Nonobese Diabetic Congenic Strain Analysis of Autoimmune Diabetes Reveals Genetic Complexity of the Idd18 and Identifies Vav3 as a Candidate 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/

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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 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 : 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 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. To the gene content of the 2.04 cM Idd18 interval was unknown. produce line 2412, mice heterozygous for the desired recombination event In this study, we have used B6 mouse genome sequencing data and and that had an additional recombination event between Idd3 and Idd10, Ensembl gene annotation to design novel polymorphic micro- resulting in a heterozygous genotype at Idd10 and a NOD homozygous satellite markers and to characterize the gene content of Idd18. This genotype at Idd3, were selected and intercrossed. enabled us to refine the 2.04 cM interval, mapped in the context of Subsequent to the completion of the studies presented in this paper, the genetic backgrounds of lines 1538 and 1101 were genotyped by ParAllele B6 protective alleles at Idd3, to a physical distance of 1.27 Mb and Biosciences (South San Francisco, CA) using their single nucleotide identify the immunologically relevant gene, Vav3, as a candidate. polymorphism (SNP) chip to interrogate 5000 SNPs. The assay demon- To test the candidacy of Vav3, we developed a novel congenic strain strated a 3.6-Mb segment of B6 DNA on chromosome 18 in line 1101, in homozygous for B6 alleles at Idd3, and an introgressed B6 segment addition to the known congenic regions on chromosome 3, whereas line 1538 only contained the known congenic regions. Line 1101 was back- spanning Idd10 and Idd18 truncated immediately upstream of crossed to remove this contaminating B6 DNA segment, and the chro- Vav3. This new congenic strain mapping (in the context of B6 al- mosome 3 B6 DNA segment was fixed to homozygosity; this new congenic leles at Idd3) has refined Idd18 to an interval of 604 kb, now strain was designated line 7754. The T1D frequencies of lines 1101 and Downloaded from designated Idd18.1, that contains only two annotated genes, Vav3 7754 were equivalent when assessed contemporaneously (Supplemental and the last exon of Ntng1, thereby highlighting the candidacy of Fig. 1). Lines 1098, 7754, 1538, and 2412 are available from Taconic Farms. Lines R1, R3, R135, 1101, and 2399 are no longer extant. Note that all the Vav3. The likelihood that Vav3 is an Idd gene was strengthened by congenic mouse strains used in this study are homozygous for NOD alleles our demonstration of extensive sequence variation in Vav3 non- at the Idd17 region, which is located between Idd3 and Idd10 (11). coding regions and allele-dependent expression of VAV3 mRNA. Diabetes frequency studies

We also tested the level of protection associated with Idd18.1 http://www.jimmunol.org/ without protective B6 alleles at Idd3. Unexpectedly, in the absence All diabetes cumulative frequency studies were conducted using female ∼ of B6 alleles at Idd3, the protection associated with Idd18.1 was not mice. The presence of T1D was tested every 10–14 d beginning at 80 d of age by the detection of urinary glucose .500 mg/dl using Diastix (Miles, observed, and a different Idd18-associated interval, designated Elkhart, IN). Studies were terminated at 214 d of age. Kaplan-Meier sur- Idd18.3, was found to be required for Idd18-mediated T1D pro- vival curves were plotted for each mouse strain, and these were compared tection. The Idd18.3 interval is 996 kb, and sequence poly- using the log-rank test (Prism4 software). morphisms highlight Fam102b as a candidate gene. Resequencing the Idd18.1 interval in the NOD mouse strain and identifying polymorphisms Materials and Methods The resequencing of Idd18.1 in the NOD mouse strain involved aligning the by guest on September 24, 2021 Oligonucleotides bacterial artificial chromosome (BAC) clone end sequences of the NOD li- Primer3 (15) was used to design primers for PCR and primer and probe brary against the B6 mouse genome sequence (18). From this, five NOD BAC sets for real-time quantitative RT-PCR (qPCR). These were synthesized by clones that formed a minimal sequencing tile path spanning the 604-kb Sigma-Genosys (Haverhill, U.K.); the probes were dual labeled with Idd18.1 interval were selected and sequenced at the Wellcome Trust Sanger TAMRA and FAM fluorescent dyes. Sequences of D3Nds and D3Mit Institute and deposited at the European Molecular Biology Laboratory microsatellite markers are available at www-gene.cimr.cam.ac.uk/todd/ (www.ebi.ac.uk/embl) (clone DN-315E9, accession number CR936848; public_data/mouse/NDS/NDSMicrosTop.html and http://mouse.ensembl. DN-164L21, CR938729; DN-127N6, CR936838; DN-250A21, CR936849; org, respectively. All remaining primers and probes used in this study are and DN-250O19, CT010461). To identify polymorphisms between NOD available in Supplemental Tables I–III. and B6 in the Idd18.1 interval, the NOD BAC clone sequences spanning the Idd18.1 interval were aligned to the B6 mouse genome sequence (NCBIm Genotyping DNA samples build 36) by using the sequence search and alignment by hashing algorithm program (19), and SNPs, microsatellites, and other insertion/deletion poly- DNA for genotyping was extracted from tail biopsies using a salting out morphisms were determined computationally and confirmed manually. The method (16). PCRs included 25 ng DNA, 0.5 U AmpliTaq Gold (Applied polymorphisms were entered into T1DBase (20, 21) and displayed graphi- Biosystems, Foster City, CA), 0.2 mM 29-deoxynucleoside 59-triphos- cally using GBrowse (22). The SNP density plots were generated by counting phates, 1–4 mM MgCl2, 10% v/v glycerol, and 62.5 ng of each primer. the number of SNPs in 10-kb windows, sliding 2 kb at a time, and plotting Microsatellite markers that were not resolvable by 4% agarose gel elec- the count at the midpoint of each window. All the annotation of the Idd18.1 trophoresis were fluorescently labeled on the forward primer, and PCR and Idd18.3 intervals can be viewed at www.t1dbase.org. products were electrophoresed on an ABI 3100 Sequencer (Applied Bio- systems) and sized using GeneScan (version 3.5.1) and Genotyper (version NOD/B6 polymorphisms in the Idd18.3 interval 3.6) software (Applied Biosystems). RFLP markers were digested with the To view B6 and NOD/LtJ variation across the Idd18.3 interval, the National appropriate restriction enzyme (New England Biolabs, Herts, U.K.) and Institute of Environmental Health Sciences (NIEHS) sponsored re- were resolved on 2–3% agarose gels. sequencing data, which was generated through the resequencing of 15 inbred strains of mouse by Perlegen Sciences (Mountain View, CA) (23), Animals was downloaded, converted into GFF format, and loaded into GBrowse. All mice were housed under specific pathogen-free conditions, and the Tissue preparation and cDNA production appropriate institutional review committee approved experimental pro- cedures. NOD/MrkTacfBR (from here on designated as NOD) mice were Whole spleen, kidney, and brain were harvested from 9-wk-old female mice purchased from Taconic Farms (Germantown, NY). The derivation of the and immediately homogenized in TRIzol using a polytron. Spleen and following congenic strains has been described previously: line 1098, thymus were obtained from 3-wk-old male mice, and single-cell suspen- NOD.B6 Idd3R450 (also known as NOD.B67) (N12) (9, 17); line 1538, sions were prepared. Erythrocytes were removed from the splenocyte NOD.B6 Idd3 Idd10 Idd18R323 (N12) (10); line R135, NOD.B6 Idd3 samples. Total RNA was extracted from the cells and homogenized tissues Idd10 Idd18R135 (N12) (10); line 1101, NOD.B6 Idd10R2 (N8) (13); line using TRIzol following the manufacturer’s instructions. One microgram of R3, NOD.B6 Idd10R3 (N9) (13); and line R1, NOD.B6 Idd3 Idd10R1 total RNA was used as the template for cDNA synthesis using SuperScript (N12) (10). Line 1100, NOD.B6 Idd3 Idd10 (N12), was developed II reverse transcriptase (Invitrogen, Paisley, U.K.) following the manu- contemporaneously with the strains described in Lyons et al. (10). facturer’s instructions. The Journal of Immunology 5077

Identification and characterization of alternatively spliced at Idd3, Idd10, the 14 proximal genes in the 1.27-Mb Idd18 region transcripts (as defined by lines R1 and R135), and NOD alleles at Vav3 and To verify the VAV3 genetic structure, the VAV3 and VAV3.1 mRNA Ntng1 (Fig. 1A,1B). Assuming Vav3 is the causative gene of Idd18 sequences, NM_020505 and NM_146139, were aligned to the B6 genomic and from the previous congenic strain mapping of Idd18 (10), we sequence spanning Idd18.1 by using the est2genome from the EMBOSS suite expected the novel line 2399 congenic mice to lose the protection of programs (24, 25). NM_020505 was searched against the mouse expressed associated with the B6 alleles of Vav3 and have a diabetes fre- sequence tag (EST) database at National Center for Biotechnology In- quency similar to NOD.B6 Idd3 congenic mice. formation (26) by using the basic local alignment search tool (27). Five ESTs (BG060768, CA870586, CA872533, BI966969, BY752269, and Diabetes frequency studies were conducted on cohorts of female BY565884) were identified and aligned against the B6 genome sequence mice from line 2399 and several control congenic strains. Line 2399 spanning Idd18.1 using the est2genome. Primers for RT-PCR were designed has a higher frequency of T1D at 7 mo of age than line 1538, which is to span the unique exon-exon boundaries present and were used to test for NOD.B6 Idd3 Idd10 Idd18, and a similar T1D frequency to lines expression in whole spleen and kidney cDNA. 1098 and 1100, which are NOD.B6 Idd3 and NOD.B6 Idd3 Idd10, Gene expression respectively, indicating that line 2399 has lost the protection as- The expression levels of VAV3 and NETRIN G1 transcripts were measured sociated with the B6 alleles of the Idd18 interval (Fig. 1C). Anal- relative to the expression of b2-microglobulin (b2m) using qPCR in an ABI ysis of the survival curves for line 2399 and the control strain, line Prism 7300 sequence detector (Applied Biosystems). All reactions were 1538, indicates that this difference is statistically significant performed using TaqMan Universal Master Mix. The expression levels are (p = 0.003). As lines 1538 and 2399 differ by the presence of B6 given as dCT, which are calculated by subtracting the cycle threshold value and NOD alleles at Vav3, respectively, these results confirm that (the cycle number at which message is first detected) for b2m from the cycle threshold value for each transcript. Lower dCT values indicate higher RNA Vav3 is a candidate gene for Idd18. From this point on, we desig- Downloaded from levels. As the cycle thresholds were in the exponential phase of amplifi- nate the Idd18 interval refined by line 2399 in the context of B6 cation, a 1 dCT difference is equivalent to a 2-fold change, and a 4 dCT alleles at Idd3 as Idd18.1. difference to a 16-fold change in RNA levels. On the basis of the different T1D frequencies of lines 1538 and 2399, the distal recombination point in line 2399 defines the Results proximal boundary of Idd18.1 to 59 kb between the microsatellite

Physical mapping of the Idd18 interval in the context of B6 markers AL845310_13 and AL845310_10 (Fig. 1B). The distal http://www.jimmunol.org/ alleles at Idd3 boundary of Idd18.1 is defined by the distal recombination point The strategy to map Idd18 that used the interaction between Idd3 in line R135 (Fig. 1B). Therefore, the Idd18.1 interval is a maxi- and Idd10/18 (14) resolved the Idd18 interval to 2.04 cM between mum of 603,824 nt between, but not including, the markers the boundaries of congenic strains R1 and R135 (10), which were AL845310_13 and AL683824_5_1 (Fig. 2). both homozygous for B6 alleles at Idd3 and Idd10, but only R135 Gene content of the refined Idd18.1 interval was homozygous for B6 alleles at Idd18 (Fig. 1A). R135 was shown to have similar levels of T1D as the control congenic strain, which The only annotated genes in the Idd18.1 interval are Vav3 and the was homozygous for B6 alleles at Idd3, Idd10, and Idd18, whereas last exon of Ntng1 (Fig. 2). Zpbp2_pseudogene is also within the by guest on September 24, 2021 R1 had lost the protection associated with Idd18 and had similar Idd18.1 interval; however, this processed pseudogene of Zona levels of T1D as the Idd3 congenic strain tested (10). To refine and pellucida binding 2 does not have an open reading frame. determine the physical length of Idd18 in the context of B6 Idd3 There are no other RNA-coding sequences annotated in the region. alleles, the recombination points of lines R1 and R135 were re- From mouse Ensembl, Idd18.1 also contains several Genscan solved using microsatellite markers designed using the public predictions; however, the predictions do not have EST evidence mouse genome sequence (28). The proximal recombination point and do not have orthologs in human or rat, suggesting that they are of Idd18 is now defined as a 147-kb region between markers chance open reading frames in the genomic sequence. NETRIN AL683823_12 and AL671917_10 and the distal recombination G1 is important neurologically (29–31), and mRNA expression is point as a 3.6-kb region between markers AL683824_5 and only detectable in brain and eye tissues (32). In addition, there is AL683824_5_1. Therefore, from mouse Ensembl (build 36), the no published function of NETRIN G1 in immune cell types, Idd18 interval, in the context of B6 Idd3 alleles, is a physical whereas VAV3 has many published functions in the immune distance of 1.27 Mb located between, but not including, the mi- system (33, 34). Although Ntng1 is unlikely to be the Idd18.1 crosatellite markers AL683823_12 and AL683824_5_1 (Fig. 1B). candidate gene, Ntng1 and Vav3 were both included in further sequence and expression analyses. Gene content of the 1.27-Mb Idd18 interval highlights Vav3 as a candidate gene for Idd18 NOD and B6 sequence comparison of Idd18.1 The annotation present in mouse Ensembl identified 16 genes and 1 To identify any sequence polymorphisms in the VAV3and NETRIN pseudogene within the Idd18 interval: 1700013F07Rik, Tmem167b, G1 genes between NOD and B6, five NOD BAC clones spanning the Taf13, Wdr47, Clcc1, Gpsm2, 4921525H12Rik, Stxbp3a, Fndc7, complete Idd18.1 interval (Fig. 2) were selected and sequenced at Prpf38b, 4921515j06Rik, Fam102b, 4930443g12Rik, Slc25a24, the Wellcome Trust Sanger Institute. In the mouse, Vav3 spans Zpbp2_pseudogene, Vav3, and Ntng1 (listed in order from the a genomic distance of 345 kb and consists of 27 exons; all the donor centromere). Only Vav3 has functions known to be associated with and acceptor splice site sequences of Vav3 are in agreement with the immune system and was, therefore, the most likely gene to the canonical GT-AG splice site (35). This is very similar to the mediate the effects of Idd18 in T1D. gene structure and size of human VAV3 (data not shown). Poly- morphisms between NOD and B6 are not present in the coding A novel congenic strain, line 2399, refines the 1.27-Mb Idd18 sequence or splice sites of Vav3, nor in the last exon of Ntng1. region to a 604-kb Idd locus, designated Idd18.1, containing However, 218 SNPs, 182 microsatellites, and 23 other insertion/ only one full-length gene, Vav3 deletion polymorphisms were identified throughout the Idd18.1 To test the hypothesis that NOD alleles at Vav3 mediate the T1D interval. Two peaks of high SNP frequency are observed in Idd18.1: susceptibility of Idd18, a new congenic mouse strain, designated the first spans intron 20 to 2.5 kb after the 39 untranslated region line 2399, was developed from line 1538. Line 2399 has B6 alleles (UTR) of Vav3 and contains 138 SNPs in 61 kb of sequence; the 5078 Vav3 IS THE Idd18.1 AUTOIMMUNE DIABETES CANDIDATE GENE

A B Mb 1538 R135 R1 1098 1100 2399 Mb R135 R1 2399

25 108.0

D3Mit93 30 D3Nds95 108.2 D3Mit167 D3Nds55 35 D3Nds36 > Idd3 D3Nds34 108.4 D3Nds76 D3Mit94 40 108.6 45 AL683823_12 D3Nds32 AL671917_10 50 108.8 D3Nds86

96 109.0 14 genes

D3Mit41 98 D3Mit286 D3Mit213 109.2 D3Mit10 bm70A7SSR18 100 AL845310_13 bm70A7SSR19 AL845310_10 Idd18* D3Mit12 Idd10 109.4 Downloaded from 2410_Micro_1 102 2410_p_SNP_5 AC102515_5

104 109.6 Idd18.1# Vav3

106 109.8 AL683824_5

AL683823_12 108 AL683824_5_1 Ntng1 http://www.jimmunol.org/ AL671917_10 AL845310_13 Idd18* 110.0 AL845310_10 110 AL683824_5 AL683824_5_1 C 112 100 D3Mit106 98 114 96 1538 n = 67 (0) 116 2399 n = 87 (11) 94 1098 n = 64 (9)

118 92 1100 n = 74 (10) by guest on September 24, 2021 % not diabetic 90 120 2399 vs 1538 p = 0.003 88 1098 vs 1538 p = 0.006 122 1100 vs 1538 p = 0.002 RS38124092 86 124 84 D3Mit370 0 30 60 90 120 150 180 210 126 Time in days

= region between in and out marker = B6 region

= B6 region in congenic strain developed for this study

= NOD region

* = 2.04 cM region defined by lines R1 and R135 (10). Now defined to 1.27 Mb.

# = 604 kb region defined by lines 2399 and R135. FIGURE 1. Refinement of the Idd18 interval identifies Idd18.1 and the candidate gene Vav3. A, The congenic strains used to define the original 2.04 cM Idd18 interval and those used in the T1D frequency study are shown. B, A closeup of the congenic boundaries in lines R1, R135, and 2399 that define the Idd18 and Idd18.1 intervals. The Idd18 interval was refined from 2.04 cM to a 1.27 Mb interval by defining the recombination points of lines R1 and R135 between, but not including, the microsatellite markers AL683823_12 and AL683824_5_1. Idd18.1 is defined as a 604-kb interval by the distal recombination points of lines 2399 and R135 between, but not including, the microsatellite markers AL845310_13 and AL683824_5_1. The locations of markers in NCBIm build 36 are shown in both A and B. C, Female congenic mice from line 1538 are completely protected from T1D, whereas 12.6% of female mice from line 2399 develop T1D at 7 mo of age (p = 0.003), thus indicating that line 2399 has lost the protection associated with the B6 alleles of Idd18.1. Lines 1098, 1100, and 2399 had equivalent levels of T1D (1098 versus 2399, p = 0.79; 1098 versus 1100, p = 0.97; 1100 versus 2399, p = 0.80). n is the number of mice in each cohort, and the numbers in parentheses indicate mice that went diabetic. The Journal of Immunology 5079

chr3 109300k 109400k 109500k 109600k 109700k 109800k 109900k B6 tile path AL845310 AL731716 AL683824

AL671857 AL671987

NOD tile path CT010461 CR936838 CR936848

CR936849 CR938729

T1DBase Curated Transcripts DIL:Zpbp2_pseudogene DIL:Vav3 DIL:Ntng1 DIL:Vav3.1 Diabetes and Inflammation Laboratory annotated EST BG060768 BI966969 BY565884 CA870586 CA872533 BY752269

B6/NOD microsatellite and insertion/deletion

B6/NOD SNPs Downloaded from

B6/NOD SNP density (SNPs per 10 kb) 50

25

0 http://www.jimmunol.org/ region boundaries AL845310_10 AL683824_5 AL845310_13 AL683824_5_1 Idd18.1 FIGURE 2. Idd18.1 annotation and sequence polymorphisms in NCBIm build 36. The B6 and NOD tile path tracks represent the sequenced B6 and NOD BAC clones. The gene content is displayed in the T1DBase Curated Transcripts tract, and the VAV3 ESTs are displayed on the Diabetes and Inflammation Laboratory (DIL) annotated EST tract. The B6/NOD microsatellite and insertion/deletion track represents all the polymorphic microsatellites and the single nucleotide and larger insertion/deletion polymorphisms. The B6/NOD SNPs track represents the location of the polymorphic NOD/B6 SNPs; black, red, blue, and green lines represent G, T, C, and A NOD alleles, respectively. Note that where multiple SNPs are located close together the lines in the B6/NOD

SNPs track may represent more than one SNP. The frequency of SNPs is more clearly displayed in the B6/NOD SNP density track. The outer and inner by guest on September 24, 2021 boundary markers of Idd18.1 are represented as red and green lines, respectively, on the region boundaries track. second overlaps the distal boundary of Idd18.1, spanning the final in intron 17), CA870586 (novel 39 UTR in intron 6), and intron of Ntng1 to 13 kb downstream of Ntng1 and contains 90 CA872533 (novel 39 UTR in intron 22). Because our initial aim SNPs in 40 kb. Twenty kilobases of the second peak, containing 46 was to identify expression differences, we did not characterize SNPs, is located in the Idd18.1 region (Fig. 2). these transcripts further. Expression of VAV3 and NETRIN G1 A B p = 0.0021 p = 0.0018 Because there was not a structural difference in VAV3between NOD 1.84 fold 8.0 1.46 fold and B6, we hypothesized that a change in RNA expression or a change in splicing efficiency of VAV3could cause susceptibility to 8.5 T1D. As well as full-length VAV3, two alternate mRNA transcripts have been published for the VAV3 gene: one identified in both mice 9.0 and humans, VAV3.1 (36), and one identified only in humans, 9.5 VAV3b (37). These transcripts are predicted to encode partial dCT VAV3 that have alternative promoters in introns of the 10.0 VAV3 gene; however, there is no evidence that these transcripts are translated into protein. VAV3.1 has a unique 59 UTR located up- 10.5 stream of exon 18 of full-length VAV3 and contains eight novel 11.0 amino acids upstream of the normal reading frame of exons 18–27. Line Line Line Line From our sequence data, polymorphisms are not present in the 1538 2399 1101 2412 novel coding sequence of VAV3.1. We found no evidence of the FIGURE 3. Differential expression of full-length VAV3 mRNA. A, novel VAV3b exon in mouse. Thymocytes isolated from line 2399 (n = 7) (NOD.B6 Idd3 10) express To fully investigate the expression of the mouse VAV3 gene, we 1.84-fold more full-length VAV3 mRNA (detected at the exon 1–2 boundary) compared with line 1538 (n = 8) (NOD.B6 Idd3 Idd10 Idd18). searched for alternative VAV3transcripts in the public EST database B, Thymocytes isolated from line 2412 (n = 8) (NOD.B6 Idd10) express at the National Center for Biotechnology Information. We identified 1.46-fold more full-length VAV3 mRNA (detected at the exon 1–2 six ESTs that gave evidence for novel splicing events not observed in boundary) compared with line 1101 (n = 8) (NOD.B6 Idd10 Idd18). Note the known VAV3 transcripts. To confirm that these splicing events that A and B have the same y-axis. Thymocytes from 3-wk-old male mice were not ESTartifacts, we used RT-PCR to amplify the novel spliced were used to avoid potential subset variations that occur as NOD mice age exons. This confirmed the expression of BG060768 (novel 39 UTR and autoimmunity progresses. Data are representative of five experiments. 5080 Vav3 IS THE Idd18.1 AUTOIMMUNE DIABETES CANDIDATE GENE

A Mb 1101 R3 2399 2412 25 D3Mit93 30 D3Mit167 35 > Idd3 D3Mit94 40 45 D3Nds32 50 B Mb 2412 R3

96 108.0

D3Mit41 98 D3Mit213 108.2 D3Mit10 bm70A7SSR18 100 bm70A7SSR19 AC093365_1 Idd10 108.4 102 AC093365_6 104 108.6

§ 106

108.8 Downloaded from Idd18.3# } D3Nds86 AC093365_1 108 22 genes AC093365_6 109.0 AL845310_13 AL845310_10 110 Idd18.1

109.2 112 AL845310_13 AL845310_10 http://www.jimmunol.org/ 114 109.4

116 109.6 Idd18.1 Vav3 Idd18* 118 109.8 120 Ntng1

110.0 122 RS38124092

= B6 region = region between in and out marker by guest on September 24, 2021 124 D3Mit370 = NOD region = B6 region in congenic strain developed for this study 126 * = 5.1 cM region defined by lines R3 and 1101 (13). Now defined to 17.1 Mb. # = 996 kb region defined by lines R3 and 2412. § = position of Idd18.2 as noted in the Discussion Section. C D 100 100 90 90 80 80 70 70 60 60 2412 n = 101 (55) 2412 n = 77 (43) 50 50 1101 n = 76 (42) 1101 n = 85 (49) % not diabetic 40 40

NOD n = 67 (60) % not diabetic NOD n = 62 (52) 30 30 2412 vs NOD p = 6.0x10-05 2412 vs NOD p = 8.3x10-07 20 20 1101 vs NOD p = 5.0x10-05 1101 vs NOD p = 2.7x10-06 10 10 0 0 0 30 60 90 120 150 180 210 0 30 60 90 120 150 180 210 Time in days Time in days FIGURE 4. The protection associated with Idd18.1 is dependent on B6 alleles at Idd3, and this finding reveals an additional region, Idd18.3. A, The congenic strains, lines 1101 and R3, used to define the 5.1 cM Idd18 interval (13), and the newly developed congenic strains, lines 2399 and 2412, are shown. Line R3 was more susceptible to T1D than line 1101 and delineated the original 5.1 cM Idd18 interval. Note that this mapping was performed using congenic strains with NOD alleles at Idd3. B, The Idd18.3 interval defined by lines R3 and 2412. The equal T1D frequencies observed for lines 1101 and 2412 (see C, D), which have NOD alleles at Idd3, B6 alleles at Idd10, and B6 and NOD alleles at Idd18.1, respectively, indicate that the protection associated with the B6 alleles of Idd18.1 is dependent on the presence of B6 alleles at Idd3. Therefore, an Idd3-independent interval, designated Idd18.3, is located in the 5.1 cM Idd18 interval defined by lines R3 and 1101. As the T1D frequencies for lines 2412 and 1101 are the same, this excludes the presence of the Idd18.3 between the distal recombination points of lines 2412 and 1101. Therefore, Idd18.3 must be located between the distal recombination points of lines R3 and 2412. This interval is 996 kb between, but not including, the microsatellite markers AC093365_1 and AL845310_10 and contains 22 genes. C, T1D frequency study performed in 2003–2004: female mice from line 1101 (NOD.B6 Idd10 Idd18) and line 2412 (NOD.B6 Idd10) differ by the presence of B6 and NOD alleles at Vav3, respectively. However, the protection associated with B6 alleles of Idd18.1 is not observed as both strains have overlapping survival curves (p = 0.99). D, The T1D frequency study described in C was repeated in 2006–2007 giving the same result (p = 0.82 for 1101 and 2412). As the congenic segments in lines The Journal of Immunology 5081

qPCR was used to measure the relative expression of full-length alleles at Idd3 and Idd10, and equal diabetes frequency of lines VAV3and the alternatively spliced VAV3transcripts compared with 2412 (NOD.B6 Idd10) and 1101 (NOD.B6 Idd10 Idd18) that also b2m. As it was not possible to specifically assess the expression of differ at Idd18.1 and have B6 alleles at only Idd10, we propose the full-length VAV3 transcript, because all the exon-exon that the T1D protection associated with the B6 alleles of Idd18.1 is boundaries of full-length VAV3 are present in alternatively spliced only observed in the context of protective B6 alleles at Idd3. transcripts, we assessed the expression of full-length VAV3 at three VAV3 differential expression is not dependent on Idd3 different locations in the transcript (the exon 1–2 boundary, the exon 10–11–12 boundaries, and the exon 21–22 boundary). The To determine whether the VAV3 expression difference, observed expression levels were essentially identical for all three qPCR as- when comparing congenic mice from lines 1538 and 2399 (Fig. says; therefore, we proceeded to assess expression levels of full- 3A), was dependent on B6 protective alleles at Idd3, we assessed length VAV3 using only the exon 1–2 boundary qPCR assay. Dif- the expression of full-length VAV3 (at the exon 1–2 boundary) in ferential expression of full-length VAV3 was observed when 3-wk-old thymocytes from male line 1101 (NOD.B6 Idd10 Idd18) comparing thymocytes from 3-wk-old male congenic mice from and line 2412 (NOD.B6 Idd10) congenic mice that have NOD lines 2399 and 1538, which have NOD and B6 alleles at Idd18.1, alleles at Idd3, B6 alleles at Idd10, and B6 or NOD alleles at respectively (Fig. 3A). Line 2399 has 1.84-fold higher expression Idd18.1, respectively (Fig. 3B). Line 2412 expressed 1.46-fold of full-length VAV3 mRNA compared with line 1538 indicating more full-length VAV3 compared with line 1101 (Fig. 3B), in- that the NOD alleles at Idd18.1 are expressed higher than the B6 dicating that the differential expression of B6 and NOD Vav3 al- alleles. This differential expression is not due to differences in leles was not dependent on the allelic status of Idd3. thymocyte cell populations, because the congenic strains tested Downloaded from An additional Idd18-associated interval, Idd18.3, is located have a similar proportion of CD4- and CD8-negative, single-pos- proximal to Idd18.1 itive, and double-positive cells (data not shown). The expression levels of the alternatively spliced transcripts ranged from 64- to The first Idd18 interval discovered was identified because of the 256-fold lower compared with the exon 1–2 boundary in full-length difference in diabetes frequency between lines 1101 (NOD.B6 VAV3 in 3-wk-old male thymocytes (data not shown). A similar Idd10 Idd18) and R3 (NOD.B6 Idd10) (Fig. 4A). Line R3 had

trend in differential expression was observed in the alternatively a higher frequency of diabetes than line 1101 and had, therefore, http://www.jimmunol.org/ spliced transcripts, although this was not statistically significant for lost the protection associated with the B6 alleles of Idd18, which all transcripts (data not shown). was defined as a 5.1 cM interval (Fig. 4A) (13). Notably, the The expression of full-length NETRIN G1 (NETRIN G1A) protection associated with the 5.1 cM Idd18 interval was not de- mRNA and spliced transcripts that contain the terminal exon pendent on B6 alleles at Idd3. Therefore, we propose that in ad- present in the Idd18.1 interval and alternative penultimate exons as dition to Idd18.1, which is dependent on B6 alleles at Idd3, compared with full-length NETRIN G1 (NETRIN G1C, D, E, and another Idd interval, which is not dependent on B6 protective G) (31, 38) was tested by qPCR. NETRIN G1A, C, D, and E were alleles at Idd3, is located in the original 5.1 cM Idd18 interval. We detected in whole brain (dCT 8, 18, 10, and 11, respectively), but designate this Idd3-independent interval, Idd18.3. Note that were absent from whole spleen and thymocytes (data not shown). Idd18.3 has not been designated Idd18.2 because another region by guest on September 24, 2021 NETRIN G1G was not detected in the tissues tested (data not has this gene name (see Discussion). shown). Physical mapping of Idd18.3 The protection associated with Idd18.1 is only observed in the The distal recombination points of lines 1101 and R3 defining the context of protective B6 alleles at Idd3 original Idd18 interval were resolved to 17.1 Mb between, but not We next assessed the protection of Idd18.1 in the absence of pro- including, the microsatellite markers AC093365_1 and D3Mit370 tective B6 alleles at Idd3 by developing a congenic strain with NOD (Fig. 4A). This region contains the Idd18.3 interval, the protection of alleles at Idd3, and the same B6 introgressed segment spanning which is not dependent on B6 alleles at Idd3. Because there is no Idd10 and the 14 genes at the proximal end of Idd18 that is present difference in T1D frequency between lines 1101 and 2412 (Fig. 4C, in line 2399 (Fig. 4A). On the basis of the increased T1D frequency 4D), this excludes Idd18.3 from the region between the distal re- of line 2399 (NOD.B6 Idd3 Idd10) compared with line 1538 (NOD. combination points of lines 1101 and 2412, which is between, but B6 Idd3 Idd10 Idd18), because of the loss of B6 protective alleles at not including, the microsatellite markers AL845310_13 and Idd18.1 (Fig. 1C), we expected line 2412 (NOD.B6 Idd10) to have D3Mit370 (Fig. 4A). Therefore, we postulate that the Idd18.3 in- an increased frequency of diabetes compared with line 1101 (NOD. terval is located between the distal recombination points of lines B6 Idd10 Idd18) (Fig. 4A) because line 2412 does not have pro- 2412 and R3, which is between, but not including, the microsatellite tective B6 alleles at Idd18.1 (Fig. 4A). However, the diabetes fre- markers AC093365_1 and AL845310_10 (Fig. 4B). The Idd18.3 quency of line 2412 was not different (p = 0.99) from line 1101 (Fig. interval is 996 kb and contains 22 genes and 1 pseudogene (Fig. 5). 4C), and both lines were equally protected from T1D as compared From the NIEHS resequencing data, there are 55 SNPs polymorphic with NOD mice (p = 6.0 3 1025 and p = 5.0 3 1025, respectively). between NOD/LtJ and B6 in the Idd18.3 interval, none of which Because the result of line 2412 was unexpected, we repeated the map to the coding regions of the 22 genes located in Idd18.3. diabetes frequency study of lines 2412, 1101, and NOD (Fig. 4D). However, this does not exclude the existence of NOD/B6 coding We observed the same result; thereby, confirming that the diabetes SNPs in these genes, because the NIEHS resequencing does not frequency of line 2412 was the same as line 1101. capture all the sequence variation because of technical and bi- On the basis of the difference in diabetes frequency observed ological issues. The highest density of SNPs within Idd18.3 is lo- when comparing lines 2399 (NOD.B6 Idd3 Idd10) and 1538 cated in and immediately upstream of Fam102b, with 8 and 24 SNPs (NOD.B6 Idd3 Idd10 Idd18) that differ at Idd18.1 and have B6 in intron 1 and 22 kb upstream of the 59 UTR, respectively (Fig. 5).

2399 and 2412 are identical except for at Idd3, this indicates that the protection associated with the Idd18.1 interval is dependent on the presence of B6 alleles at Idd3. n = the number of mice in each cohort, and the numbers in parentheses indicate mice that went diabetic. 5082 Vav3 IS THE Idd18.1 AUTOIMMUNE DIABETES CANDIDATE GENE

chr3

108.4M 108.5M 108.6M 108.7M 108.8M 108.9M 109M 109.1M 109.2M 109.3M 109.4M B6 tile path AL671854 AL672200 AL671917 AL671921 AL671899 AL683823 AL671894 AL845310

T1DBase Curated Transcripts DIL:Psma5 DIL:Mybphl DIL:5330417C22Rik DIL:Wdr47 DIL:Gpsm2 DIL:Stxbp3a DIL:Prpf38b DIL:4930443G12Rik DIL:Sort1 DIL:Celsr2 DIL:1700013F07Rik DIL:4921525H12Rik DIL:4921515J06Rik DIL:Slc25a24 DIL:AL671899.11 DIL:Tmem167b DIL:Clcc1 DIL:Fndc7 DIL:Fam102b DIL:Zpbp2_pseudogene DIL:Psrc1 DIL:Taf13 DIL:Sars

NIEHS B6/NOD SNPs

NIEHS B6/NOD SNP density (SNPs per 10 kb) 20

10

0 Downloaded from region boundaries AC093365_1 AC093365_6 AL845310_13 AL845310_10 Idd18.3 FIGURE 5. Idd18.3 annotation and sequence polymorphisms in NCBIm build 36. The B6 tile path, T1DBase Curated Transcripts, and region boundaries tracks are displayed as for Fig. 2. The location of SNPs that are polymorphic between NOD/LtJ and B6 from the NIEHS resequencing data in the Idd18.3 region are displayed in the NIEHS B6/NOD SNPs track. Note that where multiple SNPs are located close together the lines in the NOD variation track may represent http://www.jimmunol.org/ more than one SNP. The frequency of SNPs is more clearly displayed in the NIEHS B6/NOD SNP density track, which shows the SNP density per 10 kb.

Discussion ficiency has been shown to affect cytoskeletal plasticity in several Congenic strain mapping of Idd18 reveals two Idd loci, Idd18.1 cell types including fibroblasts (42) and platelets (43), and VAV3 is and Idd18.3 required for macrophage phagocytosis of apoptotic neutrophils (44), and B cell AgR endocytosis and Ag presentation (45). On the In this study, we have identified that the original 5.1 cM Idd18 basis of this evidence for a functional role of Vav3 in immune cells, interval consists of two smaller subcongenic intervals, Idd18.1 and we propose that Vav3 is the T1D etiological gene of Idd18.1. Idd18.3. Idd18.1 is 604 kb and contains only two annotated genes: In addition to its strong functional candidacy, we identified allele- by guest on September 24, 2021 the full sequence of Vav3 and the last exon of Ntng1. Interestingly, dependent VAV3 mRNA expression differences (Fig. 3). The NOD the protection associated with the B6 alleles of Idd18.1 is only allele correlates with ∼50% higher levels of VAV3 transcripts com- observed when B6 protective alleles are also present at Idd3.Inthe pared with the B6 allele; this relatively small expression difference absence of protective alleles at Idd3, we provide evidence for is characteristic of common functional variants (4, 6, 46). The ex- Idd18.3, a B6 protective interval that is 996 kb and contains 22 genes. pression difference is likely due to one or more of the 138 SNPs or Vav3 is the etiological gene for Idd18.1 based on function, 95 microsatellite and other insertion/deletion polymorphisms sequence polymorphisms, and allele-dependent mRNA present between exon 20 and 2.5 kb downstream of the 39 UTR, expression which could modulate RNA stability, transcription (by altering an Ntng1 and Vav3 are positional candidate genes for Idd18.1 (Fig. enhancer sequence), and/or efficiency of splicing (47, 48). 2). NETRIN G1, also known as LAMINET1, is a cell surface glycoprotein and forms part of the UNC-6/Netrin family. NETRIN Diabetes protection associated with the B6 alleles of Idd18.1 is G1 functions in nervous system plasticity by providing short-range dependent on Idd3 cues to neurons to promote neurite outgrowth (39, 40), and recent In this study, we have observed that the protection associated with studies have found an association between NETRIN G1 and ge- Idd18.1 is dependent on B6 alleles at Idd3. This dependency is not netic risk for schizophrenia (30, 41). The expression of NETRIN due to altered VAV3 expression caused by the Idd3-determined G1 is primarily restricted to the CNS (31, 38), and in our study, we change in IL-2 levels, because congenic strains with NOD alleles did not observe expression of NETRIN G1 transcripts in thymo- at Idd3 (lines 1101 and 2412) and B6 alleles at Idd3 (lines 1538 cytes or spleen. On the basis of the function, expression, and and 2399) were both observed to have VAV3 differential expres- phenotypes associated with disruption of NETRIN G1, Ntng1 is sion when comparing congenic strains with NOD and B6 alleles at unlikely to be a functional candidate gene for T1D. Idd18.1 (Fig. 3). This suggests that the expression of VAV3 is The VAV proteins belong to the Dbl family of Rho/Rac/Cdc42 dependent on the Idd18.1 genotype alone, whereas the T1D pro- guanine nucleotide exchange factors. Three mammalian family tective phenotype of Idd18.1 is a result of the interaction between members are known VAV1, VAV2, and VAV3, each with over- the immune pathway(s) involving Idd3/Il2 and Idd18.1/Vav3 lapping but distinct specificities for different GTPases and con- within a cell type or between cell types. The B6 protective Idd3 tribute to signaling pathways involved in cytoskeletal modulation alleles have been shown to produce 2-fold more IL-2 than the and transcriptional alterations. VAV1 is expressed exclusively in NOD susceptible alleles, resulting in an increase in the function of immune cells, whereas VAV2 and VAV3 are expressed more CD4+CD25+ T regulatory cells, a T cell subset that can prevent the broadly. The VAV family as a whole is necessary for the adaptive activation of effector T cells (4). The B6 allele at Idd3 has also immune system as triple VAV1/2/3 knockout mice have severe been shown to influence the function of APCs (49) and the de- defects in T and B cell development and function (34). VAV3 de- letion of islet-specific T cells (5). The lower VAV3 expression The Journal of Immunology 5083 associated with B6 Idd18.1 alleles likely alters the function of one associated with B6 Idd18.1 alleles (Fig. 4). Notably, Idd18.1 would or more immune cell types to enhance the dampening of the au- not have been identified if only the Idd10/18 congenic strain (line toimmune process established by the higher-expressing B6 alleles 1101, which has NOD alleles at Idd3) had been used to fine-map of IL-2. However, the lower VAV3 expression from B6 Idd18.1 the region. Similarly, the T1D susceptibility associated with alleles alleles in an environment generated by NOD alleles at Idd3 and at Idd5.4 is dependent on the presence of susceptibility alleles at B6 alleles at Idd10 does not appear to dampen the autoimmune Idd5.1/Ctla4 and is not observed if protective alleles are present at process, indicative of a functional epistasis between the effects of Idd5.1/Ctla4 (58). Congenic strain mapping enables specific gene- these loci and their haplotypes. gene interactions to be evaluated in a fixed genetic environment, We have previously shown that protective B6 alleles at the Idd3 a situation not achievable in human studies. Although gene-gene and Idd10/18 gene regions interact in a nonmultiplicative manner interactions have been documented in human T1D (59, 60), it is to provide increased T1D protection (14). Although the T1D pro- likely that most instances of masking and synergy are obscured by tection mediated by Idd18.1 depends on the allelic status of Idd3, the fact that hundreds of T1D variants are segregating simulta- other Idd loci in the Idd10/18 region could also contribute to the neously in the human population (61, 62). nonmultiplicative interaction observed in the previous study. It is There is no evidence to date for association of human VAV3 with important to note that the dependency between Idd3 and Idd18.1 T1D (61, 63) (www.t1dbase.org). However, regions synonymous has been observed using congenic strains that have B6 introgressed with Vav3 have been identified by linkage analysis in the bio- DNA at Idd10, Idd18.3, and an unpublished (H. I. Fraser and L. S. breeding diabetes-prone rat model of T1D: Iddm26 is 41 Mb, Wicker) B6 susceptibility locus, Idd18.2, located between Idd10 contains 450 genes, and overlaps Idd10, Idd18.2, Idd18.3, and and Idd18.3. A congenic strain with a small introgressed segment Idd18.1 (64); and a second unnamed region is 26 Mb, contains 196 Downloaded from (,2 Mb) spanning only Idd18.1/Vav3 is needed to assess the pro- genes, and overlaps Idd18.2, Idd18.3, and Idd18.1 (65). Identifi- tective effects of Idd18.1 alone and in conjunction with protective cation of Vav3 as a causal T1D gene in the NOD mouse and alleles at Idd3/Il2 without the effects of neighboring Idd loci. a possible T1D gene in the biobreeding diabetes prone rat helps us prioritize analysis of human T1D gene candidates that are known Fam102b as a candidate gene for Idd18.3 to function in VAV3-dependent signaling pathways controlling

Idd18.3 is a 996-kb interval immediately proximal to Idd18.1 that cytoskeletal assembly and other immune response functions. http://www.jimmunol.org/ is pivotal for the protection observed in the context of NOD alleles at Idd3 (Fig. 4). On the basis of the polymorphism frequency (Fig. Acknowledgments 5), Fam102b is the most likely candidate for Idd18.3. Although We thank Dr. Jane Rogers for assistance with B6 BAC clone sequencing. Fam102b is expressed in many tissues, its function is not known. Notably, Fam102b has high expression in dendritic cells and Disclosures macrophages (50, 51), including upregulated expression in bone The authors have no financial conflicts of interest. marrow-derived dendritic cells upon stimulation with CpG oli- gonucleotides (52). Dendritic cells have been shown to be nec- References by guest on September 24, 2021 essary for T cell-mediated T1D in NOD mice (53); therefore, 1. Kikutani, H., and S. Makino. 1992. The murine autoimmune diabetes model: Fam102b may increase T1D susceptibility via the activity of NOD and related strains. Adv. Immunol. 51: 285–322. dendritic cells. 2. Ridgway, W. M., L. B. Peterson, J. A. Todd, D. B. Rainbow, B. Healy, O. S. Burren, and L. S. Wicker. 2008. Gene-gene interactions in the NOD mouse model of type 1 diabetes. Adv. Immunol. 100: 151–175. Complications of Idd locus identification because of clustering 3. Hamilton-Williams, E. E., D. V. Serreze, B. Charlton, E. A. Johnson, of loci and interactions between alleles M. P. Marron, A. Mullbacher, and R. M. Slattery. 2001. Transgenic rescue im- plicates b2-microglobulin as a diabetes susceptibility gene in nonobese diabetic This article highlights the phenomenon of the clustering of Idd loci. (NOD) mice. Proc. Natl. Acad. Sci. USA 98: 11533–11538. The original linkage analysis peak identified on chromosome 3 (54, 4. Yamanouchi, J., D. Rainbow, P. Serra, S. Howlett, K. Hunter, V. E. Garner, A. Gonzalez-Munoz, J. Clark, R. Veijola, R. Cubbon, et al. 2007. Interleukin-2 55) is now known to consists of at least five Idd loci confirmed by gene variation impairs regulatory T cell function and causes autoimmunity. Nat. congenic strain mapping, Idd3, Idd10, Idd17, Idd18.3, and Idd18.1. Genet. 39: 329–337. In addition, we are fine mapping loci between Idd10 and Idd18.3 5. Hamilton-Williams, E. E., X. Martinez, J. Clark, S. Howlett, K. M. Hunter, D. B. Rainbow, L. Wen, M. J. Shlomchik, J. D. Katz, G. F. Beilhack, et al. 2009. and have identified at least one interval that modulates the fre- Expression of diabetes-associated genes by dendritic cells and CD4 T cells drives quency of T1D, Idd18.2 (H.I. Fraser and L.S. Wicker, unpublished the loss of tolerance in nonobese diabetic mice. J. Immunol. 183: 1533–1541. 6. Ueda, H., J. M. Howson, L. Esposito, J. Heward, H. Snook, G. Chamberlain, observations). This clustering of loci is not unique to chromosome D. B. Rainbow, K. M. Hunter, A. N. Smith, G. Di Genova, et al. 2003. Asso- 3 or T1D: the Idd4 locus on chromosome 11 is composed of two ciation of the T-cell regulatory gene CTLA4 with susceptibility to autoimmune subcongenic intervals, Idd4.1 and Idd4.2 (56), and the Sle1 interval disease. Nature 423: 506–511. 7. Vijayakrishnan, L., J. M. Slavik, Z. Ille´s, R. J. Greenwald, D. Rainbow, B. Greve, that confers protection against systemic lupus erythematosus is L. B. Peterson, D. A. Hafler, G. J. Freeman, A. H. Sharpe, et al. 2004. An au- made up of at least three subcongenic intervals (57). The dissection toimmune disease-associated CTLA-4 splice variant lacking the B7 binding of such clustered disease-causing genes requires a congenic strain domain signals negatively in T cells. Immunity 20: 563–575. 8. Lyons, P. A., and L. S. Wicker. 1999. Localising quantitative trait loci in the mapping strategy and is a complex task because in some cases the NOD mouse model of type 1 diabetes. Curr. Dir. Autoimmun. 1: 208–225. NOD alleles confer resistance to T1D, rather than the expected 9. Lyons, P. A., N. Armitage, F. Argentina, P. Denny, N. J. Hill, C. J. Lord, M. B. Wilusz, L. B. Peterson, L. S. Wicker, and J. A. Todd. 2000. Congenic susceptibility, or the clustered genes may be involved in gene-gene mapping of the type 1 diabetes locus, Idd3, to a 780-kb region of mouse chro- interactions, defined as when alleles at one locus obscure (mask) or mosome 3: identification of a candidate segment of ancestral DNA by haplotype enhance (synergize) the phenotypes conferred by alleles of a linked mapping. Genome Res. 10: 446–453. 10. Lyons, P. A., N. Armitage, C. J. Lord, M. S. Phillips, J. A. Todd, L. B. Peterson, or unlinked locus. and L. S. Wicker. 2001. 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Identification of 5084 Vav3 IS THE Idd18.1 AUTOIMMUNE DIABETES CANDIDATE GENE

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Supplemental Material

100

90

80

70

60

50 1101 n = 64 (27) 7754 n = 62 (28)

40 NOD n = 92 (68) % notdiabetic 30 1101 vs 7754 P = 0.64 -5 20 1101 vs NOD P = 1.5 x 10 7754 vs NOD P = 2.0 x 10 -4 10

0 0 30 60 90 120 150 180 210 Time in days

Supplemental Figure 1. Chromosome 18 contamination in line 1101 does not affect T1D susceptibility. A genome-wide genotype analysis of our NOD.B6 congenic strains identified a 3.6 Mb introgressed segment of B6-derived DNA on chromosome 18 in line 1101, which was believed to only have a single B6 introgressed segment on chromosome 3 spanning Idd10 and Idd18. To remove the contaminating DNA, line 1101 was backcrossed to NOD, and the resultant congenic strain was designated line 7754. To confirm that the contaminating DNA on chromosome 18 did not contain any regions affecting susceptibility to T1D, we compared the T1D frequencies for female congenic mice from lines 1101 and 7754 for 196 days. Lines 1101 and 7754 had equivalent levels of T1D (P = 0.64) confirming that the contamination on chromosome 18 did not affect T1D susceptibility, and that the protection observed in line 1101 was solely due to the B6 introgressed segment on chromosome 3. n = the number of mice in each cohort, and the numbers in parentheses indicate mice that went diabetic.

1

Marker Forward primer Reverse primer Product length NCBIm build 36 NOD B6 chromosome 3

AC093365_1 CTAATGGGGTAGGCTGACCA TTCTCCCAGACTCATGGCTT 262 259 108375214 - 108375478 AC093365_6 ACTGCTGCTTTTGATCCCTG AGGTAGCCAGATCTTGGGGT 271 273 108501985 - 108502197 AL683823_12 TTAATCCCAACACTCGGAAG GGCAGTGAATTTAAGGCTCA 296 293 108641938 - 108642177 AL671917_10 TGGGTGGTTTTGTTTGACTT CAGGCAGAATGGCAGAATAC 159 164 108788995 - 108789157 AL845310_13 AAATTTTTGCTGTGATTTCATTT AGTAATGGAAACCCTAACTAAGACAC 181 185 109312088 - 109312271 AL845310_10 TCTCCATCTCCCTCTTTCCT CACAGGGAACCAAAAGACAC 191 189 109370882 - 109371068

AC102515_5 AAGCGAACTTCTGTCCCAAC TGCCCTCTTCGCTAGTCTTT 212 214 109467623 - 109467841 AL683824_5 AAAATGTCATGGCCCAATTT TATGTGTGGAAGGTGGGACT 126 138 109912088 - 109912233 AL683824_5_1 AGGAGTTGGTCCCAATTATTTT TACTCCAACCCAAGAGGTCA 263 246 109915666 - 109915911 bM70A7SSR18 CCTCCCTCCTTCTCTCTTCC GGACTTGCCATGTGGATCTT 194 202 100402596 - 100402794 bM70A7SSR19 GCAAGAGCTTCCTTCTTCCA ATGCTGGACAAAAGCCCTTA 230 215 100411479 - 100411692 RS38124092 GCCCATATGATCCAATCACC ATGGGTGGCATTATGGCTTA RSAI cuts NOD 122337874 - 122337988

Supplemental Table 1. Novel microsatellite markers and RFLPs designed to define the boundaries of the Idd18.1 interval and other Idd intervals on chromosome 3. Sequences for the forward and reverse primers are given in the 5’ to 3’ direction. The length of the PCR products for the NOD and B6 mouse strains, where known, is given in base pairs. RS38124092 is a RFLP: the NOD product is cut by RSAI once, whereas the B6 product does not contain RSAI recognition sites.

2

EST Forward primer Reverse primer Length

BY752269 CGATGGAGTCCTGCTCTG CTGTGGATGAGCCACTGAG 163 BY565884 CGATACCTTTGGAATGAGGA TGCAGGAGGCTTAACAGTTC 153 CA870586 TGAAGGCGGAGAAGTCTATG GCAATCCTGGAGCTTTGATA 181 BG060768 CCATGATTTCAAGATGCACA TGTAGCAAGCTTTATGTCCTCA 224 BI966969 ATAGGCAAGCAACCCTGAG AGAGGGTTTGACAGCATCAC 104 CA872533 AGCTTCAGGAGAGGTTGGAT AAACCACCTTTAGCCAGGAC 207

Supplemental Table 2. Primers used to confirm whether alternatively spliced VAV3 transcripts detected in EST libraries are present in cDNA by RT-PCR.

3

mRNA Transcript Forward primer Reverse primer Probe Length

VAV3 full-length exons 1-2 CCCAGATGTCCCAGTTTCTC TTCCTCATTCCAAAGGTATCG TGAAGAACATCCGGACATTCCTGG 79 VAV3 full-length exons 21-22 GCTTCAGGAGAGGTTGGATT GGGTTGGCAAGAATAATCTACTG TTCCCAAGTGATGCTGTCAAACCC 86 VAV3 full-length exons 10-11-12 AGTATCACCTCCTCCTTCAGGA TTGTGCCAAGTCCTTCATTG TAAAACACACCCATGACCCTATGGAGAA 100 VAV3.1 TGACTGACCATGCCAATTTT GTCCATTGGTCCGTTTCTCT CTTTGTTTCAGAACAAGGGCCATTCAA 76 BG060768 CCTCATTAGAAACAGAATCCCTACA TGCTCCTGAGGGGAACAT CCAACGGAATTAACTCTGCCACAATTG 151 CA870586 GCAATCCTGGAGCTTTGATAC AGCCCAGCAACCTAAATCAC TGACTCCAACGTTTCTGTGTATTTTTCTTCTG 139 CA872533 CCTCGAGCTAAGAGTACGGTATTT CCTCTCCATGTGTACCTAAACCA CAGAAACATTTTTAATACTTACCAGGGTTGGCA 103 NETRIN G1A ACGGCTGTCAACCTAATGTCT GCACCGTAGCTTCTCACAGA ACAATGAGCTCCTGCACTGCCAG 128 NETRIN G1C CATTTCCAGTATCGGTACTAATGTC GCACCGTAGCTTCTCACAGA ACAATGAGCTCCTGCACTGCCAG 133 NETRIN G1D CCACAAGCGAGCTAATGTCT GCACCGTAGCTTCTCACAGA ACAATGAGCTCCTGCACTGCCAG 127 NETRIN G1E CTAACCCAAAACAAGCTAATGTCT GCACCGTAGCTTCTCACAGA ACAATGAGCTCCTGCACTGCCAG 131 NETRIN G1G GCAGGTCCCTCCATTCTG GTTATGCAGGCTGGGCTACT TTGTCGCAGACATTAGCTATGCACACA 115 Beta-2 Microglobulin GCTATCCAGAAAACCCCTCAAAT CTGTGTTACGTAGCAGTTCAGTATGTTC AGTATACTCACGCCACCCACCGGAGAAT 90

Supplemental Table 3. Primers and probes used for qPCR.

4