Nonobese diabetic congenic strain analysis of autoimmune diabetes reveals genetic complexity of the Idd18 locus and identifies Vav3 as a candidate gene

Abstract

We have used the public sequencing and annotation of the mouse genome to delimit the previously resolved type 1 diabetes (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, 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 3' 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 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 beta-cell destruction in NOD mice. This study also demonstrates that a congenic strain mapping approach can isolate closely linked susceptibility genes.

Figures

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 close-up 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 months of age (P = 0.0027); 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 vs 2399 P = 0.79, 1098 vs 1100 P = 0.97, 1100 vs 2399 P = 0.80). n = the number of mice in each cohort, and the numbers in parentheses indicate mice that went diabetic.

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 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 boundary markers of Idd18.1 are represented as red and green lines, respectively, on the region boundaries track.

Figure 3

Differential expression of full-length VAV3 mRNA. A, Thymocytes isolated from line 2399 (n = 7) (NOD.B6 Idd3 10) express 1.84 fold more full-length VAV3 mRNA (detected at the exon 1-2 boundary) compared to line 1538 (n = 8) (NOD.B6 Idd3 Idd10 Idd18). B, Thymocytes isolated from line 2412 (n = 8) (NOD.B6 Idd10) express 1.46 fold more full-length VAV3 mRNA (detected at the exon 1-2 boundary) compared to line 1101 (n = 8) (NOD.B6 Idd10 Idd18). Note that A and B have the same y-axis. Thymocytes from 3-week old male mice were used to avoid potential subset variations that occur as NOD mice age and autoimmunity progresses. Data are representative of 5 experiments.

Figure 4

The protection associated with Idd18.1 is dependent upon 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 utilizing 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 and D), which have NOD alleles at Idd3, B6 alleles at Idd10, and B6 and NOD alleles at Idd18.1, respectively, indicates that the protection associated with the B6 alleles of Idd18.1 is dependent upon 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 Fig. 4C was repeated in 2006-2007 giving the same result (P = 0.82, for 1101 and 2412). As the congenic segments in lines 2399 and 2412 are identical except for at Idd3, this indicates that the protection associated with the Idd18.1 interval is dependent upon 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.

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 Figure 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 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.