2701 or B*2702, suggesting that other subtype changes were compensatory. The peptide specificity of B*2701 suggests that this subtype may determine susceptibility to spondyloarthropathy.     

Sequence analysis of exons 1, 2, 3, 4 and 5 of the HLA-B5/35 cross-reacting group

The HLA-B5/35 cross-reacting group (CREG) is a set of closely related antigens including HLA-B35, B51, B52, B53 and B78. The nucleotide sequences of exon 1 through 5 of the B5/35 CREG were determined to assess the level of polymorphism. For exons 2 and 3, the previously described sequence-based typing (SBT) strategy was applied, the nucleotide sequences of exon 1, 4 and 5 were determined by allele-specific sequencing. A total of 225 unrelated individuals were HLA-B typed by heterozygous sequencing of exons 2 and 3. In the B5/35 CREG, 26 different alleles were identified, whereas 63 non-B5/35 CREG alleles were sequenced. The SBT strategy was proven to be reliable and efficient for high resolution typing of the B5/35 CREG. The nucleotide sequences of exon 1, 4 and 5 were determined for the 26 different B5/35 CREG alleles to establish the level of polymorphism. For seven different alleles, of which the exon 1, 4 and 5 sequences were hitherto unknown, the sequences were elucidated and in agreement with the known B5/35 sequences. Nineteen HLA-B5/35 CREG alleles with previously published exon 1, 4 and 5 sequences were sequenced in at least two individuals. Three new alleles were identified. The first, B*5204, showed a difference at position 200 compared to B*52011, which was previously considered a conserved position. The other two alleles, B*3542 and B*51015, showed exon 2 and 3 sequences identical to B*35011 and B*51011, but differences in exons 1 and 4, respectively. B*3542 had differences at position 25 and 72 and B*51015 showed a difference at position 636. More polymorphism might be present outside exons 2 and 3 than previously thought.     

Ambiguities of human leukocyte antigen-B resolved by sequence-based typing of exons 1, 4, and 5

The elucidation of the sequences of human leukocyte antigen-B (HLA-B)-exons 1 through 5 has led to an increase of ambiguities with alleles having identical exon 2 and 3 sequences, but differences in other exons. At the moment, 26 HLA-B alleles show such ambiguities which can be resolved by sequencing the exons in which the differences are located. Here we report a sequence-based typing (SBT) strategy for heterozygous sequencing of exons 1, 4, and 5, in addition to the previously described exons 2 and 3. The strategy was validated against a panel of 25 individuals, carrying HLA-B alleles from 33 different allele groups. Correct assignment of all HLA-B alleles was obtained for exons 1 through 5. In addition, the SBT protocol was used to resolve ambiguities in 50 individuals. The ambiguous combinations studied were B*0705/06, B*0801/19N, B*1512/19, B*180101/17N, B*270502/13/0504, B*350101/42/40N, B*390101/0103, B*400102/0101, B*440201/19N/27, and B*510101/11N/0105/30/32. In all cases, sequencing revealed the first allele to be present, except for three individuals with B*07. One of them typed B*0705; the other two were B*0706. The described SBT protocol for sequencing exons 1, 4, and 5 is a valuable tool for resolving ambiguities of HLA-B alleles with differences in these exons, as well as for studying the polymorphism of HLA-B outside exons 2 and 3.     

B*2707 differs in peptide specificity from B*2705 and B*2704 as much as from HLA-B27 subtypes not associated to spondyloarthritis

HLA-B*2707 is associated with ankylosing spondylitis in most populations. Like the non-associated allotypes B*2706 and B*2709, it lacks Asp116 and shows preference for peptides with nonpolar C-terminal residues. The relationships between the peptide specificity of B*2707 and those of the disease-associated B*2705 and the non-associated subtypes were analyzed by determining the overlap between the corresponding peptide repertoires, the sequence of shared and differential ligands, and by comparing allospecific T cell epitopes with peptide sharing. The B*2707-bound repertoire was as different from that of B*2705 as from those of B*2706, B*2709, or the two latter subtypes from each other. Differences between B*2707 and B*2705 were based on their C-terminal residue specificity and a subtle modulation at other positions. Differential usage of secondary anchor residues explained the disparity between the B*2707-, B*2706-, and B*2709-bound repertoires. Similar differences in residue usage were found between B*2707 and both B*2704 and B*2706, as expected from the high peptide overlap between the two latter subtypes. T cell cross-reaction paralleled peptide sharing, suggesting that many shared ligands conserve their alloantigenic features on distinct subtypes. Our results indicate that association of HLA-B27 subtypes with ankylosing spondylitis does not correlate with higher peptide sharing among disease-associated subtypes or with obvious peptide motifs.     

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.     

The interplay between HLA‐B27 and ERAP1/ERAP2 aminopeptidases: from anti‐viral protection to spondyloarthritis

The human leukocyte antigen class I gene HLA‐B27 is the strongest risk factor for ankylosing spondylitis (AS), a chronic inflammatory arthritic disorder. More recently, the Endoplasmic Reticulum Aminopeptidase (ERAP) 1 and 2 genes have been identified by genome wide association studies (GWAS) as additional susceptibility factors. In the ER, these aminopeptidases trim the peptides to a length suitable to fit into the groove of the major histocompatibility complex (MHC) class I molecules. It is noteworthy that an epistatic interaction between HLA‐B27 and ERAP1, but not between HLA‐B27 and ERAP2, has been highlighted. However, these observations suggest a paramount centrality for the HLA‐B27 peptide repertoire that determines the natural B27 immunological function, i.e. the T cell antigen presentation and, as a by‐product, elicits HLA‐B27 aberrant behaviours: (i) the misfolding leading to ER stress responses and autophagy and (ii) the surface expression of homodimers acting as ligands for innate immune receptors. In this context, it has been observed that the HLA‐B27 carriers, besides being prone to autoimmunity, display a far better surveillance to some viral infections. This review focuses on the ambivalent role of HLA‐B27 in autoimmunity and viral protection correlating its functions to the quantitative and qualitative effects of ERAP1 and ERAP2 polymorphisms on their enzymatic activity.     

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.     

Characterization of two new HLA-B alleles by sequence-based typing: HLA-B*0817 and HLA-B*1311

In this brief communication we report the characterization of two new HLA-B variants officially named HLA-B*0817 and HLA-B*1311. The HLA-B*0817 allele was identified in a Caucasoid male candidate for renal transplantation in the North Italy Transplant program. The nucleotidic sequence of exons 2, 3 and 4 of this novel allele is identical to that of HLA-B*0804 except for three point mutations in exon 2: from A to G at position 259, from C to G at position 261 and from G to A at position 302. These mutations are responsible for two aminoacidic substitutions [Asn (r) Glu, codon 63, and Ser (r) Asn, codon 77]. HLA-B*1311 was found in a volunteer donor belonging to National Marrow Donor Program(R). This new variant is identical to that of HLA-B*1301 except for three nucleotide substitutions at positions 353, 355 and 369 leading to two aminoacidic variations from Ile to Thr at codon 94 and from Ile to Leu at codon 95 and a silent mutation at codon 99.     

抗原处理相关转运体等位基因与HLA-B27检测呈阳性的强直性脊柱炎的相关研究

目的探讨汉族人群抗原处理相关转运体(transporter associated with antigen processing, TAP)等位基因与HLA-B27及强直性脊柱炎(ankylosing spondylitis, AS)的相关性。方法用聚合酶链反应-序列特异性寡核苷酸探针杂交技术,对48例AS患者(B27+)及123名正常对照人群(B27+27名、B27-96名)进行TAP1、TAP2等位基因分型及变异位点氨基酸表型频率分析。结果汉族人群TAP1表现型主要为Ile/Ile和Asp/Asp,而TAP2则以Val/Val、Ala/Thr和Stop/Stop占优势。TAP1和TAP2至少各有4种等位基因型,分别为TAP1*0101、TAP1*0201、TAP1*0301、TAP1*0401和TAP2*0101、TAP2*0102、TAP2*0201、TAP2*0202。研究对象中有9.9%(17/171)TAP1探针无法定型,15.8%(27/171)TAP2无法定型,呈杂交空白。病例组与对照组间TAP等位基因型分布无差异(P＞0.05)。AS(B27     

The effect of LILRB1 but not LILRA3 gene polymorphism in immunopathology of ankylosing spondylitis—A parallel to KIR genes

LILR and KIR receptors recognize HLA‐B27 and may influence immune response in ankylosing spondylitis (AS) development. Purpose of the study was to analyse LILRB1/LILRA3 polymorphisms in AS. We observed a possible protective effect of the T allele of LILRB1 rs1061680:T>C and no association with insertion/deletion polymorphisms of LILRA3 with AS.     

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.     

Susceptibility to ankylosing spondylitis is independent of the Bw4 and Bw6 epitopes of HLA-B27 alleles

We have characterized HLA-B27 alleles in a sample of the population from the Azores (n=46) with the aim of investigating the contribution of different subtypes to ankylosing spondylitis (AS). The study was carried out using PCR-SSOP and in some samples genomic sequencing was conducted. Some significant new finding have arisen from this study. First, B*2705,B*2702,B*2703,B*2707 and B*2708 alleles were found to be represented in this population. The polymorphism of B27 alleles found in a sample of the population from the Azores is higher than the Caucasian groups described. B*2703 and B*2707 have not previously been described to be represented in Caucasians and this could indicate admixtures with different populations of the world. In addition, the B*2708 allele was found to be associated with AS in a large family from the Azores. This association has not been previously reported in either ethnic group and needs to be confirmed in other population studies. This is of considerable interest since has only been described as a rare subtype underrepresented in the British population and has not been previously found to be associated with AS. B*2708 carries the sequence specifying the Bw6 epitope in contrast to most B27 alleles which carry a Bw4 sequence. Differences in this region (residues 77-83) can alter the F-pocket and affect T-cell recognition. The importance that these molecular changes can play in the pathogenesis of AS is discussed.     

Immunogenetics of two new HLA-B alleles: B*4414 and B*5708

Two new alleles, HLA-B*4414 and B*5708, were identified in north-western European Caucasoid blood donors. B*4414 differed from B*440201 by two nucleotide substitutions in exon 3 [positions 66 (T to C) and 69 (G to A)] producing two amino acid differences between the B*440201 and B*4414 specificities (tyrosine to histidine at codon 113 and aspartic acid to asparagine at codon 114). B*5708 differed from B*570101 by a single substitution (G to C) at position 247 in exon 2 causing an amino acid difference between B*570101 and B*5708 products of arginine to proline at codon 83. The likely haplotypes bearing these alleles were identified. Both alleles occurred once in approximately 25,000 random blood donors so both have a frequency of approximately 0.00002 (carriage frequency 0.004%). B*4414 and B*5708 specificities both gave 'short' serological reactivity for their expected specificities and Bw4. The likely reasons for this are discussed in relation to the epitopes of B44 and B57.     

Characterization of three new HLA Class I Alleles in Spanish Caucasians,HLA‐A*02:620, HLA‐B*27:150 and HLA‐B*07:05:01:02

Three new HLA class I alleles, HLA‐A*02:620, HLA‐B*27:150 and HLA‐B*07:05:01:02, were described in the Spanish Caucasoid population.     

Sequencing of two new HLA class II alleles: DRB3*0218 and DQB1*030202

Two novel human leukocyte antigen (HLA) class II alleles for DRB3 and DQB1 genes detected in Caucasoid Spanish individuals are described: DRB3*0218 and DQB1*030202. Both alleles have been found during routine high-resolution typing by sequencing. DRB3*0218 shows a novel DRB3 gene polymorphic position, located at amino acid residue 58, alanine to glutamic acid. This residue is shared by several DRB1 alleles, including all described DRB1*11 subtypes. DQB1*030202 differs from DQB1*030201 by a point mutation at position 319 (T to C). This nucleotide change generates a new codon at amino acid position 75 that is not shared by any other DQB1 allele.     

Mono-allelic amplification of exons 2-4 using allele group-specific primers for sequence-based typing (SBT) of the HLA-A, -B and -C genes: preparation and validation of ready-to-use pre-SBT mini-kits

Class I allelic typing based on sequencing is reliable, immutable and easy to analyse when only one allele is amplified using a specific mono-allelic technique. A strategy has been developed to selectively amplify exons 2, 3 and 4 of each allele of the three class I loci, previously identified by generic typing, in order to sequence these alleles from their intronic parts in only one direction. This procedure is based mainly on the polymorphism of exon 1 and intron 1 of the HLA-A, -B and -C genes with allele group-specific forward primers and locus-specific reverse primers so as to perform mono-allelic amplification in a 'One Step' pre-sequence-based typing (pre-SBT) PCR. The 5' polymorphism found at each locus is nevertheless not sufficient to discriminate all allelic combinations. Hence exon 2 and exon 3 polymorphism had to be used in a 'Two Step' pre-SBT PCR method to selectively amplify the two alleles in the 1.8%, 7.6% and 0.9% of unresolved combinations found in our laboratory for, respectively, the HLA-A, -B and -C loci. Preparation and validation of 'ready-to-use' aliquots of primer-mixes, pre-SBT buffer and sets of Dye terminator reaction mixtures containing locus-specific intronic primers makes the procedure easy and efficient. The SBT method is the only allelic typing technique used in our laboratory (to date, 742 HLA-A*, 802 HLA-B* and 615 HLA-Cw* alleles have been sequenced) and our successful participation in the national and international quality controls of 4 years ago testifies to the accuracy of the results.     

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,