KIR Typing Protocol
Joint HCT-KIR Project Proposal (PDF)
Combined HCT-KIR Dataform
KIR Phase II Reference Panel Data Submission Form
KIR Typing Protocol
Phase I Reference Panel: Consensus KIR Gene Assignment
| NK Receptors and HLA Polymorphism |
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BO DUPONT, M.D., D.Sc. (b-dupont@ski.mskcc.org) Memorial Sloan-Kettering Cancer Center Immunogenetics Laboratory 1275 York Avenue Room 406 K New York, NY 10021 USA Phone:(212) 639-6005 Fax: (212) 717-3128 BACKGROUND Currently, 15 expressed killer cell immunoglobulin like receptor (KIR) genes have been identified. KIRs are members of the Ig superfamily and some of the receptors directly interact with polymorphic HLA-A, -B or -C determinants. Variation in the number of KIR genes present in KIR haplotypes exists. It has also been suggested that gene duplication and unequal recombination may occur within highly homologous segments within the KIR genomic region. Recent sequencing of the KIR region would suggest that some segments within this chromosomal region contain conserved combinations of KIR genes while other segments may vary for both KIR gene numbers and their intergene relationships. GOALS The immediate goals for the collaborative research program within the 13th IHWG - NK Receptor/HLA Project are (1) to develop a robust method for defining KIR haplotypes, KIR genotypes and polymorphic variations within individual KIR genes; (2) to determine KIR haplotypes and KIR polymorphism in different populations and (3) to identify KIR compatible and KIR incompatible hematopoietic stem cell transplant donor-recipient combinations. RESEARCH PLAN The Research Plan for these studies are divided into two main phases: PHASE I STUDIES: Testing of an NK/KIR Reference Panel to establish a Standard Method for defining KIR haplotypes/genotypes and phenotypes. 49 DNA samples (Initial Reference Panel) will be provided to participating laboratories. The phase I studies will be performed during the Summer and early Fall 2001. The results from these studies will be posted on the IHWG Website and discussed during the ASHI meeting, October 13-17, 2001 in San Francisco. DNA will be shipped from Fred Hutchinson Cancer Research Center, Seattle, during the beginning of May 2001 to 20 interested laboratories. PCR protocols, primer information and data report forms will be e-mailed to participating laboratories at the same time. In case more than 20 laboratories wish to participate in this study, a second shipment of the Initial Reference Panel DNA will take place during June 2001. Three sets of DNA primers will be tested: 1. KIR Phenotyping Conserved intergenic sequence primer and KIR gene specific primers. This method should ideally identify presence/absence of KIR individual genes. 2. KIR Gene Order Primer sets testing KIR intergenic distances and limited gene order. 3. KIR Gene Polymorphism These primers are selected from Uhrberg et al., Immunity 7:753, 1997; Rajalingam et al., Tissue Antigens 57:22, 2001; Gardiner et al., J Immunol 166:2992, 2001; Vilches et al., Tissue Antigens 56:453, 2000; Vilches et al., J Immunol 164:5797, 2000 and others. It is strongly encouraged that participating laboratories include additional primer combinations based on their own experience. Any additional primers added locally should be described and reported to Dr. Dupont via e-mail b-dupont@ski.mskcc.org(b-dupont@ski.mskcc.org). PHASE II STUDIES: Application of a standard set of DNA primers and probes in participating laboratories testing DNA samples obtained from (1) local families and unrelated panel donor and (2) families of patients receiving related or unrelated hematopoietic stem cell transplants. Patients receiving HLA identical hematopoietic stem cell transplants and related, HLA mismatched transplants would be of particular interest during the early stages of the collaborative research program in order to facilitate identification of KIR identical versus non-identical donor-recipient pairs. NK/KIR REFERENCE PANEL The Initial Reference Panel will consist of a total of 49 samples. 48 samples are from 12 CEPH families including 2 parents and 2 children. One additional control DNA sample is included. This initial reference panel will subsequently be supplemented with an additional 30 samples selected from the 13th IHWG SSOP Reference Panel. Each of the CEPH families have many additional siblings and represent more than two generations. Additional samples from these families can be included in Phase II studies. The consensus assignment for the 16 KIR genes defined in the 13th IHWG KIR reference panel is shown here. REFERENCE MATERIAL DNA from B-lymphoblastoid cell lines (20 million cells per donor) will be provided to each participating laboratory. The PCR-SSP assays require "long range PCR". Standard protocols for DNA preparation as well as PCR protocols will be provided. DNA-primer information will be provided for determination of presence or absence of KIR gene as well as for detection of neighboring genes. KIR gene phenotyping protocol using intragenic primer sets will also be provided. It is recommended that participating laboratories include in their KIR typing protocol additional "local" DNA primer sets. Description of "local" DNA primer sets should be e-mailed to Dr. Dupont (b-dupont@ski.mskcc.org). The initial central data analysis will include analysis of all primer pairs for which description has been provided. KIR PHENOTYPING/GENOTYPING APPROACH KIR Phenotyping A PCR-based method for determination of KIR gene presence in genomic DNA has been developed using KIR gene-specific primers and conserved intergenic sequences (fig. 1). Genomic DNA prepared from whole blood of individuals as well as from B lymphoblastoid cell lines (BLCL) developed from family members is used for determination of the KIR phenotype (i.e. presence or absence of KIR gene in genomic DNA). Our preliminary studies have demonstrated that 10-15 different KIR genes are present in each individual. Genes 3DL3, 2DL4, and 3DL2 are present in all samples analyzed. A pseudogene (PS2), containing only exons 1-5 (corresponding to the extracellular domains of other KIR genes) is also universally present. Either 3DL1 or 3DS1 is present in all the individuals tested, supporting their identities as alleles (fig. 3).    The KIR-specific primers have been designed to delineate gene order on the KIR chromosome and accurately define intergene distances (fig. 2). Several conclusions can be made thus far: KIR 3DL3, which is present in 100% samples examined, is consistently the most upstream (centromeric) KIR gene; in contrast, KIR 3DL2 is consistently the most downstream (telomeric) KIR gene identified. All KIR genes are separated by a 2.4kb intergenic sequence, with the exception of PS2 and 2DL4, whose positions are uniquely conserved with a 14kb intergenic sequence. 3DL1 and 3DS1 are identified as alleles, occupying the same position just downstream of 2DL4. There is some sequential organization of the other KIR genes as well, with a 2DL5-like variant and 2DL1 typically occupying positions just centromeric to PS2 (fig. 4). Heterozygosity is identified within individuals, as evidenced by gene order studies (e.g. 3DL3 is identified flanking both 2DL2 and 2DS2, implying separate haplotypes).  KIR Polymorphism Identification of allelic differences within individual KIR genes is performed with KIR gene specific intragenic primer combinations using PCR-SSP methodology. It will sometimes be necessary to isolate cDNA clones for nucleotide sequencing to confirm the identity of the KIR gene. There seems to have occurred extensive gene duplication/deletion as well as intergenic sequence exchange within the KIR genomic region. It is highly likely that the number of possible alleles within each KIR gene is very large. Collection of an extensive database for KIR gene sequences will therefore be needed and an international collaboration tailored from the HLA allele sequence model will be needed. Determination of KIR gene order and KIR haplotype segregation can, however, be performed before an extensive database for KIR sequences becomes available. GENERAL DESCRIPTION OF METHODOLOGY Presence or absence of a KIR gene is performed with a KIR gene specific forward primer and a reverse primer ubiquitously found 80 base pairs downstream of the stop codon in the intergene sequence (fig. 1). The amplification products are then evaluated by agarose gel electrophoresis (fig. 3). KIR gene order is determined on genomic DNA by PCR amplification using KIR gene specific forward and reverse primers. The size of the PCR products varies according to the position of the primers (fig. 2). Additional characterization of KIR allelic variants by intragenic PCR-SSP typing is then performed using a "pre-workshop primer set" designed based on current publications. This "pre-workshop primer set" will then continuously be updated and new primer combinations will be added throughout the project period based on data available in the KIR database and results obtained from the Phase I studies. It is expected that new KIR chromosomes will be completely characterized during the project period. This information will allow us to update the gene order information and expand the number of primers used for determination of gene order, KIR haplotypes and genotypes. At present, the following informative gene order tests can be determined due to presently established fixed gene positions within the KIR genomic region: (genes shown in bold are tentatively fixed within the KIR genomic region). TIME TABLE (Spring 2001 - May 22, 2002) May 2001 Shipment of 49 DNA samples (i.e. Initial Reference Panel) to participating laboratories. Initial shipment: 20 laboratories. June 2001 (if needed) Shipment of 49 DNA samples (i.e. Initial Reference Panel) to participating laboratories. Second shipment: 20 additional laboratories. September 1, 2001 Data due for Central Analysis. October 1, 2001 Central Analysis Report. October 13-17, 2001 ASHI Meeting, San Francisco. Discussions of results from Phase I studies. November 1, 2001 Final selection of DNA primers etc. for Phase II studies. November, 2001 - March 1, 2002 Phase II Studies: (local studies). March 15, 2002 Data due for Central Analsysis. May 10, 2002 Central Analysis Report. May 12-16, 2002 Workshop Meeting, Victoria, BC - Discussions and final report. May 18-22, 2002 Congress, Seattle, WA ORGANIZATIONAL MEETINGS AND DISCUSSIONS 1. EFI Meeting, Granada, Spain Wednesday, March 28 11:30-13:30: "NK Receptors & HLA Polymorphism" - Bo Dupont, Room: Seminarios 3 and 4, Level 1 If you are not attending the EFI meeting and wish to participate in the Phase I studies, please contact Dr. Bo Dupont at b-dupont@ski.mskcc.org. 2. ASHI Meeting, San Francisco, CA October 13-17, 2001 FURTHER INFORMATION AND CONTACT PERSON Bo Dupont b-dupont@ski.mskcc.org  |