Ptpn22 and Cd2 Variations Are Associated with Altered Protein Expression and Susceptibility to Type 1 Diabetes in Nonobese Diabetic Mice

Abstract

By congenic strain mapping using autoimmune NOD.C57BL/6J congenic mice, we demonstrated previously that the type 1 diabetes (T1D) protection associated with the insulin-dependent diabetes (Idd)10 locus on chromosome 3, originally identified by linkage analysis, was in fact due to three closely linked Idd loci: Idd10, Idd18.1, and Idd18.3. In this study, we define two additional Idd loci--Idd18.2 and Idd18.4--within the boundaries of this cluster of disease-associated genes. Idd18.2 is 1.31 Mb and contains 18 genes, including Ptpn22, which encodes a phosphatase that negatively regulates T and B cell signaling. The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D. We, therefore, assessed Ptpn22 as a candidate for Idd18.2; resequencing of the NOD Ptpn22 allele revealed 183 single nucleotide polymorphisms with the C57BL/6J (B6) allele--6 exonic and 177 intronic. Functional studies showed higher expression of full-length Ptpn22 RNA and protein, and decreased TCR signaling in congenic strains with B6-derived Idd18.2 susceptibility alleles. The 953-kb Idd18.4 locus contains eight genes, including the candidate Cd2. The CD2 pathway is associated with the human autoimmune disease, multiple sclerosis, and mice with NOD-derived susceptibility alleles at Idd18.4 have lower CD2 expression on B cells. Furthermore, we observed that susceptibility alleles at Idd18.2 can mask the protection provided by Idd10/Cd101 or Idd18.1/Vav3 and Idd18.3. In summary, we describe two new T1D loci, Idd18.2 and Idd18.4, candidate genes within each region, and demonstrate the complex nature of genetic interactions underlying the development of T1D in the NOD mouse model.

FIGURE 1.

A B6 susceptibility locus, Idd18.2, and a B6 protective locus, Idd18.4, are located between Idd10 and Idd18. (A) The congenic strains used to define Idd18.2 and Idd18.4 and those used in the T1D frequency studies are shown. Lines R8 and 2410 confirm the existence of the B6 susceptibility locus, which was refined to between the centromeric recombination point of R8 and the telomeric recombination point of line 7848, and is a 1.31-Mb locus between, but not including, the microsatellite markers AC122219_3 and Susc_96.62. Idd18.4 is located between the centromeric and telomeric recombination points of line 8010, and it is a 953-kb locus between, but not including, the microsatellite markers Ptgfrn_Int1_SNP2 and Chr3-101,864. The locations of markers in NCBIM37 are shown. (B) The diabetes frequency study (conducted for 210 d in 1995–1996) indicating that congenic mice from line R8, which have a single B6 introgressed segment between Idd10 and Idd18, are more susceptible to T1D compared with NOD mice (p = 2.0 × 10⁻³). This finding suggests the presence of a B6 susceptibility locus between Idd10 and Idd18.3. (C) The diabetes frequency study (conducted for 214 d in 2003–2004) indicating that line 2410 congenic mice, which have B6-derived alleles at Idd10 and Idd18 but NOD-derived alleles at Idd18.2, are more protected from T1D compared with line 1101 (p = 3.0 × 10⁻²). Line 1101 differs from line 2410 by the presence of B6-derived alleles at Idd18.2. Therefore, line 2410 confirms the presence of a B6 susceptibility locus between Idd10 and Idd18.3. (D) The diabetes frequency study (conducted for 210 d in 2006–2007) of lines 3538 and 3539 assessing the protection associated with the Idd10 and Idd18 loci alone without B6 susceptibility alleles at Idd18.2. Lines 3538 and 3539 were much more protected from diabetes compared with NOD (p = 2.2 × 10⁻⁷, 2.9 × 10⁻¹⁵, respectively), indicating that the low levels of T1D protection observed in congenic strains with either Idd10 or Idd18 in combination with B6-derived alleles at Idd18.2 in previous congenic strain mapping studies were due to the B6 Idd18.2 susceptibility alleles masking the protection associated with Idd10 or Idd18, respectively. The diabetes frequencies of lines 2410 and 1101 were also repeated in this experiment. The protection associated with the NOD allele of Idd18.2 is clearly observed again in line 2410, as it is much more protected from diabetes than line 1101 (p = 5.4 × 10⁻⁶). The data for 3538 and NOD shown in this panel have been published previously (22). (E) The diabetes frequency study (conducted for 196 d in 2010–2011) of lines 7848 and 8010 confirm the candidacy of Ptpn22 and identify an additional Idd locus, Idd18.4, containing the candidate Cd2. Line 7848 and 8010 share a small introgressed B6 DNA segment in Idd18.4, but have B6- or NOD-derived alleles at Ptpn22, respectively. Line 7848 has a higher frequency of diabetes compared with line 8010, confirming the candidacy of Ptpn22. Moreover, the high level of diabetes protection associated with line 8010 identifies the additional B6 protective locus, Idd18.4. n = the number of mice in each cohort; numbers in parentheses indicate mice that developed diabetes by the end of the study.

FIGURE 2.

Idd18.2 and Idd18.4 annotation and sequence polymorphisms in NCBIM37. (A) The congenic boundaries of line 8010 define the Idd18.4 locus, and the centromeric boundary of line R8 and the telomeric boundary of line 7848 define the Idd18.2 locus. The T1DBase Gene Span track displays the maximum genomic interval for each gene: candidate genes are displayed in green, blue indicates other protein-encoding genes, and yellow indicates small cytoplasmic RNA genes. The NOD TilePath track represents the sequenced NOD BAC clones, and the NOD_BAC_SNP_graph represents the SNP density per 10 kb, detected by comparing the NOD BAC clone sequence to the B6 reference sequence. The NOD_NGS_SNP_graph displays the SNP density per 10 kb of SNPs detected between the NCBIM37 B6 reference sequence and NOD/ShiLtJ NGS data. (B and C) The coding sequences of the transcripts from the Idd18.2 and Idd18.4 candidate genes Ptpn22 and Cd2 are shown, respectively, in the T1DBase Curated Transcripts track. The SNPs surrounding these genes are shown underneath. These SNPs have been detected by comparing the B6 reference sequence to either the NOD BAC clone sequence (NOD Variation track) or the NOD/ShiLtJ NGS data (NGS NOD SNPs track). Black, red, blue, and green lines represent G, T, C, and A NOD alleles, respectively. The NGS data also contain ambiguous bases. R, Y, M, K, W, and S are represented as brown, orange, pink, cyan, gold and gray lines, respectively. Yellow lines are SNPs where the base information is unknown. Note that where multiple SNPs are located close together in these two tracks, the lines in the NOD variation or NGS NOD SNPs track may represent more than one SNP. There is a higher SNP density over Ptpn22; to represent this density better, the NOD_BAC_SNP_graph and NOD NGS_SNP_graph display the density of SNPs per 10 kb.

FIGURE 3.

Differential expression by genotype of full-length Ptpn22 mRNA and PEP protein. (A) Gene expression levels are displayed as dCT (see Materials and Methods); lower dCT values represent higher expression. Thymocytes isolated from male 3-wk-old line 1101 congenic mice (B6-derived alleles at Ptpn22) express 2-fold more full-length Ptpn22 mRNA compared with line 2410 congenic mice (NOD-derived alleles at Ptpn22). A similar genotype dependent expression is observed for the alternative spliced transcript, Ptpn22_K. The alternatively spliced transcripts Ptpn22_I and Ptpn22_J have the opposite genotype dependent expression, with line 2410 having the greater expression. n = 6. (B) A similar, albeit slightly lower, pattern of expression is observed for the transcripts in whole spleen from line 1101 and 2410 congenic mice: full-length Ptpn22 mRNA from line 1101 is expressed 1.4-fold more compared with line 2410. n = 6. (A and B) Results are representative of at least two independent experiments. (C) The protein expression of PEP follows the pattern of the mRNA expression. A representative Western blot analysis of P56 mouse thymocyte total lysates from line 7754 (new designation for 1101) and line 2410 congenic mice, probed against PEP and α-TUBULIN is shown. Each lane represents thymocytes collected from an individual animal. Relative ratios of PEP to α-TUBULIN were collected from individual P45-P56 mouse thymocyte total lysates in four independent experiments performed on different days by normalizing each ratio to the lowest ratio collected in each experiment. In the scatter plots, the horizontal bars represent the mean and lines represent the SD. Mann-Whitney was used to calculate statistical significance. PEP is expressed 2-fold higher in congenic mice with B6-derived alleles at Ptpn22 (lines 7754 and 7848) compared with congenic mice with NOD-derived alleles at Ptpn22 (lines 2410 and 8010; p = 2.0 × 10⁻⁴). n = 10. (D) Higher PEP expression is associated with increased negative regulation of TCR signaling. Line 7754 congenic mice with B6-derived alleles at Idd18.2 have ∼1.6-fold lower levels (p = 0.026) of phosphorylated MAPK compared with line 2410 congenic mice with NOD-derived alleles at Idd18.2 following stimulation. Each set of bars represents splenic T cells from an individual animal that were either unstimulated (unhatched bars) or stimulated with anti-CD3 Ab plus a cross-linker for 2 min (hatched bars). Phospho-p44 MAPK levels were assessed in cell lysates from 2410 (gray bars) and 7754 (open bars) congenic mice by ELISA. Three animals were tested for each congenic strain. Error bars are the SD of triplicates. The levels of phospho-p44 MAPK after stimulation were compared using Student one-tailed, unpaired t test.

FIGURE 4.

Cd2 genotype determines CD2 expression levels on B cells in spleen and bone marrow. (A) Light scatter of the splenic cells and histogram showing the gating on the splenic B220⁺ cells. (B) Histograms showing the levels of CD2 expression on the splenic B cells (B220⁺) and T cells (non B220⁺). In (B) and (E), staining with an isotype control mAb is represented by the black dotted line. In (B), (C), (E), and (F), lines 2410, 7754, 8010, and 7848 are represented by blue, green, red, and pink, respectively. (C) Scatter plots showing the MFI of CD2 on splenic B and T cells for the four strains. Thick and thin, horizontal, black lines represent the mean and the SEM, respectively. MFIs were compared based on genotype at Idd18.4, and the Mann–Whitney U test was used to determine whether these were statistically significant. (D) B220 and CD2 staining on bone marrow lymphocytes from 2410 and 7848 show the two populations, B220^lo and B220^hi, analyzed. (E) Histograms of CD2 expression on the B220^hi and B220^lo bone marrow populations comparing the four strains. (F) Scatter plots showing the MFI of CD2 on bone marrow-derived B220^lo and B220^hi populations for the four strains. Thick and thin, horizontal, black lines represent the mean and the SEM, respectively. MFIs were compared based on genotype at Idd18.4, and the Mann–Whitney U test was used to determine whether these were statistically significant. In addition to the data shown in this figure, we also performed two additional, separate experiments in which we tested mice with B6- or NOD-derived alleles at Idd18.4 in splenic B cells (n = 19 for each genotype) and in B220^hi and B220^lo bone marrow cells (n = 11 for each genotype). We observed the same expression differences as shown in this figure.