Two new alleles of HLA-B22 group in Japanese

Five different B22 split antigens, B54, B55.1, B55.2, B56 and B22N, have been reported in Japanese. Genomic DNAs were analyzed by PCR-SSCP and sequencing methods for Japanese samples serologically positive for each split antigen. In the SSCP analysis of exon 2, B55.1, B55.2 and B56 showed identical patterns, whereas B54 and B22N showed distinct patterns. In the SSCP analysis of exon 3, B54 and B55.1 showed identical patterns, whereas B55.2, B56 and B22N showed distinct patterns. Sequence analysis further showed that B54, B55.1 and B56 were encoded by B^*5401, B^*5502 and B^*5601, respectively. The other two rare antigens, B55.2 and B22N, were found to be encoded by new alleles. The exon 2 amino acid sequences encoded by B55.2 and B22N alleles were identical to those of B55- and B56-group alleles, although that of B22N included a synonymous substitution. The exon 3 sequence of B55.2 was unique among HLA-B alleles. The exon 3 sequence of B22N was identical to those of B^*1501 and B^*4601, but distinct from those of other B22-group alleles.     

Diversity of HLA-B61 alleles and haplotypes in East Asians and Spanish Gypsies

Abstract: The distribution of HLA-B61 alleles and their association with HLA-C and DRB1 alleles were investigated in six East Asian populations (South Korean, Chinese Korean, Man (Manchu), Northern Han, Mongolian and Buryat) and Spanish Gypsies and compared to our previous report on the Japanese population. The alleles were identified using a group-specific polymerase chain reaction (PCR) and genomic DNA followed by hybridization with sequence-specific oligonucleotide probes (SSOP)- Both HLA-B*4002 and B*4006 were commonly detected in the South Korean, Chinese Korean, Man, Northern Han and Japanese populations, while HLA-B*4002 was predominant in the Mongolian and Buryat populations. Strong associations of B*4002 with Cw*0304 and of B*4006 with Cw*0801 were commonly observed in these East Asian populations. In contrast, in Spanish Gypsies, only HLA-B*4006 was found and the allele exhibited a strong association with Cw*1502. HLA-B*4003 was also identified in the South Korean, Chinese Korean, Northern Han, Mongolian and Japanese populations at relatively low frequencies, and exhibited an association with Cw*0304. Moreover, the association of these B61 alleles with the DRB1 alleles revealed considerable diversity among the different populations. HLA-B*4004 and B*4009 were not observed in these populations. Consequently, the frequencies of the B61 alleles varied among the different East Asian populations, but the individual B61 alleles were carried by specific haplotypes often regardless of the ethnic differences.     

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 HLA-B44 subtyping in Spaniards. Additional evidence for heterogeneity in Caucasian populations

HLA-B44 is one of the most common HLA class I alleles in Caucasians. Exon 3 oligotyping and sequence analysis have define five B44 subtypes: B^*4402, B^*4403, B^*4404, B^*4405 and B^*4406, with variations in exons 2 and 3. We have developed a conventional DNA typing system by using a single B12-group specific amplification including exon2-intron2-exon3 in combination with 6 oligoprobes to define all B44 subtypes. 140 HLA-B44 positive unrelated Spanish Caucasians were typed. Family studies established 30 B44-bearing haplotypes. The distribution of B44 subtypes in our population was: B^*4402 32.5%, B^*4403 66.5%, B^*4404 0.5%, B^*4405 0.5%, B^*4406 not found. B^*4402 and B^*4403 represented the 99% of the B44 alleles, as described in Caucasians. However, these two major subtypes showed an inverted frequency when compared with other Caucasian populations, B^*4403 twice as frequently as B^*4402 in Spaniards. HLA-B44-associated chromosomes showed 20 different haplotypes (including HLA-A,-C,-DR,-DQ), although demonstrating clear separated haplotype composition between B^*4402 and B^*4403: B^*4402 associated to class I alleles A2 ( ) and Cw5 ( ), and B^*4403 associated to the class II allele DRB1^*0701 ( ). These findings, in addition to the validation of a complete B44 oligotyping system, revealed further evidence of antigen frequency differences among populations of the same ethnic origin.     

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.     

Conservation of genes encoding HLA-B5 and B35 cross-reactive group antigens in various races

HLA-B5 and B35 CREG antigens include HLA-B35, B51, B52, B53, and B78. Recent studies suggest that the genes encoding the HLA-B5, B35 CREG, and HLA-B58 antigens share a common ancestor. We sequenced the exons of the genes encoding HLA-B51, B53, and B58 from American black individuals and the gene HLA-B52 encoding from an Arabic individual, and compared them with previously reported sequences of HLA-B51 (B^*5101) and HLA-B52 (B^*5201) from Japanese, HLA-B53 (B^*5301) from an Algerian, and HLA-B58 (B^*5801) from a Sardinian. The sequences of the genes from the American black and Arabic individuals were identical to those from the other races. These findings support the hypothesis that these antigens have evolved prior to divergence of the major ethnic groups.     

Two new class I alleles (HLA-Cw15052 and B0706) constitute a novel haplotype in Caucasians.

HLA-Cw^*15 alleles have been previously described in association with the HLA-B specificities B51, B61 and B73. In this study, we have characterized a novel haplotype constituted by the new alleles Cw^*15052 and B^*0706.

The two variants of Cw^*1505 differ in a synonymous change in nucleotide 24 of exon 1 coding for the signal peptide. The new B^*0706 allele differs from B^*0702 and B^*0705 in single changes at exons 3 and 5, respectively, and could be considered an intermediate evolutionary step between them.

Even though strong linkage disequilibrium between the HLA antigens Cw7 and B7 has been observed, ‘HLA-C blank’-B7 haplotypes are not rare either. Cw^*15 and B7 have been found to be associated in several Caucasian individuals. Therefore, it is likely that Cw^*15 accounts for a part of the aforementioned ‘HLA-C blank’-B7 haplotypes.

With the identification of Cw^*15052, a previously proposed pathway for the evolution of Cw^*15 alleles can be completed and will be discussed.     

HLA-B35 alleles in 440 individuals from 16 different populations as assessed by DNA typing.

As part of the Anthropology Component, the distribution of HLA-B35 alleles (B^*3501 to 3513) was studied in 16 different populations by group specific amplification and SSOP hybridization. The results were as follows:

The predominant alleles in most Caucasian populations were 3501 > 3503 > 3502 > 3508. However, B^*3502 predominated in Jews, B^*3508 in Arabs, B^*3503 in Gypsies and Sardinians seem to have only B^*3501 and 3502. B^*3504, 3505, 3506 and 3509 were restricted to Amerindians, where there are still other new B35 variants to be characterized. In most individuals the different B35 alleles were found in phenotypic combinations with HLA-Cw4, suggesting that the B35, Cw4 haplotype may have existed before subtype diversification. A detailed analysis of HLA-B35 alleles in other populations might help to draw a precise picture of B35 evolution.     

Identification of the gene encoding a novel HLA-B39 subtype Two amino acid substitutions on the β-sheet out of the peptide-binding floor form a novel serological epitope

Serologic analysis suggests the existence of a novel HLA-B39 subtype (HLA-B39N) in the Japanese population. To identify this novel allele, a gene encoding HLA-B39N was cloned and the exons were sequenced. A gene encoding HLA-B39N (B^*3904) and B^*39011 differs by two nucleotide substitutions at codons 11 and 12 whereas B^*3904 and B^*39013 differ by three nucleotide substitutions at codons 11, 12, and 312. One nucleotide difference at codon 11 produces a change from serine in B^*3901 to alanine in B^*3904 whereas another difference at codon 12 changes valine in B^*3901 to methionine in B^*3904. The residues 11 and 12 are located on the β-sheet out of the peptide-binding floor and are completely buried in the molecule. These results suggest that the substitutions at these residues alter the conformation of other residues forming epitopes of alloantibodies. Analysis of HLA-B^*3901 genes in the Japanese population showed that both B^*39011 and B^*39013 were observed in the Japanese population. The present study suggests that B^*3904 may have evolved from B^*39011 rather than B^*39013.     

Epitope mapping of human monoclonal antibodies to HLA-B27 by using natural and mutated antigenic variants

The epitopes defined by three human monoclonal antibodies (mAbs) (Tr3B6, TrCG10, TrBH12) against HLA-B27 have been mapped by flow cytometry. For this purpose we used murine transfected cells expressing at their surface hybrid antigens between HLA-B7 and -B27 and, in addition, Epstein-Barr virus cells lines expressing the six HLA-B27 alleles B^*2701 to B^*2706. The results indicated that the mAbs are domain specific. TrBH12 recognizes the first external (alpha-1) domain. Residues critical for the TrBH12 epitope are located in the alpha-1 helix and include the polypeptide stretch 63–76 plus a critical amino acid at position 77. Tr3B6 binds the second external (alpha-2) domain, and one mutation (VAL¹⁵²→GLU¹⁵²) destroyed its epitope. TrCG10 also binds the alpha-2 domain.     

Peptide binding to the differentially disease-associated HLA-B2704 and B2706 and to mutants mimicking their polimorphism

Peptide binding to the disease-associated antigen HLA-B27 and its modulation by subtype polimorphism was addressed in this study. The effect of subtype changes was analyzed using a quantitative stabilization assay in which the surface expression of HLA-B27 on RMA-S cells was measured as a function of the concentration of peptides naturally presented by B^*2705, B^*2702 and their analogues. Binding to B^*2704, B^*2706 and to mutants mimicking the changes between these subtypes and B^*2705 was analyzed. Bulky aliphatic (Leu), aromatic (Phe or Tyr) or basic (Arg,, Lys) C-terminal residues contribute similarly to binding to B^*2705. For B^*2704 aliphatic C-terminal residues are the most suitable, but aromatic and even basic residues can also be accomodated. B^*2706 has strong preference for bulky aliphatic C-terminal residues and moderate suitability for aromatic ones. The effects of individual changes in the subtypes account only partially for the binding properties of B^*2704 and B^*2706, suggesting interactive effects of the changes in determining the peptide specificity of these subtypes that are not fully accounted for by simple additive effects of the individual changes.     

Nucleotide sequences of novel HLA-B35 subtypes

In recent reports we described novel hybridization patterns (HP) corresponding to 22 potentially new HLA-B locus alleles in a panel of 547 subjects studied by PCR-SSOP. Three of them correspond to new subtypes of B35. To confirm the hybridization results we have isolated DNA from PBL and performed PCR, DNA cloning and nucleotide sequencing. One of the alleles, locally called B-3505v was found in three individuals: two Hispanic, one Caucasoid. It differs from B^*3505 by 3 nucleotide substitutions that lead to changes in residues 94 (Ile > Thr), 95 (Ile > Leu) and 103 (Val > Leu). B-3505v differs from B^*3501 in residues 97 and 103. Another allele called B-3508v, was found in 7 individuals, (6 of 122 Toba Indians, 1 of 18 Pilaga Indians). It differs from B^*3509 in two silent nucleotide substitutions (codons 135 and 138) and in one substitution in residue 156 (Arg > Leu). The new allele has a hybrid sequence between B^* 3508 and B^*4801. A third subtype, locally called B-3504v, observed in two Hispanic individuals, is identical to B^*3512. B^*3512 differs from B^*3504 by 3 nucleotides and one amino acid. Substitutions in residue 95 contribute to the structure of specificity pocket F, 97 to C and E, and 156 to pockets D and E. Therefore it is possible that some of the new alleles may have different peptide binding profiles. Since differences in residue 156 have been shown to affect allorecognition and mediate GvHD, identification of such variants may have important implications in transplantation and perhaps in studies of immune responses to peptides and pathogens.     

Identification of HLA-B44 and HLA-B45 alleles using a nested arms-PCR method

The establishment of DNA based identification of HLA Class I specificities has made high resolution typing possible at the allelic level. With such methods, it is feasible to identify population variation in allelic frequencies. Working towards higher allelic resolution of the HLA Class I loci we have developed a panel of primers to identify all the known HLA-B^*44 alleles and B^*4501 using a nested ARMS-PCR approach. There are currently seven different B^*44 alleles identified: B^*4402, B^*44031, B^*44032, B^*4404, B^*4405, B^*4406 and B^*4407. The system was validated using DNAs from B lymphoblastoid cell lines which have been sequenced for their B^*44 alleles. Further typing has been performed on a panel of 29 cell lines from the IHW cell panel and some 37 samples collected as part of the anthropological study of the Orkney islanders, all of which were previously defined as being B^*44 by the 12th Workshop HLA Class I typing kit. In the Orcadian population B^*4402 was found in 28 individuals to be the most common B^*44 allele, with 18 of these individuals expressing the A^*02-Cw^*0501-B^*4402 haplotype. The remaining 9 B^*44 positive individuals were all found to express the A^*29-Cw^*1601-B^*44031 haplotype. Methods and results will be presented.     

B78Hen, a new Caucasian member within the B78 family

We have previously described an antigen B7801 (SNA) belonging into the B5 CREG, exclusively found in North and West African individuals. More recently, a new subtype B^*7802, differing from B^*7801 by two nucleotide substitutions within the class I heavy chain 1 domain has been found in a Caucasian individual (1).

Here we describe another B78 variant (B78Hen) that we observed in a Belgian German family. Serology displayed a pattern identical to the one of SNA. Careful analysis of the pedigree could rule out African ancestry. The exon 2 of B78Hen differed only by one nucleotide substitution from B^*7801 and B^*7802. In B78Hen, Asp at codon 74 of B^*7801 was substituted by Tyr, as in B^*7802. With respect to B^*7802, a silent mutation was observed in B78Hen at codon 23 (ATC to ATT). The ATT codon at that position is also found in most B51 subtypes, B^*52012 and B^*7801 and is absent in B^*51022, B^*5105, B^*7802 and B^*35.

Beside its phylogenetic implication, this finding has also practical consequences. The B78 antigens are not restricted to African subjects as it was previously thought. In view of its frequent confusion with B35 and B70 antigens, it must now be searched actively in Caucasian subjects.     

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.     

Analysis of endogenous peptides eluted from HLA-B27 variants: implications in the susceptibility to spondylarthropathies

Population studies suggest that some HLA-B27 subtypes (HLA-B^*2705, B^*2702) could be more strongly associated with the development of spondylarthropathies than others (B^*2703, B^*2706, B^*2709). Differences in the peptide binding groove could impose differences in the nature of peptides bound by these different alleles. We have eluted endogenous peptides from C1R-B^*2705 and B^*2703 transfectants. The B^*2705 HPLC profile was more complex than the B^*2703 one. Several B^*2705 and B^*2703 individual peaks were sequenced by Edman degradation and mass spectrometry. Some peptides were shared by both subtypes. One B^*2705 eluted peptide present in a major HPLC fraction was not found in the B^*2703 peptides. The corresponding synthetic peptide bound in vitro specifically to T2-B^*2705 and not to T2-B^*2703. This result emphasizes that even one amino-acid difference outside the major anchor binding pockets at position 59 between B^*2705 and B^*2703 could notably influence the endogenous peptides naturally presented. This could have consequences in terms of T cell repertoire selection and development of autoimmunity.     

Five HLA-B22 group alleles in Japanese

HLA-B22-group alleles in Japanese were identified using PCR-single-strand conformation polymorphism (SSCP) and sequence analyses. We analyzed genomic DNAs obtained from Japanese individuals positive for HLA-B22 group antigens (HLA-B54, B55, B56) including two locally proposed splits (B55.2, and B22N). In the SSCP analysis of both exons 2 and 3, we discriminated five different B22-group alleles. Each allelic pattern corresponded to each serological split antigen. Direct sequencing analysis of exon 2 and exon 3 showed that alleles encoding B54, B55.1 and B56 antigens in Japanese are encoded by B*5401, B*5502 and B*5601, respectively, and those encoding B55.2 and B22N antigens are previously unidentified alleles, B*5504 and B*5603, respectively. Full-length cDNA sequencing showed that the B*5603 sequence is identical to those of B*5501, B*5502, B*5601, and B*5602 in exons 1 to 2 except for a synonymous substitution at nucleotide position 165 in exon 2. On the other hand, the sequence of exons 3 to 7 was identical to those of some B15 and B46 alleles, suggesting that B*5603 was generated by a recombination event between one of the B55 and B56 alleles and one of the B15 and B46 alleles in intron 2. As for B*5504, the entire exon 1 to 7 sequence is identical to that of B*5502 except that the 5-half of the exon 3 sequence is identical to those of some B7, B27, B40 and B48 alleles, suggesting that an event such as gene conversion, segmental exchange, or double recombination occurred in this region.     

Subtypes of B44 in six different populations.

We have investigated the distribution of HLA-B44 subtypes in various populations, see table. Of the five B44 subtypes investigated, two are apparently quite rare because they were only found in the local central european panel (B^*4404 and B^*4406). The european populations are characterized by a relatively high frequency of B^*4402, while in the Albanian, in the Asiatic and African populations, the subtype B^*4403 is prevailing. The distribution of the B44 subtypes in the Czech and the Munich population are virtually the same. We conclude from these data, that B44 subtypes have different distributions in different populations of the world and must therefore be taken into consideration when matching for bone marrow transplantation.     

Clarification of HLA-B serologically ambiguous types by automated DNA sequencing

Abstract: Assignment of HLA-B types can be hampered by ambiguous reactivity of the typing sera resulting in inaccurate HLA-B assignments. In this study, 19 Korean samples exhibiting ambiguous serologic reactivities were characterized by DNA sequencing. Alleles identified from 7 samples were previously undetected in this population (B*1517, B*4101, B*4701, B*5001, and B*5106) and from 9 samples were common alleles in this population (B*4002, B*4003, B*4006, B*1501, B*1401, B*67012, and B*5401). Three samples were putative HLA-B homozygotes. Three major factors causing serologic ambiguity were identified: weak or false negative reactivity of typing sera (52.4%); cross or false positive reactivity of the sera (38.1%); and absence of information on the reaction patterns due to the lack of appropriate sera in the typing kit (e.g. B*4101 encoded molecule) or to the presence of recently characterized molecules (e.g. B*5106 encoded molecule) (9.5%). Overall, sequencing was helpful in clarifying ambiguous serologic reaction patterns improving the HLA typing for the Korean population.     

Diversity of alleles encoding HLA-B40: relative frequencies in united states populations and description of five novel alleles

The frequency of each B*40 allele was determined by DNA sequencing in four major United States populations: Caucasians, African Americans, Asians/Pacific Islanders, and Hispanics. Thirty-two individuals from each ethnic group, who were previously described serologically as B40, B60, or B61, were randomly selected out of a pool of 82,979 unrelated individuals for allele characterization. Out of nine different B*40 alleles identified in this study, B*4001 and B*4002 were the two most frequent B*40 alleles in all the population groups. B*4001 was the primary B*40 allele seen in Caucasians (83%) and African Americans (76%), while B*4002 was found in the majority of Hispanics (62%). The distributions of both alleles were comparable in the Asian/Pacific Islander population. These two alleles were the only B*40 alleles detected in Caucasians while four to five additional B*40 alleles were seen in the other population groups. The other B*40 alleles detected in this study included: B*4003 and B*4010 in Asian/Pacific Islanders; B*4012 and B*4016 in African Americans; and B*4004, B*4006, and B*4027 in Hispanics. Analysis revealed significant differences between Hispanics and all other groups as well as between African Americans and Asian/Pacific Islanders. This report also describes five novel B*40 alleles: B*4019, B*4020, B*4024, B*4027, and B*4028.