Nonobese Diabetic Congenic Strain Analysis of Autoimmune Diabetes Reveals Genetic Complexity of the Idd18 Locus and Identifies Vav3 as a Candidate Gene This information is current as of September 24, 2021. Heather I. Fraser, Calliope A. Dendrou, Barry Healy, Daniel B. Rainbow, Sarah Howlett, Luc J. Smink, Simon Gregory, Charles A. Steward, John A. Todd, Laurence B. Peterson and Linda S. Wicker J Immunol 2010; 184:5075-5084; Prepublished online 2 Downloaded from April 2010; doi: 10.4049/jimmunol.0903734 http://www.jimmunol.org/content/184/9/5075 http://www.jimmunol.org/ Supplementary http://www.jimmunol.org/content/suppl/2010/04/02/jimmunol.090373 Material 4.DC1 References This article cites 65 articles, 23 of which you can access for free at: http://www.jimmunol.org/content/184/9/5075.full#ref-list-1 by guest on September 24, 2021 Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! Every submission reviewed by practicing scientists • Fast Publication! 4 weeks from acceptance to publication *average Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology Nonobese Diabetic Congenic Strain Analysis of Autoimmune Diabetes Reveals Genetic Complexity of the Idd18 Locus and Identifies Vav3 as a Candidate Gene Heather I. Fraser,* Calliope A. Dendrou,* Barry Healy,* Daniel B. Rainbow,* Sarah Howlett,* Luc J. Smink,* Simon Gregory,†,1 Charles A. Steward,† John A. Todd,* Laurence B. Peterson,‡,2 and Linda S. Wicker* Wehaveusedthepublicsequencingandannotationofthemousegenometodelimitthepreviouslyresolvedtype1diabetes(T1D)insulin- dependent diabetes (Idd)18 interval to a region on chromosome 3 that includes the immunologically relevant candidate gene, Vav3.To test the candidacy of Vav3, we developed a novel congenic strain that enabled the resolution of Idd18 to a 604-kb interval, designated Idd18.1, which contains only two annotated genes: the complete sequence of Vav3 and the last exon of the gene encoding NETRIN G1, Downloaded from Ntng1. Targeted sequencing of Idd18.1 in the NOD mouse strain revealed that allelic variation between NOD and C57BL/6J (B6) occurs in noncoding regions with 138 single nucleotide polymorphisms concentrated in the introns between exons 20 and 27 and immediately after the 39 untranslated region. We observed differential expression of VAV3 RNA transcripts in thymocytes when comparing congenic mouse strains with B6 or NOD alleles at Idd18.1. The T1D protection associated with B6 alleles of Idd18.1/Vav3 requires the presence of B6 protective alleles at Idd3, which are correlated with increased IL-2 production and regulatory T cell function. In the absence of B6 protective alleles at Idd3, we detected a second T1D protective B6 locus, Idd18.3, which is closely linked http://www.jimmunol.org/ to, but distinct from, Idd18.1. Therefore, genetic mapping, sequencing, and gene expression evidence indicate that alteration of VAV3 expression is an etiological factor in the development of autoimmune b-cell destruction in NOD mice. This study also demonstrates that a congenic strain mapping approach can isolate closely linked susceptibility genes. The Journal of Immunology, 2010, 184: 5075–5084. ype 1 diabetes (T1D) is a multifactorial autoimmune NOD mouse model of T1D (1) shares several features of etiology disease, and both environmental factors and genetic loci and genetics with human T1D and has been instrumental in the spread throughout the genome govern T1D onset. The identification of genes involved in T1D susceptibility and how T by guest on September 24, 2021 variants of these genes influence the immune system (2–7). Our laboratory has previously used a congenic strain mapping tech- *Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Re- nique in the NOD mouse model, in which genome segments from search, University of Cambridge, Cambridge; †The Wellcome Trust Sanger Institute, a T1D-resistant mouse strain (B6 or C57BL/10J) are introgressed Wellcome Trust Genome Campus, Hinxton, United Kingdom; and ‡Department of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065 into the susceptible NOD background, to localize T1D suscepti- bility loci (2, 8). Four of these loci, insulin-dependent diabetes 1Current address: Department of Medicine, The Center for Human Genetics, Duke University Medical Center, Durham, NC. (Idd)3, Idd10, Idd17, and Idd18, have been identified on mouse 2Current address: Hoffmann-La Roche, Inflammation Discovery, Nutley, NJ. chromosome three, and only Idd3 and Idd10 have proposed can- Received for publication November 19, 2009. Accepted for publication February 23, didate genes (9–12). The T1D protection associated with the 650- 2010. kb Idd3 region is most likely accounted for by the differential This work was supported by a Wellcome Trust four-year studentship (to H.I.F.), a joint expression between the NOD and B6 haplotypes of the IL-2 gene grant from the Juvenile Diabetes Research Foundation and theWellcome Trust (toL.S.W. (4), and the proposed candidate gene for the 950-kb Idd10 region and J.A.T.), and Wellcome Trust Strategic Award 079895 (to the Cambridge Institute for Medical Research). The resequencing of Idd18.1 in the NOD mouse strain was per- is Cd101 (12). formed at the Wellcome Trust Sanger Institute and was funded by Immune Tolerance The initial mapping of Idd18, which is closely linked and Network Contract AI 15416, which was sponsored by the National Institute of Allergy distal to Idd10, was performed by sequentially truncating the and Infectious Diseases, the National Institute of Diabetes and Digestive and Kidney Diseases, and Juvenile Diabetes Research Foundation International. The availability of Idd18 interval of a single homozygous B6-derived congenic NOD congenic mice through the Taconic Emerging Models Program has been supported segment that spanned Idd10 and Idd18. The resultant congenic by grants from the Merck Genome Research Institute, the National Institute of Allergy strains that had lost the B6 protective alleles at Idd18 had higher and Infectious Diseases, and the Juvenile Diabetes Research Foundation International. T1D frequencies compared with the nontruncated congenic Address correspondence and reprint requests to Prof. Linda S. Wicker, Juveniles Di- abetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, strains but were more protected from T1D than NOD mice as B6 Department of Medical Genetics, Cambridge Institute for Medical Research, Univer- alleles at Idd10 provided protection (13). The determination of sity of Cambridge, Cambridge CB2 0XY, U.K. E-mail address: linda.wicker@cimr. the recombination points of these novel congenic strains local- cam.ac.uk ized Idd18 to a genetic distance of 5.1 cM (13). Subsequently, The online version of this article contains supplemental material. a second congenic strain mapping strategy to positionally clone Abbreviations used in this paper: b2m, b2-microglobulin; BAC, bacterial artificial chro- mosome; DIL, Diabetes and Inflammation Laboratory; EST,expressed sequence tag; Idd, Idd18 was used, which exploited the enhanced T1D protection insulin-dependent diabetes; NIEHS, National Institute of Environmental Health Scien- observed when B6-derived alleles are present at both the Idd10/ ces; qPCR, real-time quantitative RT-PCR; SNP, single nucleotide polymorphism; T1D, 18 and Idd3 intervals (14). This second strategy sequentially type 1 diabetes; UTR, untranslated region. truncated the Idd18 interval of a bicongenic strain homozygous Copyright Ó 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00 for two B6-derived introgressed segments: one spanning Idd10 www.jimmunol.org/cgi/doi/10.4049/jimmunol.0903734 5076 Vav3 IS THE Idd18.1 AUTOIMMUNE DIABETES CANDIDATE GENE and Idd18 and a second spanning Idd3. Notably, the protection To develop the congenic mouse strains used to test the candidacy of Vav3 associated with Idd10 is not observed in the context of B6 alleles at as Idd18, NOD.B6 Idd3 Idd10 Idd18 (line 1538) congenic mice were Idd3 (10); therefore, when B6 alleles at Idd18 were eliminated in crossed with NOD mice. The progeny were intercrossed, and tail DNA was genotyped to identify mice with a recombination event proximal to Vav3, these congenic strains, the protection observed was equal to that which would produce a chromosome that was NOD at Vav3 and B6 for the provided by Idd3 alone. T1D frequencies of novel congenic strains remainder of the Idd18 interval. One mouse with a recombination event produced from this mapping effort reduced the Idd18 interval to proximal to Vav3 was backcrossed to NOD, and progeny heterozygous for a genetic distance of 2.04 cM (10). However, at that time in 2001, the desired recombination event—and that had retained the B6 allele at Idd3—were intercrossed to produce homozygous mice for line 2399.