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

Fraser HI, Howlett S, Clark J, Rainbow DB, Stanford SM, Wu DJ, Hsieh YW, Maine CJ, Christensen M, Kuchroo V, Sherman LA, Podolin PL, Todd JA, Steward CA, Peterson LB, Bottini N, Wicker LS - J. Immunol. (2015)

Bottom Line: In this study, we define two additional Idd loci-Idd18.2 and Idd18.4-within the boundaries of this cluster of disease-associated genes.The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D.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.

View Article: PubMed Central - PubMed

Affiliation: Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom;

No MeSH data available.


Related in: MedlinePlus

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.
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fig03: 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.

Mentions: To determine whether the polymorphisms present between NOD and B6 could affect the splicing and expression of Ptpn22, we first searched for alternatively spliced Ptpn22 transcripts in EST databases and by RT-PCR. The expression of 13 different alternatively spliced transcripts (Supplemental Table IIB, IIC) was confirmed (data not shown); a contributing factor to this large number may be the rare GC-AG splice site, as noncanonical splice sites are associated with higher levels of alternatively spliced transcripts (41). The expression of the Ptpn22 transcripts from NOD- and B6-derived alleles at Idd18.2 was compared using qPCR. In addition to full-length Ptpn22, three transcripts were found to be differentially expressed: Ptpn22_I, Ptpn22_J, and Ptpn22_K (Fig. 3A). Ptpn22_I contains all exons apart from exons 12 and 13, resulting in a change of reading frame in exon 14 and a premature termination codon in exon 15. Ptpn22_J, like Ptpn22_I, excludes exons 12 and 13 but contains a novel exon 14 that includes the first 13 nucleotides in intron 14; this inclusion returns the reading frame to that of full-length Ptpn22 in exon 15. Ptpn22_K contains all exons apart from exon 15; this results in a change of reading frame and a premature termination codon in exon 17 (Fig. 2B). Full-length Ptpn22 is expressed 2-fold and 1.4-fold higher in line 1101 compared with line 2410 in thymocytes from 3-wk-old male mice (p = 1.6 × 10−5) and whole spleen from 9-wk-old female mice (p = 6.2 × 10−5), respectively (Fig. 3A, 3B), indicating that B6-derived alleles of Idd18.2 are more highly expressed than NOD-derived alleles. In the same tissue samples, the Ptpn22_I, _J and _K alternatively spliced transcripts are expressed ∼50–100-fold less than full-length Ptpn22. Ptpn22_K has a similar genotype-dependent expression, with the B6-derived alleles in line 1101 having a higher expression (3–4-fold) compared with line 2410. Conversely, Ptpn22_I and Ptpn22_J have the opposite genotype-dependent expression with B6-derived alleles expressed 2.1–5.2-fold lower in line 1101 compared with NOD-derived alleles in line 2410 (Fig. 3A, 3B).


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

Fraser HI, Howlett S, Clark J, Rainbow DB, Stanford SM, Wu DJ, Hsieh YW, Maine CJ, Christensen M, Kuchroo V, Sherman LA, Podolin PL, Todd JA, Steward CA, Peterson LB, Bottini N, Wicker LS - J. Immunol. (2015)

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.
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fig03: 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.
Mentions: To determine whether the polymorphisms present between NOD and B6 could affect the splicing and expression of Ptpn22, we first searched for alternatively spliced Ptpn22 transcripts in EST databases and by RT-PCR. The expression of 13 different alternatively spliced transcripts (Supplemental Table IIB, IIC) was confirmed (data not shown); a contributing factor to this large number may be the rare GC-AG splice site, as noncanonical splice sites are associated with higher levels of alternatively spliced transcripts (41). The expression of the Ptpn22 transcripts from NOD- and B6-derived alleles at Idd18.2 was compared using qPCR. In addition to full-length Ptpn22, three transcripts were found to be differentially expressed: Ptpn22_I, Ptpn22_J, and Ptpn22_K (Fig. 3A). Ptpn22_I contains all exons apart from exons 12 and 13, resulting in a change of reading frame in exon 14 and a premature termination codon in exon 15. Ptpn22_J, like Ptpn22_I, excludes exons 12 and 13 but contains a novel exon 14 that includes the first 13 nucleotides in intron 14; this inclusion returns the reading frame to that of full-length Ptpn22 in exon 15. Ptpn22_K contains all exons apart from exon 15; this results in a change of reading frame and a premature termination codon in exon 17 (Fig. 2B). Full-length Ptpn22 is expressed 2-fold and 1.4-fold higher in line 1101 compared with line 2410 in thymocytes from 3-wk-old male mice (p = 1.6 × 10−5) and whole spleen from 9-wk-old female mice (p = 6.2 × 10−5), respectively (Fig. 3A, 3B), indicating that B6-derived alleles of Idd18.2 are more highly expressed than NOD-derived alleles. In the same tissue samples, the Ptpn22_I, _J and _K alternatively spliced transcripts are expressed ∼50–100-fold less than full-length Ptpn22. Ptpn22_K has a similar genotype-dependent expression, with the B6-derived alleles in line 1101 having a higher expression (3–4-fold) compared with line 2410. Conversely, Ptpn22_I and Ptpn22_J have the opposite genotype-dependent expression with B6-derived alleles expressed 2.1–5.2-fold lower in line 1101 compared with NOD-derived alleles in line 2410 (Fig. 3A, 3B).

Bottom Line: In this study, we define two additional Idd loci-Idd18.2 and Idd18.4-within the boundaries of this cluster of disease-associated genes.The human ortholog of Ptpn22, PTPN22, is associated with numerous autoimmune diseases, including T1D.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.

View Article: PubMed Central - PubMed

Affiliation: Juvenile Diabetes Research Foundation/Wellcome Trust Diabetes and Inflammation Laboratory, Department of Medical Genetics, Cambridge Institute for Medical Research, University of Cambridge, Cambridge CB2 0XY, United Kingdom;

No MeSH data available.


Related in: MedlinePlus