Meta-analysis of the frequency of the <i>PTPN22 C1858T</i> SNP in ANCA Disease.

<p>Meta-analysis of the frequency of the <i>PTPN22 C1858T</i> SNP in ANCA Disease.</p

Characteristics of patients with ANCA disease enrolled in the functional studies.

*<p>Patient's sample included in western blot analysis of signaling pathways.</p

Characteristics of patients with ANCA disease enrolled in the Affymetrix microarray study.

<p>Characteristics of patients with ANCA disease enrolled in the Affymetrix microarray study.</p

Frequency of PTPN22 of protective allele (<i>A788</i>) in Caucasian ANCA patients.

<p>Frequency of PTPN22 of protective allele (<i>A788</i>) in Caucasian ANCA patients.</p

<i>IL-10</i> mRNA expression.

<p>(A) <i>IL-10</i> transcript levels are reduced in leukocytes from PTPN22 (R620W) positive patients. (A) IL-10 mRNA expression, which is mediated through the ERK pathway, was significantly lower in gain-of-function patients (<i>p</i><0.0001) by quantitative TaqMan PCR. (B) Longitudinally, the baseline level of IL-10 message in patients with the gain-of-function variant did not increase as they transitioned from active disease to remission (<i>p</i> = 0.25). (C) In contrast, patients with normal PTPN22 showed a robust increase in IL-10 as they entered remission (<i>p</i><0.0001). (D) Decreased IL-10 levels were associated with the relapsing group (n = 39, 1.8±1.15), and higher level in the non-relapse patient group (n = 14, 6.6±4.4, <i>p</i><0.0001).</p

Bioinformatics analysis of Affymetrix microarray gene expression data, comparing leukocytes with the gain-of-function genotype to those with a non-variant genotype.

<p>Principal Component Analysis (PCA) scatter plot using Partek analysis is shown in the upper left corner. PCA is mathematically defined as an orthogonal linear transformation that transforms the data to a new coordinate system such that the greatest variance by any projection of the data comes to lie on the first coordinate (called the first principal component), the second greatest variance on the second coordinate, and so on. Each dot represents a patient's expression profile; the blue color dots represent gain-of-function and red show non-variant genotypes. Analysis using the Ingenuity Pathway Tools (IPA) software utilizes a repository of biological interactions and functional annotations created from millions of individually modeled relationships. The genes in red indicate increased expression and blue represents decreased expression, comparing gain-of-function with non-variant individuals. Primary networks identified were ERK1/2, p38MAPK, and NFκB networks.</p

Frequency of PTPN22 risk-allele (<i>T1858</i>) genotype in Caucasian ANCA patients.

<p># Zheng W, 2005 (35).</p>*<p>Excluded from analysis: 6 ANCA-neg; 5 PR3+MPO dual serology: 3 p-ANCA+ANA positives.</p

Analysis of ERK1,2 and p38MAPK phosphorylation status.

<p>Bar graphs represent the ratio of intensity pERK/ERK and pp38/p38MAPK as quantitated by densitometric scanning analysis using ImageMaster VDS software. Western blot analysis for ERK1,2 and p38MAPK activation demonstrates PTPN22 gain-of-function (R620) exerts a negative effect on the ERK signaling pathway, compared to loss-of-function (R263Q) and non-variant controls. In contrast p38 mitogen-activated protein kinase (p38 MAPK) was increased with the gain-of-function (R620) phenotype.</p

Signaling pathways disrupted by the gain-of-function variant of PTPN22.

<p>(A) Signaling pathways affected by PTPN22 include SRC-family kinases (Lyn/Fyn) and RAS pathways <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Nagao1" target="_blank">[83]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Fiorillo1" target="_blank">[84]</a>. It can affect the activity of SRC-family kinases through regulation of CSK (cSRC Kinase) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Cloutier1" target="_blank">[96]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Roskoski1" target="_blank">[97]</a>. PTPN22 can affect RAS activity through binding to GRB-2 (Growth factor receptor-bound protein 2). ERK1,2 phosphorylates and activates many transcription factors, including the transcription factor Sp1 depicted here, which regulates the transcription of IL-10 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Lucas1" target="_blank">[86]</a>. (B) Changes in PTPN22 function due to the gain-of-function phenotype. PTPN22 (R620W) amino acid change lies within a domain that binds CSK, resulting in a greatly reduced binding <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Fiorillo1" target="_blank">[84]</a>. Thus CSK is available for binding and inhibiting SRC. Also, a gain-of function alteration could act as a super-antagonist of epigenetic nucleosome remodeling, based on reports that PTPs directly dephosphorylate histone tails <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0042783#pone.0042783-Rosenfeld1" target="_blank">[98]</a>. The gain-of-function phosphatase activity also affects the function of GRB2 restraint of RAS signaling and a loss of ERK1/2 phosphorylation/activation and loss of Sp1 transcriptional activity.</p

PTPN22 phosphatase activity in leukocytes.

<p>Basal level of PTPN22 phosphatase activity was high in leukocytes expressing the gain-of-function variant, (A) PTPN22 protein was active in all samples with the gain-of-function PTPN22 (R620W), while activity was undetectable in non-variant and loss-of-function control groups (<i>p</i><0.0001). Activity values were plotted against total protein captured on ELISA plate using mouse-anti-PTPN22 antibody. For mock-controls, ELISA wells were coated with normal mouse IgG in parallel (B) High basal PTPN22 phosphatase activity was present in neutrophils (<i>p</i> = 0.0004) and lymphocytes&monocytes (<i>p</i> = 0.0003) (activity calculated as fold-increase above controls). (C) High basal phosphatase activity was significantly down-regulated after PMA treatment (<i>p</i><0.0001), while there were no changes in non-variant controls (<i>p</i> = 0.75).</p