HLA-DRB3*02:61Q , a novel HLA-DRB3 allele identified in a volunteer bone marrow donor.

HLA-DRB3*02:61Q , a novel HLA-DRB3 allele identified in a volunteer bone marrow donor.

HLA-A*68:02:11, a new HLA-A*68 allele identified during family HLA typing.

A*68:02:11 differs from A*68:02:01 by a silent mutation in codon 122.

Bioinformatic databases and resources in the public domain to aid HLA research.

Research in HLA as in any other field depends on information. Different groups have generated generic and specific resources and tools to support this research. The present review describes a qualified subset of these resources, which should cover the most important starting points for research in the HLA field. It discusses access to HLA allele sequences, allele frequencies, continues with general support to access to literature, DNA and protein sequence information, structural models, teaching books, databases with phenotypic datasets, alignment tools, peptide binding, statistical tools, guidelines and ambiguity coding. Following functionalities and databases have been included: IMGT/HLA, Immuno Polymorphism Database (IPD), allele frequencies*.net, a detailed look into NCBI (National Center of Biotechnology Information) with a subset of databases and tools, focusing on literature research, sequences, user-specific support tools and applications (PubMed, GenBank, MyNCBI, blast, Gene, MapViewer, Structure, CN3D, WorkBench, and dbMHC). This is followed by a brief survey of EBI-EMBL/Ensemble, the sequence alignment tool Clustal, the peptide and ligand databases SYFPEITHI and Immune Epitope Database, and last but not least statistical packages and HLA allele coding resources PyPop, the Immuno-genomics Data Analysis Working Group and the NMDP informatics section. All databases and tools can be freely accessed. Data linked to individuals, however, might require authorization by a data access committee.

A new HLA-A*26 variant, A*2637 identified by haplotype-specific extraction and sequencing-based typing.

The novel allele A*2637 differs from the common A*260101 by a single nucleotide substitution in exon 2 (position 186 C>G) causing an amino acid exchange (SER>ARG).

A new HLA-B*44 variant, B*4453, identified by haplotype-specific extraction and sequencing-based typing.

We report the identification of a novel HLA B*44 allele, officially named B*4453, found in an Austrian patient and his two sisters.

14th International HLA and Immunogenetics Workshop: report on the HLA component of type 1 diabetes.

The type 1 diabetes (T1D) component of the 13th International Histocompatibility Workshop (IHW) obtained microsatellite (msat) and human leukocyte antigen (HLA)-DR/DQ data on case/control and family samples through an international collaboration. The aim was to detect the effects of susceptibility loci on the HLA complex independent of the primary determinants in the class II region (HLA-DR/DQ). As part of the activity of the 14th International HLA and Immunogenetics Workshop (14th IHIWS), a T1D workshop was held to present analyses of the 13th IHW data and to discuss the current status of knowledge about the genetics of T1D. These data are now available online through dbMHC, a web-based resource established by the National Center for Biotechnology. Continuing work since the 13th IHW has resulted in published work showing heterogeneity of DR3 haplotypes in data sets from the 13th IHW and Human Biological Data Interchange (HBDI). In addition, we identified markers that define DRB1*1501 DQB1*0602 haplotypes conferring reduced protection from diabetes in a Swedish 13th IHW data set. Further analyses of the 13th IHW data set not only showed some significant results but also demonstrated extensive heterogeneity reminiscent of non-HLA genes. The haplotype analysis in HBDI families identified two msats with significant effects on susceptibility and statistically significant age of onset effects at class III markers that are not because of linkage disequilibrium, with class I alleles known to affect age of onset. The above studies underscore the importance of refining our understanding of susceptibility associated with genes in the HLA complex.

The dbMHC microsatellite portal: a public resource for the storage and display of MHC microsatellite information.

Major histocompatibility complex (MHC) region Microsatellites (Msat) have been extensively used in various applications, such as disease mapping, forensics, and population genetics. A comprehensive review of HLA Msat primers has been previously published based on literature and sequence analysis, but electronic tools are lacking to make it easily accessible and actually used by the community. We have integrated data from this review, with an overlapping set of 31 Msat markers used in the 13th International Histocompatibility and Immunogenetics Workshop (IHIWS) to create a public archive that will synchronize published descriptions to a common framework. http://www.ncbi.nlm.nih.gov/projects/mhc. Currently, the dbMHC contains 389 primer pairs across the extended MHC targeting 281 distinct repeat regions (approximately 1/45 kb). Literature review and analysis of the primers reveal that over 200 synonymous names have been published for these markers. Users may view or download specific Msat data sets using the portal. Query options include name or partial name, primer sequence, neighboring genes, and/or position. Query results include locus name(s), a graphic showing of the relative location of the marker in relation to the classical HLA genes, a listing of the constituent primer pairs and name, a link to UniSTS, aliases, allele range (bp), overlapping single nucleotide polymorphisms, a link to e-polymerase chain reaction, and physical mapping information. To increase the utility of this resource, researchers using Msat markers in the HLA region are encouraged by the authors to submit new primers to the dbMHC. The minimal Msat submission consists of primers sequences, a submitter's name and contact information. Additional information recommended but not required is the laboratory protocol(s), known allele size range (bp), known aliases, and an exemplar sequence. Assigned UniSTS numbers can be used for primer pair standard identification.

The reagent database at dbMHC.

The reagent database dbMHC was built by the National Center for Biotechnology Information (NCBI) as an open resource for registration and characterization of HLA DNA-typing kits and reagents. Each reagent is uniquely identified as sequence-specific oligonucleotide (SSO) or primer (SSP), SSO mix, or SSP mix. Computerized prediction of allele reactivities, based on annealing stringency, is performed on all submissions to the reagent database. User-specified allele reactivities may be added or deleted independently of the prediction algorithm. Updates of allele reactivities are performed in synchronization with the IMGT/HLA database, in order to account for newly discovered alleles. Probe and primer sequences aligned to allelic sequences can be displayed at any time. Reagents registered in the reagent database are grouped in typing kits. Each kit or kit batch is uniquely identified. Group-specific amplification of alleles can be specified for an entire kit or for sections of each kit. Kits designed to test multiple loci are supported. Kits can be entered and updated via the web or submitted as batches in extensible markup language (XML) format. A tool for online interpretation of typing results is available. Both the reagent database and the typing kit database have been designed to facilitate the exchange of HLA typing based on raw typing data using the unique identifiers of kits or individual reagents. In addition, batch-wise reinterpretation of previous typing data can be performed either using the NCBI web site or by locally using downloaded allele-reactivity lists. Reinterpretation by the NCBI requires submission of raw typing data in XML format.

A novel HLA-DRB1 sequence, DRB1*11272.

We describe the complete exon 2 sequence of a novel HLA-DRB1 allele, DRB1*11272. This allele differs from the DRB1*11271 allele by a synonymous mutation in codon 77 where an AAT is replaced with AAC, both encode for the amino acid asparagine. The same motif at codon 77 has also been found in DRB1*1107, DRB1*1333, DRB1*0422 and in most DRB1*03 alleles. A partial exon 2 sequence of this allele has previously been deposited in the EMBL Sequence Database under the accession number AF186407.

A new h allele detected in Europe has a missense mutationin alpha(1,2)-fucosyltransferase motif II.

BACKGROUND: The FUT1 gene encodes an alpha(1,2)-fucosyltransferase (H transferase), which determines the blood group H. Nonfunctional alleles of this gene, called h alleles and carrying loss-of-function mutations, are observed in the exceedingly rare Bombay phenotype. Twenty-three distinct h alleles have been characterized at the molecular level in various populations. The FUT2 (SE) gene is highly homologous to FUT1 (H:). STUDY DESIGN AND METHODS: The FUT1 gene of an Austrian proband with the Bombay phenotype was characterized by nucleotide sequencing of the full-length coding sequence. A PCR method using sequence-specific primers for FUT2 genotyping in whites was developed. The plasma alpha(1,2)-fucosyltransferase activity was determined. The distribution of the mutations underlying 24 h alleles and 7 se alleles was analyzed. RESULTS: The proband carried a new h allele. Two nucleotide changes, G785A and C786A, in codon 262 of the FUT1 gene resulted in the replacement of serine by lysine. No alpha(1,2)-fucosyltransferase activity was detected in the proband's plasma. The proband was homozygous for the seG428A allele. Six of 17 missense mutations in nonfunctional h and se alleles occurred in highly conserved fucosyltransferase motifs. No loss-of-function mutation was observed in the aminoterminal section encompassing the transmembraneous helix. CONCLUSION: The missense mutation S262K in the FUT1 gene caused the loss of H transferase activity. The analysis of the distribution of mutations in nonfunctional FUT1 and FUT2 genes can point to functionally important domains in the H transferase.

Going back to the roots: effective utilisation of HLA typing information for bone marrow registries requires full knowledge of the DNA sequences of the oligonucleotide reagents used in the testing.

Information obtained by DNA-based HLA typing assays is more detailed and of higher quality than that obtained by conventional serological techniques. Nevertheless, it is common for data acquired in this way to be presented in the more familiar serological format. In many cases this representation can lead to significant loss of information, which may only become apparent at a later time, with the discovery of novel allele sequences. DNA-based typing methods, such as sequence-specific oligonucleotide probing (SSOP) or sequence-specific priming (SSP) generate fragmentary sequence data which is information rich. An alternative to assigning allele names to these fragments is to simply store the sequence data itself without interpretation. Bone marrow donor repositories can then be searched directly with sequence information, which though complex is more complete, rather than searching by derivative allele names.

Severe HDN due to anti-Ce that required exchange tranfusion.

BACKGROUND: Rh system antibodies are commonly encountered in blood bank practice as well as during pregnancy. Nevertheless, no examples of anti-Ce (RH7) have been reported as a cause of HDN that requires exchange transfusion. CASE REPORT: A 38-year-old woman in her fourth pregnancy was typed as blood group O D+, C-, c+, E+, e-. Anti-C and anti-e were detected in her serum during a routine prenatal work-up. Further evaluation, including flow cytometric analysis, revealed the presence of a strong anti-Ce and a weak anti-e. Her partner was typed as group A D+, C+, c-, E-, e+. A seemingly healthy male infant was delivered at 40 weeks of gestation. The infant's RBCs were typed as group O D-, C+, c+, E+, e+ with a positive DAT (titer 128). Twenty-five hours after birth, the baby had to be transferred to the neonatal intensive care unit because of rapidly rising total serum bilirubin. Despite intensive treatment, including double phototherapy, albumin infusion, and the administration of furosemide and IVIG, the total serum bilirubin level increased during the following day and exchange transfusion with 2 units of type O D-, C-, c+, E+, e- had to be performed; this resulted in a prompt decrease in total serum bilirubin without relapse. CONCLUSION: Anti-Ce caused severe HDN requiring exchange transfusion. This highlights the need for a close follow-up throughout pregnancy if unexpected RBC antibodies are present, to permit the provision of compatible blood in case of a rare antibody.

NCBI genetic resources supporting immunogenetic research.

The NCBI creates and maintains a set of integrated bibliographic, sequence, map, structure and other database resources to promote the efficient retrieval of information and the discovery of novel relationships. The connections made between elements of these resources permit researchers to start a search from a wide spectrum of entry points. These multiple dimensions of data can be roughly categorized by primary content as text or bibliographic (PubMed, PubMedCentral, OMIM, LocusLink), sequence (GenBank, Reference Sequence Project (RefSeq), dbSNP, MMDB), protein structure (MMDB) or map position (MapView). They can also becategorized by level of expert curation, which may range from validation of submissions from external groups (GenBank, PubMed, PubMedCentral,), to automatic computation (HomoloGene, UniGene), and to highly reviewed and corrected (LocusLink, MMDB, OMIM, RefSeq). Searches can be made by words (in an article title, key words, sequence annotation, database value, author) by sequence (BLAST or e-PCR against multiple sequence databases), or by map coordinates. By computing or curating bi-directional links between related objects, NCBI can represent content on the genetics, molecular biology, and clinical considerations of interest to immunogeneticists. There is also an emerging resource developed by the NCBI in collaboration with the IHWG devoted to the presentation of MHC data (dbMHC). How dbMHC will augment existing resources at the NCBI is described.

Storage and utilization of HLA genomic data--new approaches to HLA typing.

Currently available DNA-based HLA typing assays can provide detailed information about sequence motifs of a tested sample. It is still a common practice, however, for information acquired by high-resolution sequence specific oligonucleotide probe (SSOP) typing or sequence specific priming (SSP) to be presented in a low-resolution serological format. Unfortunately, this representation can lead to significant loss of useful data in many cases. An alternative to assigning allele equivalents to suchDNA typing results is simply to store the observed typing pattern and utilize the information with the help of Virtual DNA Analysis (VDA). Interpretation of the stored typing patterns can then be updated based on newly defined alleles, assuming the sequence motifs detected by the typing reagents are known. Rather than updating reagent specificities in individual laboratories, such updates should be performed in a central, publicly available sequence database. By referring to this database, HLA genomic data can then be stored and transferred between laboratories without loss of information. The 13th International Histocompatibility Workshop offers an ideal opportunity to begin building this common database for the entire human MHC.

Virtual DNA analysis as a platform for interlaboratory data exchange of HLA DNA typing results.

In 1998, the German DNA Exchange offered the possibility to report typing data as virtual DNA. Participating labs have been equipped with software based on the principle of Virtual DNA Analysis (VDA). This approach allows the combination of sequence-specific oligonucleotide (SSO), sequence-specific primer (SSP) and sequence-based typing (SBT) results. The use of all types of test kits has been allowed without any limitations, as long as basic sequence information on SSOs or SSPs was available, at least the sequence and the position of the detected motif on the sample DNA. Typing raw data of the actual SSO-SSP and, if performed, SBT information was collected. Participating labs received 20 DNA samples to type. Fourteen labs returned data on 1,250 single-locus testings. Reported data consisted of 317 SBT data, 452 SSO kits and 1,795 SSP kits with 43,312 single SSO/ SSP reactivities. One hundred and twenty-six different typing kits (unique laboratory-specific kits, commercial kits from 7 companies) have been used. In 30 (2.4%) single-locus testings, at least one single SSO/SSP reactivity has been false-positive or -negative, thus not leading to a valid result on primary evaluation. Eight of these 30 cases were due to the presence of a new DRB1*14 allele in sample no. 2. Thirty-five tests (2.8%) showed wrong allele assignments. This first attempt to collect raw typing data instead of typing interpretation on a larger scale shows the advantages of Virtual DNA Analysis like interlaboratory data exchange without loss of information, transparency of typing interpretation and reinterpretation of typing data with an updated allele database. The VDA format is a useful tool for workshops and bone marrow donor registries.

Definition of new alleles of MIC-A using sequencing-based typing.

We have sequenced exons 2, 3 and 4 of MIC-A in 23 homozygous cell lines, 22 families and 54 unrelated individuals. This has led to the definition of seven polymorphic positions in exon 2, 13 in exon 3 and 12 in exon 4, yielding a total of 33 different MIC-A allelic specificities, of which 16 have not been described before. The newly defined sequences and those of the alleles defined before were entered into a database of the SCORE program (Helmberg et al., 1998, Tissue Antigens, 51, 587) for comprehensive genotyping analysis. In the tested sample, only one genotype present in two individuals gave rise to an ambiguous genotype. If all possible combinations of the 33 alleles are considered, 10 of 636 combinations are ambiguous. The MIC-A exon 2, 3 and 4 polymorphism is characterized by diallelic single base exchanges and by a considerable degree of exon shuffling. The majority of heterozygote positions identified are non-synonymous, i.e. five of seven in exon 2, 13 of 13 in exon 3 and eight of 12 in exon 4, suggesting an important function for the MIC-A polymorphism.

HLA-B*27 typing by group-specific hybridization in microtiter plates.

We have developed and evaluated a test for HLA-B*27 based on PCR and DNA hybridization in microtiter plates. A region within exon 2 of the HLA-B gene is amplified and labeled by PCR and the amplification product is hybridized to a group-specific HLA-B*27 and a generic control oligonucleotide probe in two separate cavities of the plate. Bound sequences are detected using an ELISA-like protocol. The assay has been evaluated on 254 DNA samples routinely received for B27 testing in parallel with serological and SSP-PCR typing. Results were concordant in typing 102 HLA-B27-positive and 152 HLA-B27-negative individuals except for two samples containing HLA-B*73, which stained B27 positive in the microwell test. The new procedure is rapid and simple to perform, and the microwell format is particularly suitable for automation.

Virtual DNA analysis--a new tool for combination and standardised evaluation of SSO, SSP and sequencing-based typing results.

In order to obtain reliable information on HLA types, DNA typing with sequence-specific oligonucleotide/primer (SSO/SSP) typing sets or sequencing-based typing (SBT) is increasingly performed. The quality of the evaluation depends on the presence of a complete listing of all typed alleles as well as on the ability of detecting all corresponding alleles/allele pairs. We have developed the concept of virtual DNA analysis (VDA), which is able to combine all types of SSO/SSP/SBT results and evaluate this typing in combination according to the latest published allele sequence lists. The concept is based on the target DNA recognised by the respective typing techniques. All SSO/SSP or SBT results are transformed to a virtual sample DNA, which subsequently is analysed. Evaluation of generic or allele-specific DNA typing or the combination of both is supported. Due to this flexible approach, all kinds of SSO/SSP sets, as far as the respective SSO/SSP sequences are available, can be entered and evaluated immediately. The combination of collected data of different typing sets and procedures leads to the highest possible typing resolution. If more than one possible allele combination persists, the program reduces the result to the most specific common denominator in a stepwise manner. VDA offers the possibility of re-evaluation of former SSO/SSP/SBT results, alone or in combination. No solutions are omitted. This might be a first step towards standardisation of evaluating DNA-based HLA typing results or transfer of the respective typing data for later evaluation.

Detection and differentiation of platelet-specific antibodies by flow cytometry: the bead-mediated platelet assay.

BACKGROUND: Platelet-reactive antibodies cause a number of clinical disorders. The detection and differentiation of these antibodies are prerequisites for the adequate treatment of these disorders. The bead-mediated platelet assay described here enables the detection and differentiation of platelet-bound antibodies by the use of flow cytometry. STUDY DESIGN AND METHODS: The bead-mediated platelet assay is based on the isolation of human platelet glycoproteins by using flow cytometric standardization beads after the incubation of typed platelets with human sera. The specificity and sensitivity of this assay were tested with five sera, each containing a known platelet-reactive antibody. The monoclonal antibody-specific immobilization of platelet antigens assay was used as a reference test. RESULTS: The bead-mediated platelet assay was able to determine the glycoprotein specificity of the antibody without cross-reactions in every case. In serial dilution tests, the bead-mediated platelet assay was able to detect the antibodies at higher dilutions than the monoclonal antibody-specific immobilization of platelet antigen assay. Total test time was 3.5 hours. CONCLUSION: The bead-mediated platelet assay is a fast and reliable method for the detection and differentiation of platelet-reactive antibodies.