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December 10, 1999 Dear Colleagues, As you all know the primary aim of the IDDM Component of the 13th IHWC is to mount an international effort to identify additional genes within the HLA complex (other than those already known) with effects on disease susceptibility and resistance. During the past year we have been actively recruiting participants for the study and tried to address several critical issues pertaining to its organization. As of September 1999, 35 laboratories worldwide have accepted to participate in the study. A list of the participating laboratories can be found on the IHWG web-site (http://www.ihwg.org). Moreover, we are happy to report that 5,778 patients and 3,353 controls, all HLA-typed with typing reagents developed during previous HLA Workshops (11th and 12th), are already available for the study. According to the information provided by the participating laboratories, more than 2,000 patients and 2,000 controls may become available in the next future as HLA typing is completed on additional patients. More patients and controls may be recruited during the course of the study. We have also identified 512 families with at least one parent homozygous for relevant (i.e.disease associated) class II DR/DQ alleles for studies involving TDT analysis. Additional groups are welcome to join the study during the next few months. It is planned to close the recruitment phase by February 2,000. We are very pleased that so many investigators worldwide have accepted to participate in the study, showing to share our enthusiasm for collaborating in our common quest against the diabetes problem. An important issue that came up during the past few months was to ensure that microsatellite typing could be performed efficiently and reliably for all samples. Because a number of participants are lacking adequate facilities for microsatellite typing, we have put considerable effort into identifying a laboratory that could serve as the 13th IHWC Central Laboratory for microsatellite typing. After exploring several options, we identified a government-subsidized, non-profit typing facility. This is the National Center for Genotyping (CNG) in Paris, France, directed by Dr. Mark Lathrop. We have now finalized our agreement with the CNG in Paris, to serve as Reference Laboratory and perform microsatellite typing for the entire Disease Component of the 13th IHWC; i.e IDDM, rheumatoid arthritis, ankylosing spondylitis, celiac disease and narcolepsy. Microsatellite typing will be free to participants. Its cost will be covered by several grants that have been recently submitted to various funding agencies. The Disease Component chairpersons also met in Paris on October 11 to discuss several other key issues relative to the organization of the study. We are now entering the active phase of the study. Please find enclosed practical information on how to proceed and get the study on its way. 1. Samples to be submitted to CNG Samples from HLA-typed patients and HLA-matched controls plus samples from all available members of HLA-typed families (regardless of HLA homozygosity in parents) should be sent to the CNG. Although we had originally anticipated typing only families where at least one parent is homozygous for IDDM-associated class II alleles (DRB1*03-DQB1*020 or DRB1*4-DQB1*0302 with homozygosity for the DRB1*04 subtype), the support of the CNG will make it possible to type all HLA-typed families. Eligible families must have DNA samples from both parents and at least the proband (an affected child). 2. DNA samples for microsatellite typing The quality of the DNA extraction is very important for automated microsatellite analysis. The preferred method is a conventional phenol/chloroform extraction, but this is not mandatory, as long as you make sure to send high quality DNA. In case of low quality DNA the CNG does not guarantee the feasibility of genotyping, and will not repeat the experiment. Only 2 µg of DNA are required for each sample. DNA must be diluted in water at 10 ng/µl (please verify thoroughly the concentration), which means that each tube must contain 200 microliters. Samples must be placed into 1.5 ml screw-cap tubes. Please note that DNA samples received from the disease component will only be used for these microsatellite studies. The CNG will store unused DNA in case later studies are conducted by the disease component (of course with your permission). 3. Bar-codes for DNA samples and shipping DNA samples: In order to facilitate the shipment of DNA samples to the CNG, you will be provided with bar-codes for your DNA tubes, together with an Excel file in which each participant will enter essential data about the samples (id-number, method of extraction, amount of DNA, etc). You will then send your DNA samples to the IDDM chairpersons, together with the Excel file. European participants should send their samples to Sophie Caillat-Zucman in Paris, participants from the rest of the world should send samples to Alberto Pugliese in Miami. Sophie and Alberto will then forward all the samples received to the CNG. The deadline for shipping samples to Sophie or Alberto is February 1st, 2000. Also remember to e-mail a copy of the excel file with the sample data to: IDDM_13IHWC@yahoo.com 4. Microsatellites The CNG will type all DNA samples for the following 14 microsatellites: D6S291, DQCAR, D6S273, D6S265, D6S2222, D6S2223, D6S2239, TNFD, MIB, MICA, D6276, DQCAR 2, G55152, and DQ4. Several of these microsatellites were used by Benedicte Lie and coworkers in their preliminary studies (Am. J. Hum. Genet. 1999, 64:793-800). The MICA transmembrane microsatellite was selected because of recent data suggesting independent association of the MICA gene with Addison's disease, celiac disease and IDDM. The latter four markers were included because they would help in HLA class II typing due to strong linkage disequilibrium. 5. Participants who do their own microsatellite typing For those of you who will perform microsatellite typing on their own we have attached files with sequences of the primers for the selected microsatellites and the protocol for typing the microsatellites used by Benedicte Lie. The details for the remaining microsatellites are being worked out and you will sooon receive an updated protocol. Within a few weeks you will also receive a set of control samples that must be typed to verify the reliability of your typing. 6. Results: The CNG will need approximately 4 months to perform microsatellite typing of the whole material (approximately 30,000 samples). An intermediate analysis will be performed next summer. 7. HLA typing and SNPs The 13th IHWG (International HLA Working Group) is preparing the reagents and protocols for HLA typing (for samples not yet typed) and SNPs (single nucleotide polymorphisms). These should become available to participants during the course of year 2000. Finally, we are pleased to see how many of you have contacted us and are eager to start the study. We would also like to move as fast as possible but this is a large study involving several components, people and organizations around the world. The progress of the IDDM Component is linked to the progress of other components of the 13.IHWG, and at times this may cause delays or limit the information we can provide to you. We thank you for your patience, and rest assured that we will be in touch again and provide further updates as soon as they become available. Please don't hesitate to contact us should you have any question, and visit the IHWC web-site to familiarize yourself with the other components of this study (http://www.ihwg.org/). Thank you again for your participation. Best regards, Sophie Caillat-Zucman and Alberto Pugliese IDDM_13IHWC@yahoo.com |
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Sophie Caillat-Zucman Laboratoire d'Immunologie Hopital Necker 161 Rue de Sevres 75015. PARIS, France tel: 33 1 44 49 52 21 fax: 33 1 44 49 53 74 email: caillat@necker.fr |
Alberto Pugliese Immunogenetics Laboratory Diabetes Research Institute University of Miami School of Medicine 1450 NW 10th Avenue Miami FL 33136 USA tel: 001-305-243-5348 fax: 001-305-243-4404 email: apuglies@med.miami.edu or: apblues@yahoo.com |