Strategies to work with HLA data in human populations for histocompatibility, clinical transplantation, epidemiology and population genetics: HLA-NET methodological recommendations.

HLA-NET (a European COST Action) aims at networking researchers working in bone marrow transplantation, epidemiology and population genetics to improve the molecular characterization of the HLA genetic diversity of human populations, with an expected strong impact on both public health and fundamental research. Such improvements involve finding consensual strategies to characterize human populations and samples and report HLA molecular typings and ambiguities; proposing user-friendly access to databases and computer tools and defining minimal requirements related to ethical aspects. The overall outcome is the provision of population genetic characterizations and comparisons in a standard way by all interested laboratories. This article reports the recommendations of four working groups (WG1-4) of the HLA-NET network at the mid-term of its activities. WG1 (Population definitions and sampling strategies for population genetics' analyses) recommends avoiding outdated racial classifications and population names (e.g. 'Caucasian') and using instead geographic and/or cultural (e.g. linguistic) criteria to describe human populations (e.g. 'pan-European'). A standard 'HLA-NET POPULATION DATA QUESTIONNAIRE' has been finalized and is available for the whole HLA community. WG2 (HLA typing standards for population genetics analyses) recommends retaining maximal information when reporting HLA typing results. Rather than using the National Marrow Donor Program coding system, all ambiguities should be provided by listing all allele pairs required to explain each genotype, according to the formats proposed in 'HLA-NET GUIDELINES FOR REPORTING HLA TYPINGS'. The group also suggests taking into account a preliminary list of alleles defined by polymorphisms outside the peptide-binding sites that may affect population genetic statistics because of significant frequencies. WG3 (Bioinformatic strategies for HLA population data storage and analysis) recommends the use of programs capable of dealing with ambiguous data, such as the 'gene[rate]' computer tools to estimate frequencies, test for Hardy-Weinberg equilibrium and selective neutrality on data containing any number and kind of ambiguities. WG4 (Ethical issues) proposes to adopt thorough general principles for any HLA population study to ensure that it conforms to (inter)national legislation or recommendations/guidelines. All HLA-NET guidelines and tools are available through its website http://hla-net.eu.

Report from the killer immunoglobulin-like receptor (KIR) anthropology component of the 15th International Histocompatibility Workshop: worldwide variation in the KIR loci and further evidence for the co-evolution of KIR and HLA.

The killer immunoglobulin-like receptor (KIR) anthropology component of the 15th International Histocompatibility Workshop (IHIWS) sought to explore worldwide population variation in the KIR loci, and to examine the relationship between KIR genes and their human leukocyte antigen (HLA) ligands. Fifteen laboratories submitted KIR genotype and HLA ligand data in 27 populations from six broad ethnic groups. Data were analyzed for correlations between the frequencies of KIR and their known HLA ligands. In addition, allelic typing was performed for KIR2DL2 and 3DL1 in a subset of populations. Strong and significant correlations were observed between KIR2DL2, 2DL3 genotype frequencies and the frequency of their ligand, HLA-C1. In contrast, only weak associations were seen for 3DL1, 3DS1 and the HLA-Bw4 ligand. Although some aspects of the correlations observed here differ from those reported in other populations, these data provide additional evidence of linked evolutionary histories for some KIR and HLA loci. Investigation of allele-level variation for the B haplotype locus KIR 2DL2 showed that two alleles, *001 and *003, predominate in all populations in this study. Much more allelic variation was observed for the A haplotype locus 3DL1, with several alleles observed at moderate frequencies and extensive variation observed between populations.

Psychological care of the haemodialysis patient.

The psychological care of patients at the haemodialysis unit is becoming an increasingly important aspect of the overall treatment of patients with end-stage renal failure. As more is understood and reported regarding the impact of psychological factors on physical processes it becomes crucial that good quality psychological care is delivered to patients by nurses working at the haemodialysis unit. This study aimed to look at haemodialysis patients' perceptions of their treatment to discover if a dialysis centre in Southern England might benefit from a more formal system of psychological care. The data revealed four main themes that were of particular importance to the participants of this study: The New Self, Coping, Medical Concerns and Psychological Care. Within these areas of importance, the need for good quality information, the need for a suitable dialysis environment and the importance of social and family support were evident. Although the psychological care system that was in place at the unit appeared to be working well a more formal system of care might improve the patient experience.

Alkaline-mediated differential interaction (AMDI): a simple automatable single-nucleotide polymorphism assay.

The key requirements for high-throughput single-nucleotide polymorphism (SNP) typing of DNA samples in large-scale disease case-control studies are automatability, simplicity, and robustness, coupled with minimal cost. In this paper we describe a fluorescence technique for the detection of SNPs that have been amplified by using the amplification refractory mutation system (ARMS)-PCR procedure. Its performance was evaluated using 32 sequence-specific primer mixes to assign the HLA-DRB alleles to 80 lymphoblastoid cell line DNAs chosen from our database for their diversity. All had been typed previously by alternative methods, either direct sequencing or gel electrophoresis. We believe the detection system that we call AMDI (alkaline-mediated differential interaction) satisfies the above criteria and is suitable for general high-throughput SNP typing.

Molecular typing for HLA class I using ARMS-PCR: further developments following the 12th International Histocompatibility Workshop.

Molecular typing for HLA class I was introduced in the 12th International Histocompatibility Workshop. Following a pilot study using three methods, sequence specific oligotyping (SSO), reverse dot blot and amplification refractory mutation system (ARMS)-PCR, the ARMS-PCR method was selected for use. A great advantage of an ARMS-PCR method is that, unlike the other two methods, it can determine whether sequence motifs are in cis or in trans, as ARMS-PCR detects two cis located motifs per reaction using forward and reverse sequence specific primers. Resolution was designed to be low to medium level for HLA-A, -B and -C alleles. Two hundred and fifty class I kits and 83 HLA-A2 subtyping kits were distributed. The A2 subtyping kit used a two round nested PCR system to identify all of the A2 alleles known at the time. Typing results on control DNA samples distributed with both the kits showed a very satisfactory performance. Since the 12th Workshop, the kits have been developed with the addition of new primers and primer mixes to increase the resolution of the test.

High-throughput class I HLA genotyping using fluorescence resonance energy transfer (FRET) probes and sequence-specific primer-polymerase chain reaction (SSP-PCR).

We have developed a semi-automated HLA class I typing system utilising TET/TAMRA-labelled fluorescence resonance energy transfer (FRET) hydrolysis probes. The results from 87 individuals are in full concordance with serology and conventional gel-based systems. This assay replaces labour-intensive conventional gel-based DNA typing and has a higher allelic resolution than serology. Our approach differs from previously published fluorogenic probe typing protocols in that it provides simultaneous typing of HLA-A, -B and -C loci to medium resolution. Furthermore, by using equipment that is not specific to FRET probe analysis our system has in-built expansion capacity to 384 reactions per plate, thus making it applicable to high-throughput population screening. Automation is achieved through the use of computer software which analyses direct input from the fluorescence reader, allowing high throughput with a low inherent error rate.

Specifying the relation between novel and known: input affects the acquisition of novel color terms.

4 studies investigate how differential input affects preschoolers' abilities to learn novel color words. 3-, 4-, and 5-year-old children saw objects in novel shapes and colors and heard a novel color label for the object. Labels were presented through ostensive definition (e.g., "See, it's mauve"), corrective linguistic contrast (e.g., "See, it's not purple; it's mauve"), or an inclusion statement (e.g., "See, it's mauve; it's a kind of purple"). 4- and 5-year-old children interpreted the novel word as a shape term when ostensive information was provided but as a color term when additional information, either contrastive or inclusive, specified a relation between the novel term and a known label for that color. Furthermore, children who consistently interpreted the novel word as a color word tended to treat the novel and known labels as mutually exclusive color terms if they heard contrastive information, whereas they tended to treat the words as hierarchically related if they heard inclusion information. 3-year-olds generally did not make use of either type of information in determining the semantic domain of the novel word or the relation between terms.

Mechanisms of loss of HLA class I expression on colorectal tumor cells.

For several years this laboratory has studied the expression of HLA class I on established colorectal tumor cell lines and on fresh tumors. We review here the mechanisms by which colorectal tumor cells may lose surface expression of HLA class I molecules. Several independent mechanisms have been identified, including loss or mutations in beta 2-microglobulin genes, loss of HLA heavy chain genes, selective lack of expression of HLA alleles, and regulatory defects in HLA expression including loss of expression of the peptide transporters associated with antigen processing (TAP). The data suggest that colorectal tumor cells may evade tumor specific, HLA restricted immune attack by loss of HLA class I expression through a number of mechanisms.

Candidate regions for a testicular cancer susceptibility gene.

Epidemiological data suggest the presence of a susceptibility gene for testicular cancer in some families. Families with multiple cases of testicular cancer are rare and almost all those reported have only two affected members. We have performed a sib-pair analysis on 35 families in which there are either two or three affected brothers. These families have been typed for 220 autosomal microsatellite markers spaced 10-20 cM throughout the genome. Six regions which gave a LOD score of more than 1.0 on formal linkage analysis or a P value of 0.05 or less using a non-parametric approach are considered as candidate regions for a susceptibility gene. Of particular interest is one region on chromosome 4. Two neighbouring probes in this region both scored positively with LOD score of 2.60 on multipoint analysis. An International Testis Cancer Linkage Consortium has been formed to pool resources and will investigate these findings further with the world-wide collection of families.

A clinical and epidemiological study of human leukocyte antigen-DPB alleles in Hodgkin's disease.

An international study to investigate the role of human leukocyte antigen (HLA)-DPB alleles in Hodgkin's disease was conducted with 17 participating centers in 12 countries. A total of 741 patients and 686 controls were typed using polymerase chain reaction amplification of HLA-DPB alleles and subsequent sequence specific oligonucleotide hybridization. The frequency of HLA-DPB1*0301 was found to be significantly increased in white patients, compared with ethnically matched controls. In this population group, the DPB1*0301 allele is associated with a relative risk of 1.95 (P < 0.01). There was also a significant reduction in the frequency of HLA-DPB1*0401 in patients from Japan and Taiwan (relative risk, 0.15; P < 0.01). Clinical analysis from data on 551 patients demonstrated a significantly inferior remission duration in patients with HLA-DPB1*0901, overall (P < 0.05), and in the Japanese and Taiwanese populations (P = 0.02), where this allele is most prevalent. This analysis suggests an epidemiological as well as a possible prognostic association between HLA-DPB alleles and Hodgkin's disease.

High-resolution HLA-DPB typing based upon computerized analysis of data obtained by fluorescent sequencing of the amplified polymorphic exon 2.

To differentiate 32 HLA-DPB alleles, conventional techniques such as serology and cellular typing are inadequate for high-resolution DPB typing. The most refined DNA typing until now is SSO typing and new selected oligonucleotides can be added to this system to distinguish new allele sequences. DNA sequencing, however, reveals directly the sequence information of all polymorphic HVRs and has the advantage of being independent from exon polymorphisms. We have developed a new DNA-based typing approach that is rapid, fully automated, and therefore suitable for routine typing. The system is based upon direct sequencing of amplified DNA with fluorescent-labeled primers. The designation of alleles is obtained by a comparison of all polymorphic positions in the determined sequence with all known allele sequences retained in a database along with their heterozygous combinations. Sequence data at both constant and polymorphic positions are used for quality control. In this study, the typing results of a panel of 91 previous SSO-typed DNA samples are described. After comparison with the SSO-typing results, we conclude that with this SBT system allele assignment is reliable. The method is easy to perform since both sequencing and assignment are automated. Furthermore, the system is easily applicable to other gene systems.

TAP1 and TAP2 polymorphism in coeliac disease.

Coeliac disease is strongly associated with HLA-DQ2, but it is possible that additional major histocompatibility complex genes also confer disease susceptibility. Encoded close to HLA-DQ are two genes, TAP1 and TAP2, whose products are believed to transport antigenic peptides from the cytoplasm into the endoplasmic reticulum. Comparison of 81 coeliac disease patients with caucasoid controls revealed an increased frequency of the alleles TAP1A and TAP2A in the patient population. However, no significant difference was found when patients were compared with HLA-DR and -DQ matched controls, indicating linkage disequilibrium between TAP1A, TAP2A, and HLA-DQ2. The TAP gene products do not have a major influence on susceptibility or resistance to coeliac disease in a Northern European Caucasoid population.

Alleles and haplotypes of the MHC-encoded ABC transporters TAP1 and TAP2.

TAP1 and TAP2 are two major histocompatibility complex (MHC) genes, located between HLA-DP and -DQ, whose products form a transporter molecule involved in endogenous antigen processing. Polymorphic residues have been described in both genes and, in this study, we have identified another polymorphic site within the adenosine triphosphate (ATP)-binding domain of TAP2. We have used the amplification refractory mutation system (ARMS) polymerase chain reaction (PCR) to characterize TAP1 and TAP2 alleles and haplotypes in a reference panel of 115 homozygous typing cell lines. Of four possible TAP1 alleles, we observed three, and of eight possible TAP2 alleles, we observed five. Among 88 (homozygous typing cells) (HTCs) homozygous at HLA-DR, -DQ and -DP, 80 were also homozygous at TAP1 and TAP2. Of 27 HTCs homozygous at HLA-DR and -DQ, but heterozygous at -DP, 14 were homozygous at TAP1 or TAP2 and 13 heterozygous, consistent with recombination taking place either side of the TAP loci. Of the fifteen possible combinations of TAP1 and TAP2 alleles, we observed eleven, each at a frequency similar to that predicted by individual allele frequencies. In this ethnically heterogenous panel there is no indication that particular combinations of TAP1 and TAP2 have been maintained together.

Peptide-induced nonresponsiveness of HLA-DP restricted human T cells reactive with Dermatophagoides spp. (house dust mite).

The activation of CD4+ T lymphocytes, which play a central role in allergic inflammation, depends on the recognition of allergen-derived peptides in association with major histocompatibility complex class II gene products. In this report we demonstrate, at a clonal level, that a component of the T-cell repertoire reactive with Dermatophagoides spp. (house dust mite) in atopic individuals, is restricted by HLA-DP class II molecules. This supports the recent results emerging from genetic epidemiologic studies that indicate positive associations between the HLA-DP phenotype and immune responsiveness to a variety of common allergens. Our findings also reveal that the T cells restricted by HLA-DP recognize a species-specific epitope located in the group I allergen of Dermatophagoides pteronyssinus (residues 101-119). Furthermore, we report that the pretreatment of the T cells restricted by HLA-DP with the Der p I peptide renders them nonresponsive to an immunogenic challenge with house dust mite allergen, and the loss of antigen-dependent proliferation is associated with downregulation of membrane expression of the T-cell antigen receptor. The ability to functionally inactivate T cells restricted by HLA-DP, as well as those that recognize allergen in association with HLA-DR class II molecules, suggests that desensitization with allergen-derived peptides may have therapeutic potential in the management of allergic diseases irrespective of their HLA class II association.

Epitope specificity and MHC restriction of rheumatoid arthritis synovial T cell clones which recognize a mycobacterial 65 kDa heat shock protein.

CD4+ T cell clones specific for the mycobacterial hsp 65 were obtained from synovial fluid of a DR4 homozygous rheumatoid arthritis (RA) patient. A stimulatory epitope was defined using both deletion mutants of the mycobacterial hsp 65 and synthetic peptides and proved to be in a highly conserved region of the molecule. Despite this, however, there was no recognition by these clones of either the recombinant human homologue of mycobacterial hsp 65, P60, nor of a synthetic peptide containing an amino acid sequence from P60 corresponding to the epitope defined in the mycobacterial hsp 65. When the pattern of HLA restriction shown by the hsp-65-specific T cell clones was investigated, all clones tested proved to be restricted by HLA-DP rather than the more usual HLA-DR. Inhibition experiments suggested that this restriction also applied to the polyclonal synovial T cell response to hsp 65, but not to other antigens. Exclusive restriction of T cell recognition of an antigen by HLA-DP has not been reported previously, and strongly suggests that in this case the T cell repertoire for recognizing hsp 65 in the context of DR4 is deficient. Such an association between DR4 and the inability to respond to an immunodominant bacterial antigen may have implications for the pathogenesis of RA.

Novel detection of restriction fragment length polymorphisms in the human major histocompatibility complex.

Cosmid genomic DNA clones have been used as hybridization probes in genomic Southern blot analysis to define restriction fragment length polymorphisms (RFLPs) in the major histocompatibility complex (MHC). Using 14 different enzymes and three overlapping cosmid clones we have detected six RFLPs in a 100 kilobase (kb) segment of DNA in the class III region extending centromeric of the TNFA gene toward HLA-DR. Four of the five RFLPs, defined using the enzymes Taq I, Rsa I, Hinc II, and Hind III, and detected by the cosmid clone cosM7B, map to a 29 kb segment of DNA that includes all of the recently described G2 (BAT2) gene and a large portion of the 3' end of the G3 (BAT3) gene. The different RFLP variants were established by analyzing the DNA from three informative families and a panel of 51 HLA-homozygous typing cell lines. CosM7B detects Taq I variants of 4.3 kb, and 2.9 kb or 2.8 kb, Rsa I variants of 2.9 kb or 2.4 kb, Hinc II variants of 5.8 kb or 3.8 kb and 1.4 kb, and a Hind III variant of 4.8 kb, while cosOT2 detects Taq I variants of 4.5 kb or 4 kb. The distribution of the Rsa I, Hinc II and Taq I RFLPs detected by cosM7B, and the Taq I RFLP detected with cosOT2, within the panel of cell line DNAs was assessed by Southern blotting. The 4.3 kb Taq I variant was observed in only one cell line with the extended haplotype HLA-A29, C-, B44, SC30, DR4. The other RFLPs, however, occurred much more frequently. The 2.8 kb Taq I variant was observed in 20% of haplotypes, the 2.9 kb Rsa I variant was observed in 42% of haplotypes, and the 5.8 kb Hinc II variant was observed in 12% of haplotypes analyzed. The 4.5 kb Taq I variant detected by the overlapping cosmid cosOT2 was present in 21% of haplotypes. Analysis of the RFLP variants with each other revealed seven different haplotypic combinations. Three of the haplotypic combinations were each subdivided into two subsets on the basis of the Nco I RFLP variant they carried at the TNF-B locus. These haplotypic combinations potentially allow differentiation among different extended haplotypes such as HLA-B8, SC01, DR3, HLA-B18, F1C30, DR3, and HLA-B44, FC31, DR7. The RFLPs detected by the cosmid clones thus provide new tools which will be useful in the further genetic analysis of the MHC class III region.

Identification of HLA-DP polymorphism with DP alpha and DP beta probes and monoclonal antibodies: correlation with primed lymphocyte typing.

Thirty-four lymphoblastoid cell lines that had been previously typed for HLA-DP antigens by primed lymphocyte typing (PLT) were tested by Southern blotting and by ELISA. Using two DP beta probes and a DP alpha probe with a series of enzymes, it is possible to identify restriction fragment length polymorphism (RFLP) patterns characteristic of DPw1, -2, -3, -4, and possibly -5. ELISA typing results, based on two polymorphic DP antibodies DP11.1 and ILR1, were compared with PLT-defined and RFLP-defined types. Thus, using a range of probes and enzymes it is possible to identify DP polymorphism. The value of monoclonal antibodies for such studies is demonstrated, and the molecular data can, in some cases, pinpoint the amino acids responsible for the specificity of the monoclonal antibodies.