A comprehensive approach for typing the alleles of the HLA-B locus by automated sequencing

Abstract: We describe an approach for typing alleles of the HLA-B locus by using automated sequencing technology. The exon 2 and exon 3 nucleotide sequence of each allele is determined directly from genomic DNA in two steps. In the first step, HLA-B exon 2, intron 2 and exon 3 sequences are amplified with one or two primer pairs out of a panel of 5 primer pairs that describe all known HLA-B alleles. In the second step, templates are sequenced in 5' and 3' orientations in a PCR assay that utilizes Taq polymerase to incorporate fluorescent dye-labeled nucleotides into each new strand synthesized. Gel electrophoresis of the labeled products is performed in an automated DNA sequencer. The derived sequences are aligned against reference sequences and each nucleotide position is evaluated for homology to consensus sequence. Using this strategy, the HLA-B allele sequence is directly ascertained with precision and efficiency. The automated sequencing strategy can be readily applied in the clinical laboratory as a practical tool for high resolution typing of HLA-B alleles.     

Identification of a new HLA-B*08 variant, HLA-B*0804

The HLA-B locus is the most polymorphic locus known with currently over 100 different alleles described. Many of these alleles encode variants of the serologically-defined tissue transplantation antigens. This high level of diversity makes accurate tissue typing difficult. Here we present the sequence of a new HLA-B *08 variant, HLA-B *0804. found in Caucasian siblings JH and PF serologically typed as HLA-B51/B59 and HLA-B59/B60, respectively. Additionally, DNA-based typing by the polymerase chain reaction using sequence-specific primers (PCR-SSP) identified HLA-B *51 in JH and HLA-B *4001 in PF. However, PCR-SSP failed to identify a second allele in either of these individuals. The unusual finding of a B59 antigen in a Caucasian and the discrepant molecular typing results suggested that these individuals might express novel HLA molecules. Using denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we characterized a novel HLA-B *08 variant, HLA-B *0804. The presence of this allele was confirmed by cloning and sequencing. HLA-B *0804 differed from HLA-B *0801 by only one nucleotide substitution resulting in an amino acid replacement of phenylalanine by serine at position 67. Incidentally, this single nucleotide difference was sufficient to prevent amplification by PCR-SSP. This striking difference between both the serologically typed antigen and the PCR-SSP-identified allele compared to the sequenced allele supports the use of sequence-based typing for the analysis of HLA class I locus alleles.     

Further diversification of the HLA-B locus in Central American Amerindians: new B*39 and B*51 alleles in the Kuna of Panama

Several new HLA-B locus alleles have been discovered in South American Amerindians. By contrast, analysis of the MHC class I alleles of North American native populations has revealed few new HLA-B alleles. This suggests that the HLA-B locus is evolving rapidly in South American populations. Here we describe the HLA-B locus alleles present in individuals from a Central American tribe, the Kuna of Panama. Using a sequencebased typing technique that separates alleles by denaturing gradient gel electrophoresis (DGGE) followed by direct sequencing, we determined the HLA-B alleles from eight Kunas. Two of the HLA-B alleles present in the Kuna have been previously described in other South American Amerindian populations; one allele has been characterized in a Mexican-American. We characterized two new HLA-B alleles in the Kuna, HLA-B*3911 and HLA-B*5110 . HLA-B*3911 differed from HLA-B*3905 by only a single nucleotide substitution in exon 3. This substitution resulted in an amino acid replacement of leucine by arginine at residue 156 in the alpha 2 domain. Such a change may affect the repertoire of peptides that are bound by this molecule. HLA-B*5110 differed significantly from other HLA-B*51 alleles in that it is the result of an unusually large intra-locus recombination event of minimally 216 nucleotides. This recombination results in an allele that is part HLA-B*51 and part HLA-B*40. Thus, more dramatic recombination events may also play a role in the rapid evolution of the HLA-B locus in Amerindians.     

Allele assignment for HLA-A, -B, and -C genes to the Tenth International Histocompatibility Workshop cell lines

Development of DNA typing for Class I HLA alleles has lagged behind that of class II for a variety of technical reasons. Following the recognition of locus specific sequences in the first and the third intron, and acquiring the ability to amplify genomic DNA by intron-based PCR primer, we have devised DNA typing of class I alleles by SSOP and direct sequencing. In this study using these techniques we provide the allelic typing of HLA-A, -B, and -C genes for the B-lymphoblastoid reference cell lines from the Tenth International Histocompatibility Workshop. We also describe some common associations of the C alleles with HLA-A and HLA-B alleles.     

广东汉族人群HLA-B基因多态性研究

目的调查广东汉族人群HLA-B位点基因多态性，比较不同人群HLA—B等位基因频率分布特征。方法应用测序技术测定562名广东汉族人HLA-B位点第2、3、4外显子序列，比对数据库得到分型结果，计算HLA-B等位基因频率并与不同人群进行比较。结果共检测到59种HLA-B等位基因，其中6种等位基因频率≥5％，分别是HLA-B＊4601（14．5％），HLA-B＊400101（14．4％），HLA—B＊1502（11．5％），HLA—B＊1301（8．6％），HLA-B＊5801（8．1％）和HLA-B＊380201（6．4％）。这6种等位基因的等位基因频率合计为63．5％。同时，检测到30种等位基因频率〈0．5％的HLA-B等位基因，这30种等位基因的等位基因频率合计为4．9％。广东汉族人群HLA-B等位基因频率总体分布与中国香港华人、新加坡华人比较差异无统计学意义（P〉0．05），但与日本人比较差异有统计学意义。结论分析了HLA-B基因在广东汉族人群中的分布特征，提供了较完整的HLA-B等位基因频率分布资料，为遗传学及疾病相关性等研究提供了重要的参考数据。     

Cloning and sequencing full-length HLA-B and -C genes

Currently most available HLA-A, -B and -C DNA sequences cover exons 2 and 3 with a limited number extending to include other exons and introns. We have developed a method for the accurate determination of full-length genomic DNA sequences for HLA-A, -B and -C alleles. The method involves cloning of PCR amplified full-length HLA genes to separate alleles at heterozygous loci. The approach avoids any ambiguities from sequencing heterozygous PCR products directly and also avoids ambiguities from sequencing overlapping PCR products to achieve full-length sequence. To date we have sequenced full-length genomic sequences from representatives of all the major HLA-B and -C allele groups.     

Full-length extension of HLA allele sequences by HLA allele-specific hemizygous Sanger sequencing (SSBT)

The gold standard for typing at the allele level of the highly polymorphic Human Leucocyte Antigen (HLA) gene system is sequence based typing. Since sequencing strategies have mainly focused on identification of the peptide binding groove, full-length sequence information is lacking for >90% of the HLA alleles. One of the goals of the 17th IHIWS workshop is to establish full-length sequences for as many HLA alleles as possible. In our component “Extension of HLA sequences by full-length HLA allele-specific hemizygous Sanger sequencing” we have used full-length hemizygous Sanger Sequence Based Typing to achieve this goal. We selected samples of which full length sequences were not available in the IPD-IMGT/HLA database. In total we have generated the full-length sequences of 48 HLA-A, 45 -B and 31 -C alleles. For HLA-A extended alleles, 39/48 showed no intron differences compared to the first allele of the corresponding allele group, for HLA-B this was 26/45 and for HLA-C 20/31. Comparing the intron sequences to other alleles of the same allele group revealed that in 5/48 HLA-A, 16/45 HLA-B and 8/31 HLA-C alleles the intron sequence was identical to another allele of the same allele group. In the remaining 10 cases, the sequence either showed polymorphism at a conserved nucleotide or was the result of a gene conversion event. Elucidation of the full-length sequence gives insight in the polymorphic content of the alleles and facilitates the identification of its evolutionary origin.     

HLA-B新等位基因B*9534的测序分析及其单链扩增技术的建立

目的 分析HLA-B新等位基因HLA-B*9534的核苷酸序列,并建立HLA-B * 9534单链扩增技术.方法 采用商品化快速抽提试剂盒抽提标本基因组DNA,采用PCR技术扩增先证者HLA-B基因的第1～8外显子序列,PCR产物经双酶切后直接测序分析第2、3、4外显子.应用序列特异性引物PCR建立HLA-B*9534单链扩增技术,获得HLA-B*9534等位基因的单链产物,并对单链产物进行第2、3、4外显子测序分析.结果 先证者标本存在2个HLA-B等位基因,直接测序结果经软件分析显示与最接近的HLA-B*1518和B*4601组合存在1个碱基不匹配,即第593位A/G杂合.单链扩增技术将先证者等位基因分离后,测序得到两个等位基因为HLA-B*4601和HLA-B*9534.与最接近的HLA-B*1518的第2～4外显子序列相比,HLA-B*9534仅在第3外显子存在一个碱基的不同,即第593位A→G的改变,导致第174位氨基酸天冬酰胺改变为丝氨酸,该等位基因序列已递交GenBank(EU046491),并经世界卫生组织HLA命名委员会正式命名为HLA-B*9534.结论 发现一个新的HLA-B*9534等位基因,建立的HLA-B*9534单链扩增技术是可行的.     

Diversity is demonstrated in class I HLA-A and HLA-B alleles in Cameroon, Africa: description of HLA-A*03012, *2612, *3006 and HLA-B*1403, *4016, *4703

To examine the genetic diversity in west Africa, class I HLA-A and HLA-B alleles of 92 unrelated individuals from two areas in the Cameroon, the capital Yaoundé and the village of Etoa, were identified by direct automated DNA sequencing of exons 2 and 3 of the HLA-B locus alleles and sequence-specific oligonucleotide probe (SSOP) and/or sequencing of the HLA-A locus alleles. HLA-A*2301 (18.7%), A*2902 (10.4%), B*5301 (10.9%), and B*5802 (10.9%) were the most frequently detected alleles, present in at least 10% of the population. A total of 30 HLA-A locus and 33 HLA-B locus alleles, including six novel alleles, were detected. The novel alleles were HLA-A*03012, A*2612, A*3006 and HLA-B*1403, B*4016, and B*4703. HLA-B*4703 contains a novel amino acid sequence that is a combination of the first 5 amino acids of the Bw6 epitope and the last 2 residues of the Bw4 epitope. The addition of 6 alleles to the ever-expanding number of known class I HLA alleles supports our hypothesis that extensive genetic diversity, including previously undescribed alleles, would be observed in this African population. In the Yaoundé population, the allele frequency distribution at the HLA-A locus is consistent with distributions indicative of balancing selection. Extensive HLA-A-B haplotypes were observed in this population suggesting that only a fraction of the Cameroon HLA-A-B haplotype diversity has been observed.     

Sequence-based typing of HLA-B: the B7 cross-reacting group

The large number of polymorphic sites in the HLA-B locus makes sequencing an efficient way of detecting and analysing them. Most polymorphic sites are located in the alpha1 and alpha2 domains of the molecule, encoded by exons 2 and 3 of the gene. An HLA-B-specific sequence-based typing (SBT) strategy was designed for routine application identifying the polymorphic sites in these domains. Exons 2 and 3 were amplified separately using amplification primers located in intron 1, intron 2 and intron 3. Separate amplification of exons 2 and 3 resulted in short polymerase chain reacting (PCR) products and enabled a solid-phase sequencing approach, which made correct assignment of heterozygous positions possible due to low background. A one-step sequencing reaction was performed using fluorescent dye-labelled sequencing primers. One forward sequencing reaction was performed for exon 2, whereas for exon 3, two forward sequencing reactions were needed using two different sequencing primers located in intron 2 and exon 3. The combined sequences of exon 2 and 3 were used for automatic alignment to an HLA-B sequence database and automatic allele assignment. A total of 355 individuals with at least one allele belonging to the B7 cross-reacting group (B7, 13, 22, 27, 40, 41, 42, 47, 48, 81 and 82) were typed for HLA-B by SBT. In the B7 group 48 different alleles were identified, in the non-B7 group a further 59 alleles were sequenced, 9 new alleles were identified. The sequencing strategy described has proven to be reliable and efficient for high-resolution HLA-B typing.     

一例新的HLA-B等位基因B*5614的核苷酸序列分析

目的 研究HLA新的等位基因HLA-B*5614的分子基础。方法 样本DNA抽提采用盐析法，利用PCR方法扩增先证者HLA-B基因的第2～4外显子，PCR产物直接经TOPO转染克隆到质粒载体中分离其等位基因，对所得克隆进行第2～4外显子双向测序分析。应用序列特异性引物PCR方法证实测序所发现的突变。结果 先证者样本克隆测序得到两个等位基因，其中1个等位基因为B*1502，另一个经BLAST验证为新的等位基因，新的等位基因序列已递交GenBank(AY601726，AY601727，AY601728)。与最接近的B*5608等位基因序列相比，新的等位基因仅在第2外显子上有1个核苷酸不同，即第277位G→C，导致第93位氨基酸Cly→Arg。结论 该等位基因为新的HLA-B等位基因，被世界卫生组织HLA因子命名委员会正式命名为HLA-B*5614。     

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.     

Novel HLA-B alleles, B*8201, B*3515 and B*5106, add to the complexity of serologic identification of HLA types

Three class I alleles, B*8201, B*3515 and B*5106, have been described using DNA and cDNA sequencing. The B*8201 allele is most structurally related to B*5602, differing from it by 14 nucleotide substitutions resulting in 5 amino acid differences. The other two alleles, B*3515 and B*5106, differ from their most closely related HLA-B alleles by 2–3 nucleotide substitutions resulting in 1–2 amino acid substitutions, respectively. The majority of nucleotide substitutions marking these new alleles are observed in other HLA-B alleles suggesting that gene conversion and/or reciprocal recombination have created this diversity. All of the amino acid substitutions are predicted to alter the antigen binding site of the HLA-B molecule. The newly defined HLA-B allelic products were originally defined by their unusual serologic reactivity patterns. The B*8201 allelic product is serologically typed as a B "blank" or as a variant of B22 or B45. These patterns and the serologic reactivity of the other newly described allelic products are consistent with the protein sequence homology among specific HLA-B molecules. While serology remains a powerful tool for detecting HLA diversity, alleles generated by events resulting in the sharing of HLA sequence polymorphisms among alleles at a locus will continue to create complexity in the interpretation of typing results.     

A 25% error rate in serologic typing of HLA-B homozygotes

The microlymphocytotoxicity technique has been the accepted method for HLA class I typing since the early 1960s. However, it is often difficult to distinguish two related alleles expressed in an individual due to the cross-reactive nature of the alloantibodies used in this technique. This is especially evident at the HLA-B locus, whose more than 180 alleles fall into only 4 major interrelated cross-reactive antigen groups. To estimate the error rate in serologic typing due to the cross-reactive nature of sera, we used polymerasee chain reaction with sequence-specific primers (PCR-SSP) amplification to retype 40 individuals who were previously typed as serologic HLA-B locus homozygotes. PCR-SSP revealed that 10 of these 40 individuals (25%) were actually heterozygous at their HLA-B locus The HLA-B locus alleles of 9 of these 10 discrepant individuals were further analyzed by denaturing gradient gel electrophoresis followed by direct sequencing. The sequence analysis confirmed that all nine individuals were indeed HLA-B locus heterozygotes. This surprisingly high error rate in serologic definition of HLA-B molecules argues for the use of rapid DNA-based techniques in HLA class I typing, even in the setting of solid organ transplantation.     

Complete subtyping of the HLA-A locus by sequence-specific amplification followed by direct sequencing or single-strand conformation polymorphism analysis

Abstract: A variety of reasons related to the HLA class I system has complicated the application of molecular approaches to HLA class I typing. Here we present a PCR-based HLA-A typing strategy considering the sequence variations of the two most polymorphic exons which allows complete subtyping of the HLA-A locus. The method is based on a sequence-specific amplification identifying the serologically defined HLA-A specificities. The PCR products generated by these group-specific primers bear the sequence information necessary for a postamplification specificity step. The primer pairs are located within one exon, either exon 2 or exon 3, which avoids amplification of polymorphic intron sequences allowing subsequent single-strand conformation polymorphism analysis and facilitating direct sequencing. Using this method we investigated 48 cell lines and 153 clinical samples. 23 PCR reactions are performed per individual for the assignment of the serological specificities A1-A80. The reproducibility was 100% in all cell lines and 85 clinical samples typed on two separate occasions. With the exception of 13 out of 231 possible serological combinations all homozygous and heterozygous combinations of A1-A80 can be distinguished by specific amplification patterns. Comparing the PCR based typing results with those of serology in 12% a discrepancy was found. Solid-phase sequencing or SSCP analysis of the group-specific PCR fragments allowed complete subtyping of the HLA-A locus. This strategy can identify all 48 HLA-A alleles based on the sequence variations of the 2nd and 3rd exon. 1128 homozygous and heterozygous allele combinations are possible for the HLA-A locus. Only 4 out of these 1128 allele combinations remained unresolved.     

Complete generic and extensive fine-specificity typing of the HLA-B locus by the PCR-SSOP method

Abstract: This study describes sequence specific oligonucleotide probe (SSOP) typing of hypervariable regions in exons 2 and 3 of HLA-B locus genes. A single HLA-B specific PCR-product spanning from bp 84 in exon 2 to bp 241 in exon 3 was used for dot blot hybridization to forty-seven chemiluminescent labeled oligonucleotide probes. Thirty-one of these probes were derived from four hypervariability zones in exon 3 of HLA-B genes and covered most known sequence polymorphisms within these regions. In addition, sixteen probes derived from polymorphic regions in exon 2 were used to discriminate alleles not unequivocally characterized by the exon 3 based probes. This SSOP panel gave rise to eighty-six distinct hybridization patterns that could be used to unequivocally define all WHO-designated serological HLA-B specificities except for HLA-B54 in all homo- and heterozygous combinations. Furthermore, sixty-six out of ninety-seven molecularly defined HLA-B subtypes were characterized by unique hybridization patterns in all homozygous and most (possibly all) heterozygous combinations. The reproducibility of these results was confirmed by analysis of forty-four Workshop reference cell lines and of seventy-eight randomly chosen samples (one-hundred forty-seven alleles) from unrelated individuals serologically typed in the laboratory. For sixty-five samples (one-hundred-thirty-three alleles), molecular typing confirmed the results obtained by serology and allowed molecular subtype assignment for ninety-one alleles tested. A serologically blank allele could be defined by molecular analysis in three cases. The method presented here for molecular typing of the HLA-B locus can be used as an alternative to biochemical methods such as one-dimensional isoelectric focusing for assignment of serologically cross-reacting HLA-B molecules as well as for subtype characterization of a large variety of HLA-B alleles.     

HLA class I variation in Australian Aborigines: characterization of allele B*1521

Abstract: Traditional methods of serological typing have largely used antisera of Caucasoid origin, which can overlook HLA heterogeneity in non-Caucasoid populations. Therefore, we have used molecular techniques to evaluate potential polymorphism in HLA class I molecules of Aborigines from the central desert and northern coast of Australia. The DNA sequence of common Aboriginal HLA-A and B antigens were compared with serological reaction patterns which suggested new polymorphisms. Although serological data indicated that long and short variants of A34 may exist, regardless of the serological pattern, all individuals carried the A*3401 allele. Therefore, the variation in A34 reaction pattern observed serologically was not attributable to primary sequence variation in the HLA A*3401 allele. Similarly, there was no detectable polymorphism in the sequences of selected HLA-B alleles, even though some of these alleles showed unusual serological reaction patterns. However, a new allele of B15 (B*1521) was detected in two individuals carrying this serotype. The cells from both of these individuals showed ambiguous reaction patterns with monospecific B62 and B75 sera. cDNA sequencing of the HLA B15 gene from these cells revealed a B15 allele that differed from B*1502 by a single nucleotide change. This change occurred at position 272, resulting in a C to G substitution at residue 67 in the consensus B15 cDNA sequence. Hence, the Australian Aborigines as an ethnic group show very little primary sequence polymorphism within the class I loci, consistent with the results obtained from previous serological studies.     

Six mamu-A locus alleles defined by a polymerase chain reaction sequence specific primer method

Rhesus monkeys are relevant models for human diseases and transplantation. In each case, a complete understanding of these models requires knowledge of macaque MHC. Due to high polymorphism and multiple genes per haplotype, it has been difficult to develop a rapid typing method for rhesus monkey MHC class I. We developed a simple and rapid PCR-SSP strategy for rhesus monkey Mamu-A locus typing. Fifty-two rhesus monkeys were included in the study. Six rhesus monkey allel-specific primer pairs were designed based on published Mamu-A locus gene sequences. Allele-specific PCR products ranged in size from 346 to 788 bp; 5' and 3' Mamu-A locus allele specific primers were located in the second and third exons, respectively. Specific PCR product gel purification was followed by direct sequencing, without subcloning, in both directions. Our data showed variability in the number of Mamu-A alleles ranging from 1 to 4 per genotype. The highest frequencies were observed for Mamu-A*02, -A*04, and -A*03 alleles. Thus, we report here the first PCR-SSP typing method for Mamu-A*02, -03, -04, -05, -06, and -07 array of class I alleles. This technique appears to be a highly reproducible and discriminatory method for detecting this subset of class I A locus genes in rhesus monkeys.     

HLA-DQA1 genotyping by bi-directional sequencing of PCR-amplified DNA spanning exon 2

We describe a simple, reliable technique for HLA-DQA1 genotyping based on direct DNA sequencing of PCR amplified fragments from genomic DNA. The alleles of DQA1 can be divided into two subsets, with one subset demonstrating a 3 base deletion in exon 2 relative to the other. Typing heterozygous individuals who possess one allele from each sub-group can be difficult using a direct sequencing approach, since the two overlapping DNA sequences move out of phase by 3 nucleotides once the point of deletion is reached. The complete sequence is obtained by performing two separate sequencing reactions with fluorescent dye primers, coming from either end of the template. This allows all heterozygous positions in exon 2 to be unambiguously assigned.     

HLA-B16 antigens: sequence of the ST-16 antigen, further definition of two B38 subtypes and evidence for convergent evolution of B*3902

Abstract: The ST-16 antigenic specificity of the HLA-B locus is defined as a B39 variant of Mexican-Americans. Nucleotide sequencing of cDNA shows the ST-16 allele (B*3905) differs from B*39011 by a single substitution that substitutes tyrosine for aspartic acid at position 74 of the mature class I heavy chain. The complete coding region sequence for the common caucasoid allele encoding the B38 antigen has been determined. This B*3801 allele differs from B*3802 at two nucleotide substitutions within the Bw4 sequence motif. B*3801 and B*3802 may have been derived independently from B*39011 by conversion events with B alleles donating distinctive Bw4 motifs. A novel allele B*39022 derived from a Colombian Indian differs from the B*39021 allele of Japanese origin at two widely separated silent substitutions. Comparison of sequences for the known B16 alleles suggest that B*39021 and B*39022 were independently derived by recombination from B*39013 and B*39011 respectively.