Common And Well-documented Research Articles

HLA-DPB1 allele frequencies in the West Midlands region of the United Kingdom: A critical evaluation against the common, intermediate and well-documented allele catalogues CWD 2.0.0, EFI CWD and CIWD 3.0.0.

The Birmingham H&I laboratory performed HLA typing on 456 potential deceased solid organ donors in the UK between 2014 and 2016. Accurate DPB1 typing is essential for determining HLA compatibility in transplantation, thus we report HLA-DPB1 for potential deceased solid organ donors. To correctly interpret HLA typing data, laboratories must understand both international and local HLA allele frequencies. In this analysis, we determined HLA-DPB1 allele and genotype frequencies for these 456 donors. HLA-DPB1 diversity was evaluated against the common and well-documented (CWD) alleles 2.0.0 catalogue, the European Federation for Immunogenetics (EFI) CWD catalogue and the common, intermediate and well-documented (CIWD) 3.0.0 catalogue. Additionally, we determined which alleles are common in our local deceased donor population. We observed 27 HLA-DPB1 alleles with DPB1*04:01 being the most frequently observed (allele frequency=0.4342). All alleles detected locally were present in CIWD 3.0.0, however, DPB1*124:01 and *135:01 were not present in CWD 2.0.0 and DPB1*104:01 and *135:01 were not present in EFI CWD. Twenty of 27 DPB1 alleles identified were defined as locally common and also listed as common in CIWD 3.0.0 representing 62.5% of common alleles in the subset of CIWD 3.0.0 from individuals of a European geographic, ancestral or ethnic background. The alleles HLA-DPB1*16:01 and *20:01 are locally common but not listed as common in EFI CWD and DPB1*104:01 is not listed as common in CWD 2.0.0 catalogue. Our analysis showed that the detected alleles and locally common alleles within our population were aligned with the CIWD 3.0.0 catalogue.

Improvement in HLA-typing by new sequence-specific oligonucleotides kits for HLA-A, -B, and -DRB1 loci.

Polymerase chain reaction sequence-specific oligonucleotide is commonly used for HLA-typing. We replaced our LabType SSO HD (HD) kits with LabType SSO XR (XR) kits (One Lambda, Inc., Canoga Park, California) for HLA-A, -B, and -DRB1 following acquisition of a LABScan3D analyzer. The XR kits have more bead regions than the HD kits, allowing for an extended number of probes and exon coverage. They are claimed to improve typing resolution and to diminish the number of allele ambiguities, including common and well-documented (CWD) and null alleles to be resolved. We retrospectively selected patients who had their first HLA-typing performed with the HD kits and their second determination with the XR kits between 2015 and 2016. Forty-two patients were selected for HLA-A typing comparison, and 48 for HLA-B and 41 for HLA-DRB1. XR kits significantly decreased the number of allele ambiguities for HLA-A and -B. On the other hand, the improvement was limited for the HLA-DRB1 locus. The XR kits did not resolve all the CWD HLA allele ambiguities, which may be important for organ and/or hematopoietic stem cell transplantations. The XR kits eliminated 88%, 62%, and 27% of null allele ambiguities for HLA-A, -B, and -DRB1 loci, respectively. In conclusion, the XR kits allow for a significant improvement of HLA-typing resolution for HLA-A and -B loci in comparison with HD kits. In contrast, the number of oligonucleotides in the XR HLA-DRB1 kit should be extended to include exon 3 at the very least. It could also be interesting to include oligonucleotides allowing HLA-DRB3, 4, and 5 typing.

Common and well-documented HLA alleles of German stem cell donors by haplotype frequency estimation.

We present a catalog of common and well‐documented (CWD) alleles of the German population for the six HLA loci A, B, C, DRB1, DQB1, and DPB1. This study is based on a sample of over 5 million volunteer adult hematopoietic stem cell donors from the 26 German donor centers. To establish the catalog, allele and haplotype frequencies were estimated with a validated implementation of the expectation‐maximization algorithm. CWD criteria similar to existing CWD catalogs were applied in order to be able to put our findings into the context of relevant existing references. Overall, 2155 HLA‐A, ‐B, ‐C, ‐DRB1, ‐DQB1, and ‐DPB1 alleles were identified as CWD in the German donor population representing about 20% of the HLA alleles at two‐field resolution in the IPD‐IMGT/HLA Database release v3.25.0 from July 2016 for these six loci. We found a substantial concordance of CWD alleles between the three catalogs and showed the contribution of the German donor population to the CWD alleles domain. In conclusion, the definition of CWD criteria that allow interoperability, scalability, and flexibility will be crucial for the development of a worldwide CWD catalog.

HLA common and well-documented alleles in China.

The American Society for Histocompatibility and Immunogenetics HLA common and well-documented (CWD) catalog, CWD 2.0.0 catalog and European Federation for Immunogenetics (EFI) CWD catalog have been published, which are useful for improving the accuracy of HLA genotyping in laboratories. Here, we studied the Chinese HLA CWD catalog. A total of 812 211 unrelated volunteer donors from the China Marrow Donor Program (CMDP) were analyzed. Six hundred seventy-six alleles at the HLA-A, -B, -C, -DRB1, and -DQB1 loci were defined as CWD alleles in the Chinese population, including 159 common and 517 well-documented alleles. The distribution of HLA alleles in the Chinese CWD catalog is different from that in the EFI CWD catalog. Thirty-two percent (215/676) of CWD alleles in the Chinese CWD catalog are shared with those in the EFI CWD catalog. Fifty-six percent (380/676) of alleles in the Chinese CWD catalog are not found in the EFI CWD catalog, while 655 alleles in the EFI CWD catalog are neither common nor well-documented alleles in the Chinese CWD catalog. The Chinese CWD catalog described in this study may help to improve high-resolution histocompatibility testing for CMDP-accredited laboratories in China. However, to accommodate an increasing number of HLA alleles, this Chinese CWD catalog should be regularly updated.

Patterns of non-ARD variation in more than 300 full-length HLA-DPB1 alleles

Our understanding of sequence variation in the HLA-DPB1 gene is largely restricted to the hypervariable antigen recognition domain (ARD) encoded by exon 2. Here, we employed a redundant sequencing strategy combining long-read and short-read data to accurately phase and characterise in full length the majority of common and well-documented (CWD) DPB1 alleles as well as alleles with an observed frequency of at least 0.0006% in our predominantly European sample set. We generated 664 DPB1 sequences, comprising 279 distinct allelic variants. This allows us to present the, to date, most comprehensive analysis of the nature and extent of DPB1 sequence variation.The full-length sequence analysis revealed the existence of two highly diverged allele clades. These clades correlate with the rs9277534 A → G variant, a known expression marker located in the 3′-UTR. The two clades are fully differentiated by 174 fixed polymorphisms throughout a 3.6 kb stretch at the 3′-end of DPB1. The region upstream of this differentiation zone is characterised by increasingly shared variation between the clades. The low-expression A clade comprises 59% of the distinct allelic sequences including the three by far most frequent DPB1 alleles, DPB1*04:01, DPB1*02:01 and DPB1*04:02. Alleles in the A clade show reduced nucleotide diversity with an excess of rare variants when compared to the high-expression G clade. This pattern is consistent with a scenario of recent proliferation of A-clade alleles.The full-length characterisation of all but the most rare DPB1 alleles will benefit the application of NGS for DPB1 genotyping and provides a helpful framework for a deeper understanding of high- and low-expression alleles and their implications in the context of unrelated haematopoietic stem-cell transplantation.

P065 Updating the common and well documented allele list

Aim The goal of this study was to update the 2012 common and well documented (CWD) allele list using data from 3940 class I and 4124 class II assignments obtained by next generation sequencing (NGS) of two registry (USA and Argentina) populations and 360 donor-recipient pairs from the CIBMTR repository. Methods DNA was prepared from blood, buccal swabs, or cell lines. HLA-A, -B, -C alleles were amplified with primers annealing in the 5’ and 3’ UTRs. The sequences of exons and introns were determined by next generation sequencing (NGS) with an Illumina Miseq using an in-house protocol and Conexio Assign software analysis. Class II NGS sequencing focused on exons 2 and 3; however, some alleles had additional exons and introns characterized. Alleles that were on the well-documented list and alleles not considered CWD based on Mack et al. Tissue Antigens 2013 were identified. Results The majority of alleles detected had previously been designated as common. For example, from the Argentine registry samples, 92% of 2944 HLA-A alleles identified were common. In the total study population, 77 class I alleles previously designated as WD and 97 alleles not defined as C or WD were observed from 1 to >5 times. Thirty-seven of the 174 class I alleles occurred greater than 5 times, e.g., previously undefined A*02:17:02, B*44:02:01:03, and C*17:01:01:05. The majority (90%) of the 19 not CWD alleles were intron variants e.g., A*03:01:01:05, B*39:01:01:03, C*06:02:01:03. Forty-five class II alleles fell into the WD or not defined categories; 29% occurred >5 times. Class II alleles occurring >5 times included DRB1*04:06:02, DQB1*03:01:01:03 and DPB1*04:02:01:02. Conclusions The NGS data expand our knowledge of the frequency of specific full length HLA alleles. The majority of the uncommon alleles characterized in this study are synonymous variations of common alleles.

P016 Extending the IMGT/HLA database: Whole GENE sequencing of 247 distinct HLA-DPB1 alleles

Aim Our current understanding of sequence variation in HLA-DPB1 is largely limited to exons 2 and 3, as only few alleles have been characterized at full length. To close these gaps in the IMGT/HLA database and to better understand patterns of polymorphisms in the HLA-DPB1 gene, we selected samples from the DKMS donor pool that represent all common and well-documented (CWD) alleles and performed phased full-length sequence characterization. In addition we characterized alleles that are currently not defined as CWD but occur at moderate to high frequencies in the DKMS donor populations. Methods We performed long-range PCR targeting the whole DPB1 gene using primers flanking the UTR-regions. The 12 kb amplicons were sequenced by two strategies on different platforms: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on PacBio® RS II instruments. Phase-defined allelic consensus sequences were generated from the long-read data and polished with the high-fidelity short reads using the DR2S software (DKMS Life Science Lab). Results We successfully sequenced the full-length DPB1 sequences of 331 samples (662 haplotypes) covering 40 common, 14 well-documented, and 40 not-CWD-defined alleles. In total, 247 distinct allelic variants were found considering also variation outside of exons 2 and 3. The 12 previously known full-length DPB1 alleles were each confirmed at least once. Most novel variation was found to reside in the intronic regions of DPB1. A phylogenetic analysis of the 247 alleles confirmed the existence of two major evolutionary branches characterized by two haplotype groups downstream of exon 2 with little internal variation and no evidence of historical recombination. These haplotype groups are tightly linked to the expression marker rs9277534 in the 3’ UTR. Conclusions We present the full-length sequences of 247 unique DPB1 alleles covering all common and all well-documented (CWD) variants. We have successfully submitted these alleles to IMGT/HLA, thus providing full genomic extension of 84 known DPB1 alleles and adding 151 novel DPB1 alleles to the database. Together, these alleles represent more than 99.9% of the DPB1 alleles encountered in the DKMS donor populations.

P089 How accurate is RT-PCR typing for HLA-DPB1 alleles?

Aim RT-PCR is a rapid method to determine HLA antigens and many laboratories utilize this assay for deceased donor typing. However, HLA-DPB1 allele level results are typically reported to UNOS without confirmation by high-resolution typing methods. Our aim was to compare the accuracy of RT-PCR to sequence based testing (SBT) in order to asses the frequency of incorrect reporting. Methods HLA-DPB1 typing on 79 deceased donors previously typed using RT-PCR reagents (Linkage Bioscience) was confirmed using SBT methods. DPB1 typing results obtained by the different methods were compared. All discrepant results were verified by LabType SSO. Results In 99% (78/79) of the cases, RT-PCR produced the same allele pairing results obtained by SBT; in the remaining case, discrepant results were obtained between RT-PCR and SBT. LabType SSO determined that the SBT result was inaccurate. The manufacturer suggested that the erroneous SBT result was most likely due to the lack of exon 3 sequence information for the allele in question. In 43% (34/79) of the cases, RT-PCR detected an ambiguous combination that included multiple common and well-documented (CWD) alleles. In 21 of the ambiguous combinations, the alternative allele was in the same G group as the reported allele; in 13 cases, the alternative allele was in a different G group and three were reported incorrectly. In the remaining non-ambiguous cases, one incorrect tying was chosen based on frequencies of the alleles, however, the CWD allele was later ruled out by SBT (Fig. 1). Download : Download high-res image (88KB) Download : Download full-size image Conclusions In general, RT-PCR methods are an efficient and accurate way to determine HLA-DPB1 alleles prior to organ allocation. However, in some situations, higher resolution methods such as SBT or SSO may be required to resolve common ambiguities and ensure that accurate typing results are reported. A procedure for revising reported alleles in the UNOS database may need to be adopted to help quantify the true prevalence of HLA-DPB1 alleles in the population.

Common and well-documented HLA alleles over all of Europe and within European sub-regions: A catalogue from the European Federation for Immunogenetics.

A catalogue of common and well-documented (CWD) human leukocyte antigen (HLA), previously established by the American Society for Histocompatibility and Immunogenetics (ASHI), is widely used as indicator for typing ambiguities to be resolved in tissue transplantation or for checking the universality of any HLA allele in the world. However, European population samples, which are characterized by a substantial level of genetic variation, are underrepresented in the ASHI catalogue. Therefore, the Population Genetics Working Group of the European Federation for Immunogenetics (EFI) has facilitated data collection for an European CWD catalogue. To this end, 2nd-field HLA-A, -B, -C,- DRB1,- DQA1,- DQB1 and -DPB1 data of 77 to 121 European population samples (21 571-3 966 984 individuals) from 3 large databases, HLA-net/Gene[VA], allelefrequencies.net and DKMS, were analysed. The total number of CWD alleles is similar in the EFI (N = 1048) and ASHI (N = 1031) catalogues, but the former counts less common (N = 236 vs 377) and more well-documented (N = 812 vs 654) alleles than the latter, possibly reflecting differences in sample numbers and sizes. Interestingly, approximately half of the CWD alleles reported by EFI were not reported by ASHI and vice-versa, underlining the distinct features of the two catalogues. Also, although 78 common alleles are widely distributed across Europe, some alleles are only common within specific sub-regions, showing regional variability. Although the definition of CWD alleles itself is affected by different parameters, calling for current updates of the list, the EFI CWD catalogue provides new insights into European population genetics and will be a very useful tool for tissue-typing laboratories in and beyond Europe.

P001 On the occurrence of non-cwd alleles in the german dkms donor database

The list of Common and Well-Documented (CWD) alleles comprises alleles that have been either observed at pertinent exons at least 5 times via DNA typing technologies (SBT, NGS) in unrelated individuals (well-documented) or have a frequency higher than 0.001 in populations of at least 1500 individuals (common). We compared the current CWD list 2.0.0 to the DKMS Germany database of more than 4.5 M unrelated stem cell donors. Using results with allelic resolution for HLA-A, -B, -C, -DRB1, -DQB1, and -DPB1, we determined the number of occurrences for each allele. As the CWD list is based on IMGT/HLA release 3.9.0 (July 2012), we first analyzed typing results prior to this date. We then included typing results into the analysis up to April 2016. As a rule, whenever a two-field allele in the CWD list was represented in our database by three or four field allele designations, we treated it as CWD. Whenever a two- or three-field allele in the database had a longer equivalent, it was treated alike. Based on alleles known in HLA release 3.9.0, we found 396 (991) individual alleles typed prior to 2012 (April 2016) that were observed more than 5 times in our database, but not listed as CWD. Of the 396 (991) non-CWD alleles, 18 (60) alleles are part of a G-code in the CWD list. Including alleles introduced after HLA release 3.9.0 in our analysis, additional 134 alleles met the CWD requirement. By using the CWD list, for example, the multi-allele code HLA-A*25:AC might be reported as A*25:01 and A*25:03 (non-CWD) only in a footnote although it was observed 97 (176) times in our analysis. Some registries also accept results from CT as high resolution ignoring ambiguous pairs where at least one partner is non-CWD. Our results suggest that the application of the CWD list in laboratories to omit ambiguous results or to not include non-CWD alleles when transmitting CT results should be avoided. Given the increasing availability of high-resolution NGS-based phased typing results, the CWD list should be updated in an automated fashion from data available on BMDW. Download : Download high-res image (249KB) Download : Download full-size image

P089 Enhanced resolution in rapid HLA typing of deceased donors: A single center experience using a new Taqman-based qPCR approach

Aim Organ donation is a highly orchestrated process with the initial donor testing, including donor HLA typing, needing to be completed as rapidly as possible. The need for increased donor HLA typing resolution is continually growing. However, true high-resolution typing is time and resource prohibitive for deceased donors. Real-time PCR-based HLA typing assays have improved turn-around-times and are increasingly able to provide higher-resolution typing. Here we examine the performance of a new Taqman-based qPCR approach as compared to an endpoint melt curve based assay. Methods 80 DNA samples were HLA typed by first generation QTYPE (Olerup) and LinkSeq 1575 (Linkage Biosciences) for HLA-A, B, C, DRB1, DRB3/4/5, DQA1, DQB1, DPA1 and DPB1. Comparisons were made between assays for the number of common and well-documented (CWD) alleles (based on the IMTG/HLA 3.19.1 database) and the number of single antigen bead (SAB) specificities that could be resolved from the CWD typing. Results Average Roche Lightcycler 480 II run time was 42 min for the QTYPE assay and 75 min for LinkSeq. Compared to the endpoint melt curve-based LinkSeq assay, the TaqMan-based QTYPE assay defined CWD alleles at an equal or better resolution for HLA-A 13/18 loci, HLA-B 17/27 loci, HLA-C 13/15 loci, 17/17 HLA-DRB1, 3/3 HLA-DRB3/4/5 loci, 6/7 HLA-DQB1 loci, 4/6 HLA-DQA1 loci, 3/3 HLA-DPA1 and all HLA-DPB1. The number of SAB alleles that each CWD allele string was able to detect was determined using both Immucor and One Lambda SAB reagents. QTYPE resolved the multi-SAB antigens: A30, A34, A68, B13, Cw10, DR1, DR11, DR13, DR14, DR16, DRB3, DRB5, and DQ2 to a single SAB allele, while the LinkSeq assay detected CWD alleles that covered 1 + SAB alleles. A2, B44, DR4, DQ6 covered 1 + SAB in both tests, but QTYPE, on average, had better resolution for these SAB antigens. Other SAB were covered by 3 or more CWD alleles. All DPB1 were uniquely resolved by both assays except for DPB1 ∗ 04:02/105:01 in some LinkSeq tests. Conclusion The advantageous multiplexing capacity of the TaqMan based qPCR HLA typing permits greater resolution and, in general, resolves SAB alleles more consistently than the endpoint melt curve based assay. With increasing numbers of CWD HLA alleles, the TaqMan approach provides a greater potential for expansion and CWD allele discrimination. J. Lunz: Grant/Research Support; Company/Organization; Olerup. M. Wetmore: Employee; Company/Organization; Olerup. B. Passey: Employee; Company/Organization; Olerup SSP AB. D. Freedom: Grant/Research Support; Company/Organization; Olerup. G. Hill: Employee; Company/Organization; Olerup.

OR33 Allelic diversity and haplotype structure of HLA-DPB1 based on full-length characterization of most CWD alleles

Aim Currently, full-length sequences are known for only 12 out of 644 HLA-DPB1 alleles, covering 6 of 54 common and well-documented (CWD) alleles. To better understand the patterns and types of polymorphisms in the HLA-DPB1 gene, we full-length sequenced a set of donors from the DKMS Life Science Lab sample repository selected with the goal to represent all common and well-documented (CWD) alleles. Methods Primers were developed flanking the UTR-regions of DPB1 resulting in a 12 kb amplicon. After long range PCR two redundant sequencing strategies were employed: Shotgun sequencing on Illumina MiSeq instruments and Single Molecule Real Time (SMRT) sequencing on PacBio RS II instruments. Phase-defined consensus sequences were generated from PacBio data and confirmed with short reads using the DR2S software (DKMS Life Science Lab). Results We successfully sequenced the full-length sequences of 173 samples (346 haplotypes) covering 37 common, 14 well-documented, and 3 not-CWD-defined DPB1 alleles. An additional 84 allelic variants were found based on variation outside of exons 2 and 3. Nine previously known full-length DPB1 alleles were confirmed at least once. For these alleles 45 novel intron variants were found. Novel variation is present most densely within the 5 ′ UTR and introns 1 and 2. A phylogenetic analysis of the 138 unique DPB1 alleles shows the existence of two major evolutionary branches characterized by a 4 Kb block at the 3 ′ end which is composed of only two haplogroups with no evidence of historical recombination. Conclusion Here we present full-length sequences of 138 unique DPB1 alleles covering most common and all well-documented (CWD) alleles. We observe distinct differences in recombination rates within DPB1 indicating differential evolutionary forces acting on different parts of the gene.

P143 Determining the serologic equivalent of a relatively rare common allele DRB1∗11:17

Aim Many common and well-documented (CWD) alleles have not been assigned official World Health Organization (WHO) approved serologic equivalents, which often makes the reporting of time-sensitive deceased donor typing and virtual crossmatch very challenging. Here we report the characterization of the serologic reactivity of a relatively rare CWD allele, DRB1∗11:17. Methods A 31 year-old African American female deceased donor was presented for HLA typing using the real-time (RT) PCR and PCR-SSP methods at the low-intermediate resolution. High resolution typing was retrospectively performed by SBT. Serologic specificity was investigated by way of surrogate flow cytometric crossmatch (FCXM) using donor lymphocytes and selected patient sera with well characterized antibody specificities as defined by single antigen solid-phase assays. Results Donor DR typing was confirmed by all assays as DRB1∗11:01, DRB1∗11:17. According to the NMDP database, DRB1∗11:17 has an overall frequency 0.04% in African Americans, where most commonly found, and accounts for 0.27% of all DR11 alleles reported. While there is no official WHO assigned serologic specificity for DRB1∗11:17, the artificial Neural Network (NN) analysis predicted it to react as DR14. Of the 9 surrogate FCXMs performed using selected sera containing antibody reactivity to DR14 (5 to DRB1∗14:01 only; 2 to DRB1∗14:02 only; 2 to both DRB1∗14:01/14:02) at various strengths (2000–14000 MFI), 8 resulted in positive B-cell crossmatches. No noticeable difference was observed between the reactivity to DRB1∗14:01 vs 14:02. The one negative crossmatch could be attributed to a rather weak DR14 reactivity ( ⩽ 4 aa substitutions as compared to DRB1∗14:01G, indicating a higher sequence homology to DRB1∗14 than to DRB1∗11 alleles. Conclusion In this study, the serologic specificity of DRB1∗11:17 has been confirmed as DR14, which is consistent to that predicted by the NN analysis.

P026 Four unrelated individuals sharing a haplotype with a rear allele DQB1∗06:123

In 2013, one laboratory from Saudi Arabia reported a rare allele of DQB1 locus to IMGT using two different cells. At our laboratory, we observed four unrelated individuals typed for DQB1 locus by RSSO as DQB1∗06:29/123. Since DQB1∗06:29/123 has the same beads reaction by RSSO, Sequence based typing for the four individuals confirmed the typing to be DQB1∗06:123. In addition, the four unrelated individuals shared the same haplotype (A∗02; B∗50; C∗06; DRB1∗15:01; DQB1∗06:123). This particular allele may be included in the common and well-documented (CWD) alleles. As per the 2012 published CWD HLA alleles an allele that is detected three times or more in unrelated individuals with a specific haplotype as discussed above should be included in the well-documented category.

Uncommon HLA alleles identified by hemizygous ultra-high Sanger sequencing: haplotype associations and reconsideration of their assignment in the Common and Well-Documented catalogue

Although the number of HLA alleles still increases, many of them have been reported being uncommon. This is partly due to lack of full length gene sequencing, especially for those alleles belonging to an allele ambiguity in which the first discovered allele has been assigned as the most frequent one. As members of the working group on Common and Well Documented (CWD) alleles and since we implemented full length group-specific sequencing as standard method routinely, we have investigated the presence of presumably rare alleles in our collection of HLA typing data. We identified 50 alleles, that were not previously encountered as Common or Well Documented. Sixteen of them should be added to the CWD catalogue, since we encountered them in 5 or more unrelated individuals. Another 11 could be added, based upon our results and the data present in the IMGT database and the rare allele section of the allele frequencies database. Furthermore, tight associations were observed between several different alleles even at the level of synonymous and non-coding sequences. In addition, in several cases the uncommon allele was found to be more frequent than its common counterpart.

Commom, well-documented and rare HLA alleles observed in the brazilian bone marrow registry

Aim We intend to characterize alleles with 5 or more counts in non related donors of the Brazilian Bone Marrow Registry (REDOME) that are not listed in common and well documented (CWD) alleles version 2. Methods Identification and comparison of alleles and NMDP alleles codes which first allele in the subtype are not in CWD version 2 from the REDOME data base at February 15, 2015. Results We identified 30,395 HLA typings with 1575 alleles or NMDP alelle codes of not common or not well documented among the 3,227,387 donors. A total of 1213 donor registries corresponding to 158 alleles in A locus, 306 in B locus, 47 in C locus, 136 in DRB1 locus and 37 in DQB1 locus had less of 5 counts. In a few cases (28/283) it was possible to have access to the outputs and from these, 11 were probably misinterpreted results, although in one case A∗02:90 was also reported in Chinese donor registry. We also identified 259 alleles with 5–25 counts, 116 alleles with 26–100 counts, 29 alleles with 101–500, and 4 with more than 500. Eighty of these alleles or NMDP allele were most likely misinterpretation due to SSO bead adjustments corresponding to 9582 donors. Actual SSO output reviewed interpretation and SBT confirmed typings are show in Table 1. Conclusion Sequencing based typings were on going to confirm these Brazilian CWD HLA alleles and are displayed at www.imunogenetica.org (in Portuguese). Download : Download full-size image

LBP19: EXTENDED COVERAGE BY NEXT GENERATION SEQUENCING METHODS REFINES THE CHARACTERIZATION OF THE COMMON AND WELL DOCUMENTED HLA ALLELES

The accurate identification of the true allele frequency of HLA genes in various ethnic groups may be a challenging task. The current methodology to catalog the common and well-documented (CWD) HLA alleles is based on the 3rd or 4th field made from assumptions for gene segments that were not tested by Sanger Sequencing methods. We examined the allelic frequencies in all HLA loci by leveraging the power of Next Generation Sequence (NGS) and long range PCR to interrogate the entire sequence of the class I genes and most of the extent Class II genes. In a HLA typing study of 6,000 subjects we unexpectedly found several examples of alleles with common occurrence that were thought to be rare, and represent the most common allele of the 1st-2nd fields allele group. For example, we found that the alleles B ∗ 40:01:02 and DRB1 ∗ 08:01:03 represent the common alleles of their groups while the alleles with the lowest digit number (B ∗ 40:01:01, DRB1 ∗ 08:01) were absent. Extending the sequences also failed to find alleles thought to be common, while other alleles appeared to replace them; DRB3 ∗ 02:24 that associates tightly with DRB1 ∗ 14:01:01 is common while the closely related allele DRB3 ∗ 02:01 thought to be common, was not found; these alleles differ in exon-3, that had not been tested frequently. Our study also showed that several subtypes of the allele groups A ∗ 29:02, B ∗ 44:02, B ∗ 18:01, B ∗ 52:01, C ∗ 05:01, C ∗ 06:02, C ∗ 07:01, C ∗ 07:02, DQA1 ∗ 01:01, DQA1 ∗ 01:02, DQA1 ∗ 01:03, DQA1 ∗ 05:01, DQA1 ∗ 05:05, DQB1 ∗ 03:01 and DQB1 ∗ 05:01 differing at non coding regions or by silent substitutions at areas not tested frequently, are very common. Intriguingly, the alleles differing in areas not tested routinely in previously used assays, show distinctive associations with alleles of contiguous or close loci and contrary to previous predictions indicate that the evolutionary processes in HLA are more complex and modern populations show strongly conserved common haplotype blocks. Our findings show that HLA diversity may have arisen from opposing mechanisms such as allele diversification and convergent evolution.

A Study on Polymorphisms and Common and Well-Documented Alleles of HLA-DPA1 and-DPB1 in Southern Chinese Han Population

Objective To explore the polymorphisms of human leukocyte antigen (HLA)-DPA1 and-DPB1,and determine the common and well-documented (CWD) alleles in southern Chinese Han population.Methods From February 2010 to September 2011,a total of 1 730 cases of peripheral blood samples from unrelated healthy voluntary blood donors of Chinese Han population in Shenzhen blood center,were collected in this study by compater random number table.All these samples were subjected to HLA-DPA1 and-DPB1 sequencing-based typing at exon 2 in both directions.In-house group specific primers were designed and utilized to identify the ambiguous allele combinations.Products of sequencing reactions subjected to sequence cleaning by EtOH/ethylene diamine tetraacetic acid (EDTA) method.The purified products were run electrophoresis on the ABI 3730 DNA sequencer,then obtained sequences were imported into the Assign 3.5 software,and the HLA-DPA1 and-DPB1 allelic genotype were assigned.Results In 1 730 unrelated healthy southern Chinese Han individuals,a total of 7 HLA-DPA1 alleles were identified,including 5 common alleles of which the frequencies were more than 0.1％.Frequencies of each common allele were 54.65％ of DPA1 * 02 ∶ 02,31.24％ of * 01 ∶ 03,10.26％ of * 02 ∶ 01,3.64％ of * 04 ∶ 01,and 0.12％ of * 01 ∶ 04.Two rare novel alleles were observed in less than 3 times.Thirty three HLA-DPB1 alleles including 20 common alleles with a frequency more than 0.1％,and 2 well-documented alleles with a frequency less than 0.1％ but were detected more than 3 times and 11 rare alleles of which two were novel alleles were determined.Conclusions Technique for simultaneous HLA-DPA1 and-DPB1 sequencebased typing at exon 2 and assay for determining ambiguous allele combinations using the in-house group specific primers have been established in this study.The information of allele frequencies and CWD alleles for HLA-DPA1 and-DPB1 in southern Chinese Han population were obtained. Key words: Human leukocyte antigens; Sequencing-based typing; Group specific primer; Common and well-documented alleles

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Most HLA alleles described in the IMGT/HLA database are very rare. The Common and Well Documented (CWD) allele list was created in 2007 to identify the subset of HLA alleles likely to be observed again, and the CWD list was revised in 2013 (CWD2). Our aim was to identify which protein-level CWD2 alleles were estimated from US registry HLA data. We evaluated allele frequency coverage of CWD2 “Common” and “Well Documented” categories in estimated haplotype frequencies for A,C,B,DRB1,DQB1 loci derived from all DNA-based registry typings in 26 US populations (http://bioinformatics.nmdp.org/FullRegistryHaplo/). We also identified CWD2 alleles not present in the US frequencies, the most frequent non-CWD2 alleles, and percentage of single-pass SBT requiring additional typing to meet CWD2-based typing resolution requirements. Common CWD2 alleles constituted a minimum of 91.1% of allele frequency at any locus in any population. Inclusion of Well Documented CWD2 alleles reached 99.5% coverage. 5 CWD2 alleles, all Well Documented category, were not observed in US frequencies (B*40:22N, B*52:21 C*07:67, DRB1*04:12, DRB1*14:14), however all 5 were estimated in a preliminary 2012 global BMDW dataset. Additionally, there were haplotype frequency estimates for many non-CWD2 alleles (514 A, 257 C, 707 B, 276 DRB1, 49 DQB1). The estimated percent of single-pass SBT typings with CWD2 genotypic ambiguity was at most 16.6% at any locus in any population, up from 9.6% for CWD1. Available US haplotype frequencies offer near complete coverage of CWD2 alleles, and offer additional advantages of providing population-specific allele frequency and haplotype linkage information. Publication of global BMDW haplotype frequencies is a goal of an international collaborative effort by the World Marrow Donor Association (WMDA) Registry Diversity working group, and will cover all CWD2 alleles when complete. The most frequenct non-CWD alleles may predict future additions to the living CWD list.

Common and well‐documented HLA alleles: 2012 update to the CWD catalogue

We have updated the catalogue of common and well-documented (CWD) human leukocyte antigen (HLA) alleles to reflect current understanding of the prevalence of specific allele sequences. The original CWD catalogue designated 721 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, and -DPB1 loci in IMGT (IMmunoGeneTics)/HLA Database release 2.15.0 as being CWD. The updated CWD catalogue designates 1122 alleles at the HLA-A, -B, -C, -DRB1, -DRB3/4/5, -DQA1, -DQB1, -DPA1 and -DPB1 loci as being CWD, and represents 14.3% of the HLA alleles in IMGT/HLA Database release 3.9.0. In particular, we identified 415 of these alleles as being 'common' (having known frequencies) and 707 as being 'well-documented' on the basis of ~140,000 sequence-based typing observations and available HLA haplotype data. Using these allele prevalence data, we have also assigned CWD status to specific G and P designations. We identified 147/151 G groups and 290/415 P groups as being CWD. The CWD catalogue will be updated on a regular basis moving forward, and will incorporate changes to the IMGT/HLA Database as well as empirical data from the histocompatibility and immunogenetics community. This version 2.0.0 of the CWD catalogue is available online at cwd.immunogenomics.org, and will be integrated into the Allele Frequencies Net Database, the IMGT/HLA Database and National Marrow Donor Program's bioinformatics web pages.