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II. Background
HLA haplotypes. Human MHC haplotypes that demonstrate significant non-random association are usually referred to as common, conserved, extended or ancient haplotypes. These may be specific to a given ethnic population or shared by several different populations. The degree of linkage disequilibrium or conservation observed is frequently related to the number of loci included in the haplotype or haplotype fragment under analysis. Non-random linkage association usually occurs between the HLA-C, B, segment and the DRB1 segment. Extended haplotypes that span the entire MHC complex from HLA-A to HLA-DP are very infrequent, and those haplotypes that have been described generally consist of segments, blocks or fragments of the region between HLA-B/C and DR.
Current studies. Most available haplotype data is derived from studies of unrelated individuals in whom the putative haplotype is defined by statistical association analysis. These statistically derived haplotypes are generally limited to two or three HLA loci. Very little confirmatory data based on family segregation analysis have been available to validate the accuracy of these mathematical models. Furthermore, very little of the available haplotype data has been generated by DNA-based HLA typing methods. Previous collaborations have assembled haplotype data from family studies, but the original data has been reduced to summary tables for efficient cataloguing and storage. Lacking committed resources for a sustainable and comprehensive database, it has not been possible to maintain the actual raw, untransformed data.
Genomic analysis of HLA. DNA-based HLA typing results are generally stored as interpreted allele assignments. There are major drawbacks, however, to storing assigned allele data instead of the original raw typing data. The allele assignment itself is strictly dependent on the allele database used. Thus, a given typing result will, over time, show a varying degree of ambiguity depending on the number of new alleles taken into account. Once interpreted, typing results without raw data cannot be updated as additional alleles are discovered. These drawbacks can be avoided if the data are stored in raw (uninterpreted) form. The raw typing data must be linked to critical sequence information about the SSO probes and SSP primers used to generate the data. Based on the overall reactivity pattern of the individual reagents, a retrospective "virtual DNA analysis" can be constructed.
Establishment of a permanent human MHC database (dbMHC). This database is currently under construction at the National Center for Biotechnology Information (NCBI) and will be made a public resource following conclusion of the 13th IHWS.
III. Research Plan
A. Study Overview The study will be divided into four phases:
Phase I - Collection and reporting of available family HLA data. Data forms are available upon request from the IHWG Secretary or as an excel download.
Phase II - Generation of new DNA-based typing data
a. Collect cells and DNA from informative families, and establish cell lines whenever possible.
b. QC and Validation of local methods and reagents. Request Reference Panel DNA for QC testing. Participating laboratories will be "qualified" for genomic data submission by testing a set of coded DNA samples selected from the IHWG HLA Reference Panel (maintained by the IHWG Cell and Gene Bank).
c. Begin typing families for Priority Level I genes and markers.
d. Individual investigators and labs should identify and join one or more of the Human MHC Haplotype Working Groups (WG).
e. Report phase II data to the IHWG Database.
Phase III - Typing
a. Begin typing families for Priority Level II and Level III markers.
b. Begin the special studies recommended by individual Working Group.
c. Exchange cells or DNA with collaborating laboratories (optional).
d. Submit selected cells or cell lines expressing well-defined haplotypes to the Cell and Gene Bank.
e. Report phase III data to the IHWG Database.
Phase IV - Data Analysis
a. Working Groups begin data analysis. The IHWG Database will provide each Working Group with the appropriate set of data.
b. Working Groups convene at the 13th IHWS meeting in Victoria BC for:
- Review of the common data reported to the IHWG Database
- Presentation of data from individual laboratories
- Discussion of overall results
- Planning of future research.
B. Selection of Informative Families
The following selection criteria should be used in identifying families for study:
- Nuclear family with: i) both parents; ii) a minimum of four members; and iii) a sufficient number of offspring to allow for an unambiguous identification of the four parental HLA haplotypes.
- Sufficient demographic data should be available to describe the presence or absence of HLA-associated disease (since certain HLA haplotypes are known to be disease associated).
- Sufficient cells and/or DNA should be available for future studies. Whenever possible, B-lymphoblastoid cell lines should be established (the IHWG Cell Bank will establish cell lines from selected family members upon request).
- Institution approved informed consent has been obtained.
C. Genetic Loci Studied
Participants are encouraged to analyze cells and/or DNA from these selected families for the major HLA-linked genes. The priorities for the genomic analysis are outlined as follows:
1. Priority Level I (required). Typing must be performed using a "qualified" DNA typing system (see Methods and Reagents). Minimal requirement is for completing an intermediate level of resolution (equivalent to the best serology). Labs are encouraged whenever possible to complete high resolution DNA typing and definition of alleles.
a. Class Ia: HLA-A, B and C
b. Class IIa: HLA-DRB1, DRB3, DRB4, DRB5, DQB1
2. Priority Level II (optional).
a. Class IIa: HLA-DQA1, DPA, DPB
b. Class Ib: HLA-E, F and G
c. TNFa, TNFb, TAP, BAT-1, HSP-70 (see Polymorphic Genes and Markers within the Human MHC)
d. Microsatellite markers (see Microsatellite Markers for the Human MHC)
3. Priority Level III (optional).
a. MICA, MICB
b. Other defined genes and genomic markers:
(1) GABAb1R, IKBL, RxRbR, Notch-4, C2, C4 and B
(2) SNPs (protocols, reagents and possibly kits will be available by early 2002)
D. Human MHC Haplotype Working Groups
During previous International Workshops, Haplotype Working Groups have been organised to help recruit and analyze haplotypes in selected populations according to geographic and ethnic criteria. The same scheme will be used for the 13th IHWS (see
IHWG Classification of Ethnic Groups). It is anticipated, however, that investigators may also choose to establish additional working groups appropriate for their research interest, such as linkage disequilibrium studies with microsatellites or SNPs, or studies focused on a specific locus (e.g., MICA and MICB).
IV. Methods and Reagents
A. Collection of available HLA data
Participants are allowed to submit data generated with commercial, local or IHWG methods and typing reagents. Methods and reagents will be classified as qualified or non-qualified as defined below:
1. Qualified methods and reagents. Methods and reagents approved by the IHWG, or other local/commercial reagents that have been SCORE qualified (see Data Collection; and Informatics Support/Score Software).
2. Non-qualified methods and reagents. Local or commercial methods and reagents that are not documented in the SCORE SSP/SSOP sequence database.
Data collection forms can downloaded from the IHWG website as Microsoft Excel files, or forms can be requested directly from the IHWG Secretariat (206-667-6990; secretary@www.ihwc.org). Completed forms should be submitted as e-mail attachments to database@ihwg.org.
B. Demographic Data
1. Geographic location and racial/ethnic origin
2. Essential health data for at least the proband (medical history for family members is optional). Collecting and reporting health histories is contingent on having appropriate informed consent.
C. Informatics - Establishment of a Central SSO/SSP Database and Data Collection Tools
The shared resources developed for the IHWG and the 13th International Histocompatibility Workshop provide an ideal platform for establishing a common accessible sequence polymorphism database. Before this can be achieved, it is necessary to establish a centralized probe/primer database. This database should contain the sequence motifs detected by the primers and probes used in each individual typing system or typing kit. This resource will be essential for the establishment of a sequence database, and will also serve to assist individual typing labs to meet the accreditation guidelines of ASHI and EFI.
All individual laboratories, institutions or companies actively involved in the production of typing reagents are encouraged to submit data describing the essential characteristics of their reagents (i.e. primer and probe sequences) to this database. Laboratories using homemade reagents will be provided detailed instructions on how to submit this information.
D. Access to SCORE Software
Participants will be supplied with software (SCORE) for virtual DNA analysis of HLA data generated with SSO/SSP typing reagents. The software will be used for data collection, storage, interpretation and exchange. To validate reagents and methods, participating laboratories will be asked to type the IHWG SSOP/SSP Reference Cell Panel using their routine typing method, and to enter raw typing results into the SCORE software. Following data entry, the program allows one to interpret any combination of raw typing data with an updated allele database. The allele database will be updated in the future as the sequences for new alleles are published. The program will create an export file that can be mailed (email or floppy disk) to the IHWG Database in Seattle. Raw typing data will be compiled and evaluated. Evaluation will focus on results obtained from typing the Reference Panel DNA according to:
1. Quality of individual SSO/SSP probes/primers;
2. Robustness of the same reagents when comparing results from independent testing performed in different laboratories;
3. Evaluation of SSO versus SSP typing techniques;
4. QC data will be reported to individual submitting participants. The identity of individual laboratories will be coded and remain confidential.
System Requirements: PC; Windows 95/98/00/NT; CD-ROM drive or Internet access; specification of typing kits used in your laboratory.
E. Expenses
SCORE software and the SSOP/SSP/SBT Reference panel DNA are free to participants: (1) willing to carry out the studies described here, and (2) willing to report data to the IHWG Database. Shipping costs for the cell panel and software, cost of other reagents and typing expenses must be covered by each participating laboratory.
F. Acknowledgements
Active participants contributing data, cell lines, and/or assisting data analysis will be recognized on the IHWG web site, and in any publications or reports. Acknowledgements can include individual investigators and/or laboratories as preferred.
V. Time Line
January 2002
- Registration of participating laboratories (Please complete and return the Registration Form)
- Selection of informative families and submission of available family HLA data (IHWG data entry forms)
- Distribution of IHWG-SCORE software
- Distribution DNA from the IHWG SSO/SSP Reference Cell Panel for QC and Validation testing
- Submission of QC data (IHWG SSP/SSOP Reference Panel)
- Analysis of QC data, and reporting of results to individual labs
- Availability of protocols (methods and reagents) for selected SNPs, and possibly reagent kits.
- Begin submission of updated family/haplotype data (newly generated DNA-based genomic data)
April 1, 2002
- Last submission of data prior to the 13th IHWS meeting (May 2002)
May 12-16, 2002
- 13th IHWS meeting Victoria, Vancouver Island, British Columbia, Canada
May 18-22, 2002
- XIII International Congress of Histocompatibility and Immunogenetics, Seattle, Washington, USA
VI. Organising Committee
Co-chairs: John Hansen (Seattle) and Edmond Yunis (Boston)
Committee members: E. Albert (Munich); C. Alper (Boston); Z. Awdeh (Boston); M. Carrington (Bethesda); F. Christiansen (Perth); B. Dupont (New York); H. Erlich (Alameda); G.B. Ferrara (Genoa); M. Fernandez-Vina (Silver Springs); E. Gazit (Israel); D. Geraghty (Seattle); R. Hartzman (Silver Springs); W. Helmberg (Bethesda); A. Johnson, (Washington, DC); T. Juji (Tokyo); J.T.D. Lee (Taiwan); D. Middleton (Belfast); W.H. Park (Seoul); E. Petersdorf (Seattle); G. Thomson (Berkeley); and M. Tilanus (Utrecht).
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