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Sequencing of polymerase chain reaction (PCR)-amplified genomic DNA encompassing the putative proximal promoter and the coding region was used to identify KIR2DL5 alleles from 77 unrelated Caucasian individuals. PCR and sequencing were used to link each new allele to its neighboring KIR locus to identify 2DL5A or 2DL5B loci. Allele 2DL5A*001 was found in 24 of the 37 2DL5 positive individuals; 2DL5B*0020101 and 2DL5A*0050101 were also observed. Two new alleles, 2DL5B*008 and 2DL5B*009, contained substitutions altering the amino acid sequence of the leader and transmembrane region, respectively. Two other novel alleles, 2DL5B*0020102 and 2DL5A*0050102, contained alterations of the 5′ upstream region, bringing the number of unique promoter sequences to six. Promoter activity of the alleles was compared using luciferase reporter assays. Our results support those recently published, in which the promoter of 2DL5B*0020101 was shown to be more active in vitro compared to 2DL5A*001, and also provide additional information about the transcriptional activity of the promoters of the newly characterized alleles related to two altered transcription factor binding sites.

The relative importance of various human leukocyte antigen (HLA) loci and the resolution level at which they are matched has not been fully defined for unrelated donor transplantation. To address this question, National Marrow Donor Program data from 3857 transplantations performed from 1988 to 2003 in the United States were analyzed. Patient-donor pairs were fully typed for HLA-A, -B, -C, -DRB1, -DQB1, -DQA1, -DPB1, and -DPA1 alleles. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 (8/8 match) was the minimum level of matching associated with the highest survival. A single mismatch detected by low- or high-resolution DNA testing at HLA-A, -B, -C or -DRB1 (7/8 match) was associated with higher mortality (relative risk, 1.25; 95% CI, 1.13-1.38; P < .001) and 1-year survival of 43% compared with 52% for 8/8 matched pairs. Single mismatches at HLA-B or HLA-C appear better tolerated than mismatches at HLA-A or HLA-DRB1. Mismatching at 2 or more loci compounded the risk. Mismatching at HLA-DP or -DQ loci and donor factors other than HLA type were not associated with survival. In multivariate modeling, patient age, race, disease stage, and cytomegalovirus status were as predictive of survival as donor HLA matching. High-resolution DNA matching for HLA-A, -B, -C, and -DRB1 alleles is associated with higher rates of survival.

Two new human leukocyte antigen-B alleles, B*4069 and B*5409, were identified in China.

Sequencing of PCR amplified genomic DNA including most of the coding region was used to identify killer cell immunoglobulin-like receptor 2DL1 alleles from three families and 77 bone marrow transplant patients and donors. Alleles 2DL1*00302 and *002 were frequently observed in addition to two other known alleles and four new alleles, 2DL1*00402, 2DL1*007, 2DL1*008, and 2DL1*009.

Over 10 million volunteer donors and cord blood units are typed for human leukocyte antigen (HLA) within hematopoietic stem cell registries and cord blood banks. Identification of matched donors in countries different from that of the searching patient is facilitated by Bone Marrow Donors Worldwide and the World Marrow Donor Association (WMDA). A survey of new volunteer donor typing among WMDA member registries has identified the methods and resolution of HLA testing and the level of quality control used to evaluate the accuracy of typing results. The diversity of HLA assignments within registries has generated a number of informatics challenges. Registries are using population genetics to enhance registry diversity and to facilitate the identification of matched donors.

Since the advent of the European Marrow Donor Information System in the first half of the last decade, fully automated data exchange between registry computer systems has been playing an ever-increasing role in the international search for unrelated donors of blood progenitor cells. This exchange, however, was hampered by different local conventions used to present HLA data and complicated by the need to extend the official WHO nomenclature to accommodate the registries' information systems and to cross-validate HLA data obtained with different methods and/or at different loci. The guidelines presented here have been developed by the World Marrow Donor Association to standardize the nomenclature to be used and the validation checks to be applied in the international electronic exchange of HLA-typing data among unrelated volunteer hematopoietic stem cell donor registries and umbilical cord blood banks. Two reference web sites have been designated to maintain and update the approved HLA nomenclature and all the ancillary information needed by the conventions described here.

Aim We explored the impact of using next generation sequencing (NGS) of the entire gene in 300 samples carrying potential new or rare HLA class I alleles. Methods Samples had been previously typed for exons 2 + 3 by SBT sequence based typing (Sanger) and/or by (SSO) sequence specific oligoprobes. Targeted class I loci were amplified using locus-specific PCR primers described by Hozomichi et al. Sonication was used to obtain 400 base pair fragments; an Illumina True Seq Nano kit was used for end repair and adaptor/index ligation. Libraries were pooled and paired end sequences were obtained with a MiSeq. AssignTM MPS software (Connexio, Genomics) was used for analysis. Results Of the 600 alleles sequenced, 69 (11.5%) novel alleles (HLA-A: 27, B: 31, C: 11) differed in the antigen recognition domain (ARD)-encoding exons 2 and/or 3. Ten additional alleles (1.7 %) carried previously unobserved differences in non-ARD-encoding exons (exon 1:2, exon 4:5, exon 5:2, exon 7:1). Thirty three alleles (5.5%) appeared to exhibit novel sequences only in introns. At HLA-A∗03:01:01:01 a novel insertion of 19 bp was observed in intron 2 in two different cells. One hundred and fifty seven (26%) alleles had incomplete sequences in the IMGT/HLA data base, of which most of these (121) had intron sequences identical to the closest related allele. The remaining 331 alleles were primarily common and well documented alleles. Conclusion NGS provided complete, unambiguous, high resolution allele assignments. In this more comprehensive analysis, there was less variation from the IMGT reference database than expected. Most of the introns of alleles with incomplete sequences were conserved when compared to their putative ancestral allele. L. Hou: Other (Identify); Company/Organization; Intellectual property. B. Tu: Other (Identify); Company/Organization; Intellectual property. C. Masaberg: Other (Identify); Company/Organization; Intellectual property. B. Beduhn: Employee; Company/Organization; National Marrow Donor Program. D. Goodridge: Employee; Company/Organization; Connexio Genomics. J. Ng: Other (Identify); Company/Organization; Intellectual property. C.K. Hurley: Other (Identify); Company/Organization; Intellectual property.

Aim NMDP high resolution (HR) testing programs require typing for common or well documented (CWD) null alleles in alignment with Mack et al., Tissue Antigens. 2013: 81(4): 194–203 (CWD 2.0). The aim of this study was to determine the frequency of CWD 2.0 Class I null alleles in NMDP HR typing programs and whether these alleles occur outside of their expected haplotypes. Methods Labs performing HR typing routinely test exons 2–4, then additional regions depending on where a CWD null allele polymorphism is located. Typing methods include SSO, SBT, and NGS. HR Class I typing results from historic adult donor registry samples and CBU samples performed in 2013–2015 were queried for reports of CWD null alleles. Results Four different Class I CWD null alleles were reported a total of 35 times in 41,864 tests. B∗51:11N is expected in the A∗02∼DRB1∗04 haplotype but was observed in A∗03:01∼C∗07:02∼DRB1∗04:02. C∗04:09N was observed with its most common haplotype A∗23:01∼B∗44:03∼DRB1∗07:01 in 53% of cases and with B∗44 in 100% of cases. The common A∗02:53N was not reported; it occurs in up to 0.10% of Chinese populations. The remainder of the CWD nulls were not observed. G group frequency is the null allele frequency within the two digit G group typing. Conclusion Although the frequency of Class I CWD null alleles in the queried HLA results is low, they may warrant continued routine typing. Failure to identify a null allele results in a hidden mismatch between donor and patient. However, lab costs increase due to the additional typing required to detect polymorphisms located outside of exons 2–3. As exon 7 is not routinely tested, typing for C∗04:09N only in the presence of B∗44 may be a future consideration for NMDP HR contracts and policies. Download : Download full-size image

Aim We have developed an allele-level resolution next generation sequencing (NGS) strategy for HLA class I alleles and used it to explore HLA diversity in a registry donor population. Methods Long-range amplification of full-length HLA-A, -B, -C loci was performed in separate polymerase chain reactions using DNA from a buccal swab. Amplicons from one individual were then pooled and sheared by sonication. A library was constructed using Illumina’s TruSeq Nano kit and the DNA fragments tagged with one unique index combination. Libraries from 96 individuals were combined and sequenced simultaneously in a single 500 cycle (V2) paired-end run using an Illumina MiSeq. Data analysis used Conexio Genomics AssignTM MPS software. Results The typing protocol was validated with 552 donor registry samples previously typed by Sanger sequencing and probe hybridization at single genotype G level resolution, yielding a concordance of 99%. NGS yielded primarily single allele-level genotypes. Excluding amplification failures and inability to obtain an NGS assignment (4% loci), 32 discrepant NGS allele assignments were attributed to poor PCR amplification or loss of reads during assembly by software. Complete exon sequences identified 53 known A alleles (48 common or well-documented (CWD)), 89 B alleles (83 CWD), 52 C alleles (47 CWD). Ten potential new alleles with exon variation outside of exons 2 + 3 and over 25% of samples with potential intron variation require further study. The ability to make assignments based on specific regions of the sequence (e.g., exons only) was critical so that typings could be rapidly transmitted to the registry prior to a more comprehensive analysis of the diversity. Conclusions The protocol for NGS of class I alleles provides accurate results and should facilitate high volume high resolution typing of donor registry populations. The extent of new variation in exons is limited but the potential for intron variation is high. C. Hurley: Other (Identify); Company/Organization; Intellectual property.

Based on unusual probe hybridization patterns, two human leukocyte antigen (HLA)-A, five HLA-B, and two HLA-C alleles (A*240208, A*3211Q; B*1822, B*3938Q, B*4606, B*4607N, B*5139; Cw*0321, Cw*0734) were identified in individuals from the Singapore Bone Marrow Donor Program and Singapore Cord Blood Bank. Eight of the nine alleles encode amino acid substitutions altering the antigen-binding region including three alleles with changes altering a cysteine at codon 164 (A*3211Q, B*3938Q, B*4607N). This substitution either eliminates a key disulfide bond or results in a stop codon, both likely affecting the expression of the HLA molecules. Only one allele (A*240208) carries a synonymous substitution.

The frequencies of DRB1*12 alleles were determined in four US population groups by DNA sequencing. Only DRB1*120101 (or DRB1*1206 or *1210) and DRB1*120201 alleles were identified, the latter primarily in the Asian American population. Additional testing of a subset of samples to detect the presence of DRB1*1206 found all of the alleles to be DRB1*120101 (or DRB1*1210). Retesting of six samples previously typed as DRB1*1206 found only DRB1*120101 (or DRB1*1210).

During 2005, a total of 174 cord blood units with their paired maternal samples from the Singapore Cord Blood Bank were typed for HLA-A, -B, -C at intermediate resolution and DRB1 at allelic resolution. Analysis of allele segregation in mother and child assigned 185 different four locus (HLA-A, -B, -C, -DRB1) haplotypes in Chinese, 66 in Malays, and 34 in Asian Indians. Very few four locus haplotypes were shared among population groups. To evaluate the frequencies of four locus haplotypes, the Expectation Maximization algorithm was used with HLA assignments from 536 unrelated Chinese volunteers from the Singapore Bone Marrow Donor Program registry. The paired maternal and cord blood study identified 75 different B-C associations in Chinese, 52 in Malays, and 24 in Asian Indians. Common B-C associations may be shared among population groups; for example, B*4001g-Cw*0702g was common in Chinese and Malays, whereas B*1502g-Cw*0801g and B*3501g-Cw*0401g were found in all three groups. The high diversity of four locus haplotypes originates from multiple combinations of both HLA-A and -DRB1 alleles with each B-C haplotype.

Twenty-three novel human leukocyte antigen-B alleles are described: B*070204, *0738, *0742, *0821, *130202, *1312, *1575, *1598, *1599, *270507, *2728, *350104, *3558, *3811, *3931, *3932, *4045, *4107, *420501, *4812, *510106, *5520, and *5616. Thirteen of the variants are single-nucleotide substitutions from their most homologous allele, eight resulting in amino acid changes (B*0742, *1312, *1598, *1599, *3558, *3931, *4107, and *5616) and five with silent substitutions (B*070204, *130202, *270507, *350104, and *510106). Three alleles (B*0738, *4812, and *5520) differ by five nucleotide changes, altering four amino acids. The remaining seven alleles differ from their most similar alleles by two to three nucleotides, altering from one to two amino acids.

The allelic diversity and associated human leukocyte antigen (HLA) disparity of 1775 bone marrow recipients and their unrelated donors, matched for six of six (1361/1775,77%), five of six (397/1775, 22%), or four of six (17/1775, 1%) HLA-A, -B, -DR antigens, were retrospectively evaluated. The comprehensive HLA analysis included the class I (A, B, C) and II (DRB1, DQA1, DQB1, DPA1, DPB1) loci. Most (>66%) of the predominantly Caucasian study population carried one or two of five to seven common alleles at each HLA locus. In spite of this limited diversity, 29% of the six of six antigen-matched transplants carried allele mismatches at HLA-A, -B, and/or -DRB1, and 92% carried at least one allele mismatch at one of the eight HLA loci tested. Of the 968 HLA-A,-B,-DRB1 allele-matched pairs, 89% carried mismatches at other HLA loci, predominantly at DP loci. The substantially greater than expected HLA allelic disparity between donor and recipient suggests extensive haplotypic diversity and underscores the importance of enhancing approaches to mitigate the deleterious effect of HLA mismatches.

Ten novel HLA-DRB1 and one DRB3 alleles are described. Eight of the variants are single-nucleotide substitutions, four resulting in an amino acid change (DRB1*1145, *1148, *0828 and *1514) and four with silent substitutions (DRB1*040504, *130103, *160502 and DRB3*020204). Two alleles differ by two nucleotide changes altering one (DRB1*1447 and *1361) amino acid and one allele alters three nucleotides and two amino acids.

Altered TGF-beta1 expression due to polymorphisms affects a wide variety of normal cellular and disease processes such as T cell activation and proliferation, tumor progression, and asthma. In this study, a comprehensive examination of function and diversity was undertaken for the TGFB1 promoter region and exon 1 (-2,665 to +423). The known TGF-beta1 promoter was extended to encompass 463 bases by the identification of a strong enhancer activity for a distal segment (-2,665 to -2,204). Ten novel polymorphisms and 14 novel alleles were identified. Most single nucleotide polymorphisms (SNPs) appear to be randomly associated except c.-768_-769insC and c.+74G > C and a set of five novel polymorphisms present in a single allele in persons of African descent. The TGFB1 alleles clustered into three phylogenetic groups based on the common functional SNPs c.-1347C > T (commonly known as -509C-T) and c.+29T > C (commonly known as +869T-C) suggesting three phenotypic groups. Two SNPs unique to African-Americans affect the TGFB1 regulatory region. The c.-1287G > A SNP in the promoter alters the binding affinity of two unidentified transcription factor complexes which translates into a significant difference in reporter gene expression and the c.-387C > T SNP in the 5' UTR alters the binding of Stimulating protein 1 and 3. Thus, TGFB1 possesses a highly polymorphic, extensive regulatory region that likely impacts the pathogenesis of numerous TGF-beta1 related diseases.

The KIR2DL4 gene including a portion of exon 1 through exon 9 was sequenced from two families and eight cell lines from the International Histocompatibility Workshop (IHWS). Two known alleles and eight variants were detected. Overall, there were five synonymous and three non-synonymous changes when the variants were compared to the coding sequences of the most closely related known alleles plus a common frameshift change in five of the variant alleles. Alignment of the new variants with all known alleles showed that the regions encoding the extracellular region and the cytoplasmic tail were the most polymorphic. Two non-synonymous changes, P146H and L161V, occurred in an extracellular immunoglobulin-like domain. Five of the eight variants had a single adenine deletion in the exon encoding the transmembrane region, potentially resulting in a truncated protein lacking the cytoplasmic tail. The distribution of the deletion variant among many KIR2DL4 alleles may explain the high frequency of this variation in the population. Four of the eight consanguineous IHWS cell lines were found to be heterozygous for KIR2DL4 carrying two alleles that differed from one another by a few nucleotide substitutions. Analysis of intron sequences in the families revealed the nature and distribution of interspersed repeat elements which comprise 46% of the KIR2DL4 nucleotide sequence and consist of 12 elements including six SINEs (13.73% of the total length), one LINE (12.41%), and five LTR elements (19.51%). The results revealed the presence of extensive diversity in the KIR2DL4 gene. This is the first extensive report providing both exon and intron data in related individuals.

Outcome of unrelated donor marrow transplantation is influenced by donor-recipient matching for HLA. Prior studies assessing the effects of mismatches at specific HLA loci have yielded conflicting results. The importance of high-resolution matching for all HLA loci has also not been established. We therefore examined the effects of HLA matching (low or high resolution or both) on engraftment, graft-versus-host disease (GVHD), and mortality in 1874 donor-recipient pairs retrospectively typed at high resolution for HLA-A, -B, -C, -DRB1, -DQ, and -DP. Mismatches at HLA-A, -B, -C, and -DRB1 each had similar adverse effects on mortality. Only HLA-A mismatches demonstrated significant adverse effects on GVHD. These adverse effects on outcome were more evident in transplants with low-resolution versus only high-resolution mismatches. Mismatches for HLA-DQ or -DP did not significantly affect outcome. When high-resolution mismatches at HLA-A, -B, -C, and -DRB1 were considered together, adverse effects on survival and GVHD were observed. We therefore conclude that matching for HLA-C should be incorporated into algorithms for unrelated donor selection. High-resolution mismatches at HLA-A, -B, -C, and -DRB1 adversely affect outcome, but less so than low-resolution mismatches. When clinical circumstances allow, high-resolution class I typing may help optimize donor selection and improve outcome.

This special report details the World Marrow Donor Association's recommended procedures regarding the international search for an unrelated donor for hematopoietic stem cell transplantation. The responsibilities of the national hubs, transplant center and donor registry staff are outlined for all actions associated with the preliminary search, formal search, donor confirmatory typing and final donor selection.

World Marrow Donor Association standards are aimed at enhancing the quality of unrelated volunteer donor hematopoietic stem cell registries assisting transplant physicians in the international search for unrelated donors for their patients. The standards cover: (1) general organization of registries; (2) donor recruitment; (3) donor characterization; (4) information technology; (5) facilitation of search requests; (6) second/subsequent donations; (7) collection/processing/transport of stem cells; (8) follow-up of patient/donor; and (9) financial/legal responsibilities.

Today, more than 50 registries of HLA-typed potential adult hematopoietic stem cell donors have been established in 40 countries and include more than 7.5 million volunteers. HLA testing of new volunteers includes HLA-A, -B and often -DR typing at low to intermediate resolution. Searching patients are tested for these same loci, preferably at a higher level of resolution. Over 95,000 patient searches are received by registries annually resulting in approximately 4660 unrelated transplants. In 2001, nearly one-third of transplants involved a patient in one country receiving stem cells from a donor in another. The diversity of the HLA system complicates the search process, requiring sophisticated registry algorithms for matching, and expertise in allele and haplotype frequencies and associations to design search strategies. Within registries, HLA frequency data have been used to evaluate optimal registry size and composition.

The frequencies of 29 HLA-DRB1*04 alleles were determined for five major U.S. populations found within a hematopoietic stem cell volunteer donor registry. One hundred sixty-one DRB1*04 positive individuals from each of the self-described groups, Caucasians, African-Americans, Asian/Pacific Islanders, Hispanics, and Native Americans, were randomly chosen from a database of 82,979 unrelated persons. Subjected to polymerase chain reaction-sequence-specific oligonucleotide probe (PCR-SSOP) typing, these 805 individuals carried a total of ten different DRB1*04 alleles, ranging from DRB1*0401 to DRB1*0411 with DRB1*0409 conspicuously absent from all five groups. The distribution of DRB1*04 alleles varied among the groups, with DRB1*0401 being predominant in Caucasians, African-Americans, and Native Americans. DRB1*0404 and DRB1*0407 were the two most commonly observed alleles in Hispanics, whereas DRB1*0405 and DRB1*04031 were most common in Asian/Pacific Islanders. The remaining 18 DRB1*04 alleles known at the time of the study were not observed. Although not observed in the frequency study, seven previously unreported DRB1*04 alleles are also described.

This paper describes seven novel HLA-B alleles. Five of these new alleles contain polymorphic motifs previously reported in HLA-B alleles, suggesting an origin resultant from a gene conversion mechanism. B*0723 contains a polymorphism previously unreported in class I HLA molecules. B*4105 contains a nucleotide substitution previously unreported in class I HLA molecules, which encodes a protein sequence previously reported only in HLA-C locus alleles.

One hundred sixty-one DRB1*03 positive individuals from each of five U.S. population groups (Caucasoids, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans) were randomly selected from a database of 82,979 individuals. DRB1*03 alleles were identified by polymerase chain reaction-sequence-specific oligonucleotide probe typing. A total of six DRB1*03 alleles out of 21 known alleles were detected. DRB1*03011 was the predominant DRB1*03 allele in all populations. Caucasoids were found to be the least diversified; only DRB1*03011 was observed. African Americans carried DRB1*03021 at a high frequency. This allele was observed in three other populations. DRB1*0304 was found in Asians/Pacific Islanders and DRB1*0305, DRB1*0307 and a new allele, DRB1*0316, was found in Hispanics. A subset of individuals was also typed for DRB3 alleles. DRB3*0101, DRB3*0202, and DRB3*0301 were detected and seven DRB1-DRB3 haplotypes were defined. Testing of other individuals not included in the DRB1*03 frequency study identified a variation of a common extended haplotype, A1, B8, DR3, which carries DRB1*0304 and two previously unreported DRB1*03 alleles, DRB1*0311 and *0320, are also described.

We have sequenced a cDNA clone encoding an HLA-B71 (B70) allele from an African American individual. Serologic definition of B70 allelic products is very difficult due to extensive cross-reactivity of the alloantisera with B35, B15, and B5 antigens. The new sequence most closely resembles the sequence of B∗1503, differing only by three amino acids at positions 63, 67, and 116. The B71 sequence differs from alleles of the B15 antigenic group (serologically defined as B62) by 7–8 amino acids and from members of the B35 family by 10 to 12 amino acids. B71 may represent an evolutionary intermediate, sharing elements common to both B35 and B15 allelic groups.

One hundred sixty-one individuals from each of five US population groups, Caucasians (CAU), African Americans (AFA), Asians/Pacific Islanders (API), Hispanics (HIS), and Native Americans (NAT), were randomly selected from a volunteer bone marrow registry database consisting of 14,452 HLA-DRB1*11 positive individuals. This sampling provided at least an 80% probability of detecting a rare allele that occurred at 1% in the DRB1*11 positive population. Samples were typed for DRB1*11 alleles by polymerase chain reaction-sequence specific oligonucleotide probe typing (PCR-SSOP). A total of 10 DRB1*11 alleles out of 27 possible alleles were detected. The distribution and diversity of DRB1*11 alleles varied among populations although DRB1*1101 was the predominant DRB1*11 allele in all populations. Caucasians were the least diversified; only four common alleles (DRB1*1101-*1104) were observed. As well as the four common alleles, other groups also carried one or two other less frequent alleles including DRB1*1105 (API), *1106 (API), *1110 (AFA), *1114 (HIS), *1115 (NAT), and *1117 (AFA). A subset (418) of these individuals were also typed for DRB3 alleles. Most (97.6%) showed a strong association of DRB1*11 with DRB3*0202.

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.

A subunit vaccine forPlasmodium falciparummalaria will need to contain well-defined T helper cell epitopes that induce protective immune responses to the parasite. One major barrier to the use of subunit vaccines is the requirement for T helper cell epitopes to be presented by the HLA class II molecules that are present in the population being vaccinated. Since the majority of malaria studies have focused on HLA-DR, little information on the role of HLA-DQ in the binding and immune response to malarial epitopes is available. This study used an in vitro peptide-binding assay to predict the extent of HLA-DQ binding of four conserved T helper cell epitopes identified from asexual-stage malaria vaccine candidate antigens. Epstein-Barr virus (EBV)-transformed human B-cell lines expressing 14 different DQ molecules (DQ2.1, -2.2, -4.1, -4.2, -5.1 to -5.3, -6.1, -6.2, -6.4, -7.1, -7.3, -8, and -9) representing all broad serological specificities, including common DQ molecules present in populations in areas where malaria is endemic, were used in the binding assay. Moreover, an HLA-DQ transgenic mouse model was employed to evaluate the correlation between the in vitro DQ binding of the peptides and the generation of in vivo immune responses following peptide immunization. This study identified two broad DQ-binding peptides, ABRA#14 and SERA#9. ABRA#14 also induced T-cell proliferation and Th1-associated cytokine production in DQ8+transgenic mice. The combination of peptide binding to EBV-transformed cell lines and DQ transgenic mice provides a method for identifying additional T-cell epitopes for inclusion in a vaccine.

The frequencies of 30 HLA-DRB1*13 alleles and 15 DRB3 alleles were determined for the 5 major U.S. ethnic populations: Caucasians, African Americans, Asian/Pacific Islanders, Hispanics, and Native Americans. A random sampling (163) of DRB1*13-positive individuals from each self-described ethnic group was selected out of a pool of 82,979 unrelated individuals, providing at least an 80% probability of detecting a rare allele that occurred at 1%. These 815 samples were subjected to allele-level SSOP typing and/or DNA sequencing which identified 11 different DRB1*13 alleles. DRB1*1301 and DRB1*1302 were the most common alleles seen in the five major ethnic groups while DRB1*1304 was not detected among Caucasians and DRB1*1305 was not detected among African Americans. DRB1*13 allele diversity was surprisingly more limited among African Americans compared to both Caucasians and Asian/Pacific Islanders. To determine the extent of DRB1*13-DRB3 associations, 504 of these samples expressing only one DRB3-associated DRB1 allele were subjected to PCR-SSOP typing and 14 DRB1*13-DRB3 haplotypes were detected. The distribution revealed that African Americans were significantly different from Caucasians, Asian/Pacific Islanders, and Hispanics. Allele frequency studies such as this further support previous findings that the distribution of HLA types can differ significantly among different ethnic populations.

The World Marrow Donor Association has formulated guidelines for establishing the extent and quality of histocompatibility testing for unrelated donor registries, umbilical cord blood banks, and transplant centers involved in international exchange of hematopoietic stem cells for allogeneic transplantation. Registry and cord blood bank guidelines suggest that, at a minimum, initial HLA typing should be performed for three HLA loci, HLA-A, -B, and -DR, at low resolution/split antigen level. DNA-based testing methods should be utilized for HLA-DR typing. DNA-based testing for HLA-A and -B should replace serologic testing of new volunteer donors and cord blood units as robust protocols and reagents become available to the laboratories. Transplant center guidelines for typing of patient, family and to confirm the HLA types of potential unrelated donors should include, at the minimum, typing HLA-A, B, and -DR loci using primarily DNA-based testing methods at allele level resolution for DRB1 and low resolution/ split antigen level for HLA-A and -B. It is strongly recommended that the typing of a patient and the selected donor be performed using the same set of reagents, methodology, and interpretation criteria with fresh tissue samples to ensure HLA identity. Guidelines for laboratory accreditation, approaches to quality assurance and quality control for HLA testing, and suggestions for the format of the HLA database of donor types are also outlined.

The frequency of each A∗28 allele was determined by PCR-SSOP typing in 5 major U.S. ethnic populations: Caucasians, African Americans, Asians/Pacific Islanders, Hispanics, and Native Americans. The percent of serologically defined A28-positive individuals in the 5 populations ranged from 2.7–17.9%. Fifty-nine individuals who were previously serologically typed as A28, A68 or A69 were randomly chosen for allele-level typing from each ethnic group from a database of 82,979 consecutively typed unrelated individuals. The most common A∗28 allele for Caucasians, Asians/Pacific Islanders, Hispanics, and Native Americans was A∗68012, while A∗6802 was found in the majority of African Americans. Only four and three A∗28 alleles were seen in Caucasians and African Americans, respectively, while five to six A∗28 alleles were seen in the other population groups. The A∗6804 and A∗6806 alleles were not observed in any of the five ethnic groups.

Eleven novel human leukocyte antigen (HLA) alleles were identified during routine sequence-specific oligonucleotide probe (SSOP) typing (LABType®; One Lambda Inc., Los Angeles, CA) of volunteers for a hematopoietic stem cell registry in Beijing, China. The new alleles were detected when one or more probes gave an unexpected reactivity pattern.

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.

Unrelated volunteer donors (69) recruited by the National Marrow Donor Program were HLA typed by DNA-based methods for both the HLA-A and -B loci. Each donor had been previously typed by serology by at least two independent laboratories. Of the 69 samples, all serologic laboratories were in concordance for HLA-A in 62 typed samples and for HLA-B in 48 typed samples. Of the serologically concordant samples, 5 samples typed for HLA-A and 7 samples typed for HLA-B received DNA and serology types differing in their level of resolution. One sample typed for HLA-A and 3 samples typed for HLA-B by DNA methods gave different results from their serologic assignments. Of the samples exhibiting disparities among the different serologic typing laboratories, the DNA-defined types of 7 samples typed for HLA-A and 18 samples typed for HLA-B were consistent with at least one of the serologic assignments. The DNA types for the remaining 3 HLA-B typed samples did not agree with the serologic assignments and their alleles were subsequently sequenced. One of these sequences was a previously undefined allele, B*1537. Sharing of polymorphic sequences among HLA allelic products creates difficulties for consistent serologic assignments of some types complicating the process of identifying potential donors from bone marrow registries. Thus, the use of DNA-based typing techniques for characterization of donor class I types should allow a more consistent definition of types and should speed the donor selection process.

Antigens bearing the B62 serologic specificity are a heterogeneous group being encoded by at least 10 alleles and are widespread in most populations including the Korean population (10.5%). This study characterized a new allele encoding a B62 molecule with extra B52 serologic reactivity from a Korean family and unrelated individuals. Based on the DNA sequence, it appears that the single nucleotide substitution at codon 171 (TAC-->CAC), resulting in an amino acid change from tyrosine to histidine, is responsible for creating the extra reactivity. B*1538 was confirmed by PCR-SSP using a primer annealing to codon 171 in two additional unrelated individuals also exhibiting the same serologic reaction pattern. The haplotype associated with the novel allele, A31-B*1538-Bw6-Cw3-DRB1*1101-DRB3*02-DQB1*0301, was identified in the family members and two unrelated individuals. The novel B*1538 allele and its associated haplotype adds to the HLA diversity in this population.

ABSTRACT: Cells expressing HLA molecules in the B15 family were identified by serologic typing in routine testing of volunteer donors of various ethnic backgrounds for a bone marrow registry. DNA sequencing was used to identify HLA-B15 alleles associated with each serologic type and to examine the diversity within the B15 antigen family. Alleles which appeared predominantly in each B15 serologic cluster included: B15 with no defined serologic subdivision (B∗1501), B62 (B∗1501), B63 (B∗1516, B∗1517), B75 (B∗1502, B∗1521), and B76/77 (B∗1513). Other B∗15 alleles were also found associated with the serotypes and some of these alleles (e.g., B∗1501 and B∗1516) were found in two or more serologic clusters illustrating the complexity of this family. The B15 unsplit and B75 groups were the most complex exhibiting 16 and 7 alleles, respectively, within each serotype. Five new B∗15 alleles (B∗1530, B∗1531, B∗1533, B∗1534, B∗1535) and 5 other new HLA-B alleles (B∗38022, B∗3910, B∗4010, B∗51012, and B∗5108) were also identified.

Four previously unreported DR52-associated DRB1 alleles have been characterized through DNA sequencing, contributing to the diversity of the HLA system. DRB1*1424 is nearly identical to DRB1*1402 in the second exon, except that it contains the "I---A" motif found at codons 67-71 common to the DRB1*15 alleles. DRB1*1425 contains the "A--H" motif, found in DRB1*1401 at codons 57-60, in a sequence otherwise identical with the second exon of DRB1*1307. Compared with DRB1*11012, DRB1*1323 contains three predicted amino acid changes at codons 58 (ala-->glu), 67 (phe-->ile), and 71 (arg-->glu). The sequence of DRB1*1324 is identical to exon 2 of DRB1*1103, except that DRB1*1324 does not contain the GAG at codon 58 characteristic of the DRB1*11 alleles. These new alleles may have arisen through gene conversion, and they contribute to the complexity of the DR6 family.

The increased use of DNA-based typing techniques has improved the accuracy and reliability of HLA types assigned to patients requiring an allogeneic bone marrow transplant and their potential donors, facilitating better donor selection. The benefits of this technology, which will completely replace serologic typing within the next few years, have not yet been fully exploited since the typing information obtained in the form of DNA sequence polymorphisms is presently converted to an HLA allelic type for submission to a registry. Furthermore, within the registry, the allelic type may be further converted to a serologic antigen for use in the search and match process. Because the current nomenclature system makes selection of donors who are likely to provide optimal matches difficult, it is critical that DNA-based HLA typing data be recorded by registries as sequence polymorphisms tested as positive or negative rather than interpreted HLA types and that searching and matching utilize these polymorphisms. Until this transition is complete, we will not fully utilize this powerful HLA typing technology to best serve those patients seeking unrelated donors.

T-cell recognition of alloantigen involves both the MHC molecule and its associated peptide ligand. To understand the relationship between the specificity of alloantigen recognition and the structure of TCR molecules, we have investigated TCR gene utilization by sequencing TCR genes from well-defined allospecific T-lymphocyte clones. Alloreactive TLC consisted of a panel of clones primed to recognize DR1-related alloantigens. Our sequencing results revealed extensively diverse, but nonrandom, usage of TCR AV and BV gene segments and essentially no conservation in CDR3 or junctional sequences. Such observations are consistent with allospecific TCR that interact with MHC molecules on a generic level while recognizing specific peptides. They also reduce potential enthusiasm for anti-TCR therapy in allograft rejection.

Definition of peptide binding motifs for DR molecules has proven difficult as the peptides that bind to a DR molecule have shown extensive variability at putative motif positions. Recent studies suggest that specific peptide anchor residues (motif positions) and specific DR residues can differ in importance for peptide binding to a DR molecule. To assess further the relevance of individual peptide anchor residues, the binding of serial alanine-substituted analogs of influenza virus hemagglutinin (HA) 306-318 and human myelin basic protein (MBP) 152-165 to a panel of transfected wild-type DR molecules was examined. This analysis included DR molecules from a wide range of allelic families and, unlike most earlier studies, multiple members of single DR allelic families. The data show that different peptide residues serve as critical anchors for binding to different DR molecules. For example, MBP binding to DR(alpha, beta 1*0303) required peptide residues F154 (i), R159 (i + 5) and R162 (i + 8). In contrast, MBP binding to DR(alpha, beta 1*0102) required peptide residues I153 (i) and L156 (i + 3). More importantly, the combination of critical anchor residues in HA and MBP differed for binding to a single DR molecule [e.g. V309 (i) for HA and I153 (i) and L156 (i + 3) for MBP binding to DR(alpha, beta 1*0102)]. Although the location of the binding pocket in each DR molecule compared to the DR (alpha, beta 1 *0101) crystal is expected to be similar and suggests a common extended DR binding motif, the present results suggest that the relative importance of individual peptide anchor residues and of the corresponding DR binding pockets will differ for each DR/peptide complex.

A system for intermediate level identification of the HLA-B locus alleles was devised. This system can be extended to identify individual alleles in any sample. The first step used primers which amplify all HLA-B alleles. This amplicon was subjected to SSOP hybridization to allow intermediate level typing of samples. In the second step, group-specific primers were utilized to obtain specific amplification of groups consisting of a few alleles. The oligotypes within each group were identified by the use of SSOP. The separation of groups of alleles by amplification allowed the use of a limited number of probes to identify oligotypes present in a sample. Additional probes can be added as new alleles are identified, increasing the flexibility of the system. HLA typing software was developed to determine the resolution of the system and to identify HLA oligotypes. PCR-SSOP methods are in wide use and have been extensively validated. The procedures reported here will be relatively easy to implement for large-scale DNA-based typing of the HLA-B locus.

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.

We have discovered a previously unpublished HLA-DRB1 allele, observed in a patient (SB), his mother, and one sibling. The undefined allele gave sporadic positive reactions with sera in the DR52-associated group. SSOPH analysis utilizing both generic and group specific primers and probes also gave ambiguous results. SB typed clearly as a DRB1*0301 (paternal allele) but the DNA from SB also bound probes specific for DRB1*14 and DRB1*11. Sequencing revealed that the undefined allele was similar to a DRB1*14 allele with a segment of sequence found in DRB1*11 alleles. The patient was MLC reactive with donors who express DRB1*0301, *1401 and *0301, *11 and was nonreactive solely to DRB1*0301 (Dw3) homozygous typing cells.