Novel HLA-A and HLA-B alleles

Abstract: Nine novel HLA-A and HLA-B alleles are described: A*2609, A*6803, A*6806, B*1539, B*1540, B*2712, B*4103, B*5109, and B*5603. Most appear to have arisen by gene conversion events. B*5603 appears to have arisen by a reciprocal recombination event joining exon 2 of a B*55/*56 allele with exon 3 of a B*15 allele. Serologically, the antigen encoded by this allele types with broad B22- and Bw6-specific alloantisera. Also unique, the antigen encoded by B*2712 does not react with B27-spedfic alloantisera but does react with Bw6-specifk alloantisera.     

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.     

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.     

Immunogenetic study of a new HLA allele,B*2723

A novel HLA-B*27 allele (B*2723) detected by irregular serological and PCR-SSP typing results was identified by nucleotide sequencing of exons 2 and 3. B*2723 differs from B*27052 by nine nucleotides which encode seven amino acid changes at positions 63 (Glu to Asn), 67 (Cys to Phe), 69 (Ala to Thr), 70 (Lys to Asn), 71 (Ala to Thr), 74 (Asp to Tyr) and 77 (Asp to Ser) in the alpha1 helix. All these substitutions are possessed by B*35 alleles suggesting that B*2723 was created by a gene conversion-like event involving B*27052 and a B*35 allele. Using the HLA-A*26 and DRB1*12 alleles of the B*2723-bearing haplotype as 'markers', two further examples of B*2723 were found in 29,851 blood donors. Therefore, B*2723 has a 'minimum' gene frequency of 0.000034 (phenotype frequency 0.0067%) in blood donors resident in Wales. In all three families, B*2723 was present on a haplotype with: A*26; Cw*0202; DRB1*1201/6/7; DRB3*02; DQA1*05; DQB1*0301. The B*2723 product failed to react with HLA-B27 antisera and reacted weakly or not at all with Bw4 antisera. Lack of the ECAKA motif at amino acid positions 63, 67, 69-71 probably accounts for lack of the B27 specificity while the amino acid combination 74Y, 77S, 80T, 81L may cause aberrant Bw4 reactivity.     

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.     

Serologic ambiguity and allelic frequency of the HLA-B40 family in the Korean population

The most frequently identified HLA-B type in Koreans is HLA-B40 (13.4%). Due to the lack of mono-specific alloantisera and cross reactivity of sera used as typing reagents, discrimination between the serologic splits of B40, B60 and B61, has been a problem in tissue typing laboratories. In this study, an efficient PCR-SSP typing system was established to distinguish B60 and B61 and to assess the difficulty in serologic assignment for these types. The SSP system was also used to elucidate the frequency of B40 alleles (B*4001-B*4008) encoding B40 molecules in the Korean population. Eighty eight unrelated individuals identified serologically as B40 positive were selected from 358 consecutive volunteers from the unrelated bone marrow registry. Seven sets of PCR that amplify exons 2 and 3 of the HLA-B gene using 10 sequence specific primers (SSP) were used for discrimination between B60 and B61, and for B40 allelic typing. A clear discrimination of B60 and B61 was possible in all samples including 48 serologically ambiguous samples (B60 – 14/48; B61 – 34/48) and 5 potentially B40 homozygous samples (B60/B61 heterozygotes – 4/5; B60 homozygote - 1/5). Therefore, the use of a focused SSP approach enhances serologic definition of HLA types in routine clinical testing. In allelic typing, all B60 samples (26) appeared to be B*4001, but B61 samples revealed more heterogeneity (B*4002 – 36/58, B*4003 – 4/58, B*4006 – 18/58). In addition, B*4003 seemed to be closely associated with the A24-Cw3-DRB1*02 haplotype (3/4). The characterization of allele frequency as well as haplotypic association will be helpful in determination of the optimal size of the volunteer marrow donor pool in the Korean population.     

Sequence-based typing of exons 1-5 of a new HLA-B allele, B*3927*

Anew human leucocyte antigen-B (HLA-B) allele, B*3927, was detected in three individuals of a Caucasian family by routine typing with sequence-specific primers (SSP). Serological typing showed B27 Bw4 and B39 Bw6, whereas SSP detected only B*27 as well as the Bw4 and Bw6 motif. The sequence of exons 1-5 of the new allele was determined by allele-specific amplification and sequencing. The new B*39 allele showed one nucleotide difference with B*390101 at position 299 in exon 2. Codon 100 changed from GAG to GTG, resulting in an amino acid substitution from glutamic acid to valine at position 76 of the mature protein. The haplotype carrying the B*3927 allele was A*010101, B*3927, Cw*120301, DRB1*0101 and DQB1*050101.     

Identification of a novel allele HLA-B*7805 in a Japanese female

A new HLA-B*78 allele, B*7805, was identified in a healthy Japanese female. The results of her serological HLA class I typing showed an unusual Bw4/Bw6 pattern with strongly positive reactivity to anti-Bw6, i.e. A24, -, B52, -, Bw4, Bw6. In DNA typing, she was typed as A*24, -, B*52, B*78-like, Cw1202, -, (Bw4, Bw6). Cloning and sequencing of exon 2 and exon 3 of her B locus genes revealed a new allele B*7805. The cloned B*7805 differed from B*78021 by three nucleotide substitutions in exon 2 at position 259 (A to G), 261 (C to G) and 272 (A to C), and contained sequences defining Bw6 motif in the region of codon 77 to 83.     

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 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.     

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.     

A nonsense mutation in exon 3 results in the HLA-B null allele B*5127N

A new HLA-B null allele has been identified within the B*51 group by combined serological and molecular typing of an Italian Caucasoid family. Serological data indicated that the proband typed homozygous for A2 and B60. Confirmatory typing using sequence specific oligonucleotide hybridization (SSPOH) detected a second B allele within the B*51 group. Allele specific typing (SSP) for B*51 subtypes, including the known B*5111N allele, was performed, and typing results were consistent with B*5101, suggesting the presence of a new null variant. Cloning and sequencing of this allele identified a B*5101 variant with a nonsense mutation in exon 3. This new null allele has been designated B*5127N. The combined use of serologic and DNA-based typing methods facilitates the identification of null and low-expression alleles. An overview of null alleles of class I HLA is presented.     

HLA-B*4012: a new allele with unique serological features

We have defined the new allele HLA-B*4012, which had been isolated from a black individual. It was initially recognized as a serologically unique allele when typing her father for renal transplantation. The HLA class I phenotype was A*0201,*6602; B*4001,*4012; Bw6; Cw*0304,*1505. Sequencing from exon 1 through intron 3 of B*4012 was performed. B*4012 is identical to B*4001 and B*4010 in exon 3, and in the 3' part of exon 2, but it is unique in that exon 1 and the 5' part of exon 2 are identical to B*1503, B*1509, B*1510, B*1518, B*1523, and B*1529. The generation of this allele is best explained by a recombination event in exon 2 (break point between nucleotides 205 and 222 from the beginning of the coding region) of B*4001 or B*4010 with one of these B*15 variants as a donor allele. Its unique serological feature (B48, B60, B70, and B72 reactivity) is consistent with the sequence data of its donor alleles.     

Identification of two new HLA-B22 variants, HLA-B*5509 and B*5606

In our recent study using high-resolution HLA-B locus typing by sequence-based typing (SBT) we identified 9 new alleles in a total of 355 unrelated individuals (4). Three of them concerned an allele belonging to the B22 group. One of them, B*5607, showed the unusual presence of a Bw4 sequence motif, as described previously (5). In this report the other two B22 variants are described; one belonging to the B55 specificity and named B*5509; the other one being a B*56 allele and assigned B*5606, which brings the total number of alleles belonging to the B22 group to 18.     

Identification and sequencing of HLA-B*0714 and B*2718 alleles and novel exon 1 sequences of B*0709 and B*2714 alleles in potential bone marrow donors

Sequence specific oligonucleotide probe hybridization and sequence specific primer PCR typing of volunteer bone marrow donors suggested the presence of variants of known HLA-B alleles in two individuals. PCR products encompassing HLA-B locus exons 1, 2, and 3 were prepared, subcloned and sequenced. A Hispanic individual had a novel B*07 allele (B*0714) and a Chinese individual had a novel B*27 allele (B*2718). In two other individuals, a previously unknown sequence of exon 1 was determined for HLA-B*0709 (African American) and B*2714 (Native American). These findings further illustrate the substantial genetic variation present at the HLA-B locus within human populations. We discuss the structural variation in the protein sequence for these HLA-B alleles and its potential functional effects.     

A new HLA-B*27 allele (B*2719) identified in a Lebanese patient affected with ankylosing spondylitis

Eighteen different HLA-B*27 alleles (B*2701-B2718) have so far been recognized by the WHO Nomenclature Committee for Factors of the HLA System. Frequency and disease association of these alleles with spondyloarthropathies differ among ethnic groups. We describe here a novel HLA-B*27 subtype identified in a Lebanese patient suffering from ankylosing spondylitis (AS). This new variant differs from the common HLA-B*2705 DNA sequence at five different nucleotide positions. These nucleotide changes lead to three amino acid differences in the alpha2 domain; Thr to Ile at position 94, Leu to Ile at position 95 and Asn to Arg at position 97. Since this novel allele is encountered in an AS patient, the associated sequence changes are not expected to affect significantly neither the presentation of a putative arthritogenic peptide nor the conformation-dependent recognition by effector cells.     

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.     

HLA-DQA1 and MLC among HLA (generic)-identical unrelated individuals

We modified a previously published PCR-RFLP for DQA1 typing (1) and examined the predictive value of HLA-DQA1 in mixed lymphocyte cultures (MLC) among matched (HLA generic types) pairs of unrelated individuals. There were 61/102 (60%) pairs with positive MLC, one-third of which could be predicted by DQA1* typing alone. DQA1 matching and MLC reactions were classified into 3 groups: 1) DQA1 mismatches showing positive MLC: 19/102 (19%); 2) DQA1 matches showing negative MLC: 41/102 (40%); 3) DQA1 identical showing positive MLC: 42/102 (41%). Five different HLA haplotypes that result from non-random association of HLA generic types (high delta haplotypes) were overrepresented in the individuals tested. One of these haplotypes carrying HLA-B7, DR2 was found associated with three different DQA1 alleles (*0201, *0103, *0102). The remaining four high delta haplotypes were associated with one DQA1 allele in all independent examples tested: HLA-A1, B8, DR3 with DQA1*0501; HLA-A26, B38, DR4 with DQA1*0301; HLA-A2, Bw62, DR4 with DQA1*0301 and HLA-A1, Bw57, DR7 with DQA1*0201. Forty per cent of the negative MLC were explained in part by the excessive number of individuals carrying two of these four haplotypes, which probably carry determinants in linkage disequilibrium with HLA. Nineteen per cent of HLA-identical (generic types) unrelated pairs show positive MLC reactions and all of them are DQA1* mismatched, suggesting that DQA1* allele typing should be used to screen samples prior to performing MLC.     

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*4703: sequence confirmation, serology and distribution

This study has confirmed the nucleotide sequence of exons 2 and 3 of the B*4703 allele, discovered by an unusual HLA-B47 and Bw6 serological pattern, in two subjects of Black/Japanese and Caribbean Black descent. Titration studies on 25 HLA-B47 cross-reactive sera, stimulated by B13, B27, B44 and B60, and nine Bw6 antisera/monoclonal antibodies, showed that the B*4703 product can be distinguished from the established HLA-B47 specificity. The phenotypes of these donors and an International Cell Exchange donor suggests an association between B*4703 and Cw*0701/ 06 in Black subjects. No examples of B*4703 (or B*4702) were found in 10,194 PCR-SSP HLA-A,B typed Welsh Bone Marrow Donor Registry panel members indicating a phenotype frequency of <0.0098% in this primarily Northern European Caucasoid population.