International Histocompatibility Working Group (IHWG)
March 20, 2000
Dear colleagues,
Many of you have already contacted the organizers of the 13th IHWC about the Anthropology/Human Genetic Diversity component. As you know, the goal of this component of the 13th IHWG is to generate and analyze HLA allele and haplotype frequency data from a variety of human populations. These population data will prove valuable in a number of areas, such as the detection and inference of selective pressures, and the phylogenetic reconstruction of evolutionary relationships between modern human population, and will prove clinically relevant for fields such as disease susceptibility and transplantation. The number of alleles at a given locus and the degree of allelic diversity observed in most populations make the HLA loci uniquely informative for phylogenetic analyses of human populations.
Unlike previous Workshops, the population data generated during the 13th IHWG will be maintained in an on-going and accessible human MHC database, to be curated by the NIH (NCBI). In addition, the analytic programs used by the Biostatistics Core will be available for use by participating laboratories. If you have already contacted the Anthropology/ Human Diversity Component, we thank you for your interest. If not, and if you have population samples that you would like to type as part of the Workshop, we invite you to participate in this international project. The goal of the Workshop is to supply standardized HLA typing reagents, reference cells and quality control (QC) samples to participating labs. If you have interesting samples but cannot carry out the typing in your lab, we will try to identify another lab that can perform the typing. Please contact us and provide information about the nature of the population and the numbers of individual samples. You can also access information about 13th IHWG projects, protocols and reagents via the home page.
- Henry A. Erlich, PhD
Roche Molecular Systems, Inc.
1145 Atlantic Avenue
Alamada, CA 94501
510-814-2918
510-814-2910 (fax)
henry.erlich@roche.com
Human Subjects
It is important that the studies and activities of the 13th IHWG proceed in accordance with Federal regulations and/or International guidelines and regulations for the protection of human subjects. Each participating laboratory will need to check with their local institutional review board or ethics committee to determine the necessary steps to be taken to assure compliance with these regulations and guidelines. The workshop has resources, such as sample consent forms and letters of certification of compliance to assist individual participating laboratories comply with these guidelines. If you need help or have questions, please contact Karen Hansen of the Fred Hutchinson Cancer Research Center Institutional Review Board (Phone: 206-667-4867) email: khansen@fhcrc.org or Eric Mickelson, email: emickel@fhcrc.org (Phone: 206-667-4922).
Typing Reagents
The 13th IHWG Technology Development Core SSOP Committee (chaired by Carolyn Hurley) has prepared a protocol and selected probes for achieving a high resolution analysis of HLA. The reagents for class I are new, while the reagents for class II are essentially the same set of probes developed for the 11th and 12th International Workshops. 13th IHWG SSO probes (either labeled or unlabeled) and primers for both class I and class II dot blot typing can be obtained from a commercial vendor (according to Workshop specifications). See the protocols section of the web site for specific information about primer and probe sequences and recommended reagents. If you are interested in obtaining these reagents, please use our order form (Reference Cell Panels/Quality Control Test Panels). A special set of reagents (in the reverse lineblot format) will be prepared for the Anthropology/Human Diversity component by the Human Genetics research group at RMS. A limited number of research prototype tests for HLA-A, B, and C will be available. This format involves the use of biotinylated primers, an immobilized array of probes on a nylon membrane (lineblot strip), and a streptavidin-HRP detection system. We will provide the protocols and the reagents (PCR premix, strips, and detection reagents) as well as trays for strip hybridization and color development, and software for the interpretation of genotypes from the probe reactivity pattern. For both the dot blot and the reverse lineblot formats, the lab will require a PCR thermal cycler and a water bath. We would like to receive the typing data in terms of the probe reactivity pattern, as entered into the interpretation software.
Reference Cells
Panels of well-characterized reference cells for use as positive and negative primer and probe controls will be provided by the IHWG Cell and Gene Bank. DNA from these reference cells will be available by April 1, 2000 and can be ordered using the Reference Cell Panels/Quality Control Test Panels form. A panel of approximately 55 cells will be used as controls for SSOP assays.
Qualification of Participating Labs and QC
To assure high-quality data, a reference QC cell panel (n = 15) from the QC Laboratory will be sent out to participating labs. This reference cell panel may be ordered on the same form as for the reference cell panels Reference Cell Panels/Quality Control Test Panels. We ask that you type these samples and send in the results prior to proceeding with the population samples. We will provide feedback and, if there are problems, suggest possible corrections. In addition, an additional 15 QC samples will be provided for typing along with the population samples to help "filter" the data.
Reverse Lineblot Format
- The HLA-A test has 56 probes on a single strip (both exon 2 and exon 3)
The HLA-B test has 83 probes on two strips (one exon 2 and one exon 3)
The HLA-C test has 36 probes on a single strip (both exon 2 and exon 3)
The PCR-premix has two primer pairs for each locus, one for exon 2 and one for exon 3.
The trays have multiple wells to carry out the hybridization, wash, and detection steps in a water bath.
After hybridization, wash, and detection, positive probes (those that hybridized to the labeled amplicon) show up as a blue line on the nylon membrane strip.
The probe reactivity pattern seen on the strip is entered into probe pattern interpretation software to yield the genotype (see below).
To defray the costs of making and distributing the reverse lineblot reagents, we would ask to be reimbursed for the HLA-A, B, and C typing reagents ($35 for the 3 tests per sample) as well as shipping costs. The reverse lineblot tests will be distributed in lots of 50 tests. Thus, the cost of 100 tests (two lots) for A, B, and C (6 lots total) would be around $3500. All class II loci will be typed with the Workshop SSO probes, and SSO probes for class I are also available. If you are interested in receiving the reverse lineblot typing reagents, please send a letter to me, indicating your interest, the nature of your samples, and a statement certifying that these reagents will be used only for the research purposes of the 13th IHW. IMPORTANT!! We will also need a statement certifying that the samples have been collected in compliance with local standards of ethical conduct regarding human subjects. MS Word document (Letter of Certification)
Tentative Schedule
- SSO probes and primers for class I and class II -- available from spring 2000
- Reverse lineblot typing systems for A, B, and C -- available from May 2000
- Reverse lineblot test results on reference QC panel sent from participating lab to Workshop -- starting by end of June 2000
- Feedback regarding preliminary typing of ref panel to labs -- starting by end of July 2000
- Typing of population samples and submission of data to IHW -- runs from Aug-Dec, 2000
- Analysis of data with Biostatistics Core -- runs from Jan-Jun 2001
Level of Resolution/Ambiguity
As you are all only too familiar, PCR/SSO DNA typing often results in ambiguities, that is, a given probe reactivity pattern is consistent with more than a unique pair of alleles (genotype). This problem becomes greater as the number of identified alleles increases. Capturing the data as a pattern of probe reactivities means that the allele assignations can be updated in the future. Amplifying the alleles of a heterozygote sample separately and the use of additional probes can usually resolve, or at least, minimize this ambiguity. It is the goal of the Anthropology/Diversity component to generate data at as high a level of resolution as is practically possible. The level of resolution determined by the reverse lineblot reagents for A,B, and C typing will be the minimum level of resolution for submitting class I data for the Anthropology/Diversity component. Ideally, a subset of the samples that show ambiguities will be typed to higher resolution but it may not be practical to do this on all samples. Primers and SSO probes will be available for higher resolution typing.
Nature of Analyses
The analyses of the population data will be carried out in collaboration with the Biostatistics core. Allele and haplotype frequencies will be estimated and patterns of linkage disequilibrium will be analyzed. Hardy-Weinberg equilibrium will be examined and Ewens-Watterson (homozygosity F-statistic) analyses of selection pressures will be carried out. The partition of variance into within and between population variation (Fst, q) will be analyzed and the relationship (genetic affinities) between populations will be examined with various phylogenetic methods (neighbor joining, UPGMA, AMOVA, etc.) as well as principal component analysis.
We look forward to working with you over this coming year.
Best regards,
Henry A. Erlich, Ph.D, Chair and
Steven J. Mack, Ph.D.
Anthropology/Human Diversity Component
International Histocompatibility Working Group
