PMC6102291_01
CNS changes in the patient. Brain magnetic resonance imaging demonstrating white matter periventricular lesions, as shown in the a fluid-attenuated inversion recovery (FLAIR) image, and b T2-weighted image. c Haematoxylin & eosin/luxol fast blue staining of the brain biopsy shows (I) normal architecture with preservation of grey-white differentiation. Immunohistochemical staining with glial fibrillary acidic protein demonstrates white matter and subpial gliosis (II, III). Images were taken at varying magnification with scale bars as shown
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Fig1
Magnetic resonance imaging of the brain and the spinal cord demonstrated diffuse abnormalities in the periventricular region and the white matter, with extensive wellerian degeneration in the brainstem affecting mainly the pyramidal tract (Fig. 1a, b).
Abnormal STAT1 function and gene expression. a IL-17 (left panel), IFN-gamma (middle panel) and IL-2 (right panel) secretion by PBMCs following phytohemagglutinin (PHA) stimulation for 48 h. Levels of indicated cytokines were determined by ELISA in triplicate samples. NS not stimulated. Western blot of activating STAT1 phosphorylation (anti-pTyr701) following stimulation with b IL-27 (10 microg/microL) and c IFN-alpha (8.5 ng/muL) in patient and control T-cell lysates. Anti-actin was used as a loading control. d STAT1 phosphorylation in STAT1 wild-type (WT), H629Y- and R274G-transfected U3A cells, following stimulation with IFN-gamma (100 ng/mL). e Increase in phosphorylated STAT1 in the nuclear fraction of transfected U3A cells following IFN-gamma stimulation. Anti-tubulin was used as the cytoplasmic marker and anti-RCC1 as the nuclear marker. Cyt cytoplasmic, Nuc nuclear. f Venn diagram of 1.5-fold change in IFN-gamma-inducible genes. Diagram shows microarray analysis of upregulated genes following IFN-gamma stimulation (100 ng/mL for 8 h) in U3A cells transfected with WT, H629Y or R274G STAT1. g U3A cells transfected as indicated with WT, H629Y or R274G STAT1 were stimulated for 8 h with IFN-gamma (100 ng/mL) and mRNA levels of CXCL10, CXCL9, GBP1 and IRF1 were determined by q-PCR. Expression data, normalised to the levels of the house-keeping gene GAPDH, are presented as means and standard deviations from a total of 3-6 experiments. *P < 0.05 versus WT or R274G; **P < 0.01 versus WT or R274G (Student's t-test)
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Fig2
IFN-alpha and IL-27 stimulation (Fig. 2b, c). Increased tyrosine phosphorylation was confirmed in STAT1-deficient U3A fibrosarcoma cells transfected with the H629Y mutant (Fig. 2d), comparable to phosphorylation in the R274G-transfected U3A cells, indicating that nuclear transit was not altered. The analysis revealed 24 IFN-gamma-upregulated inducible genes in the H629Y mutant as compared with far greater (83 genes) triggered by the R274G mutation, while WT showed an increase of only 22 genes (Fig. 2f and Table 2). As shown in Fig. 2g, the rise in CXCL10 in the R274G variant was far greater than the increase recorded in the H629Y mutant.