PMC6491679_01
PMC6491679_02
Identification of a heterozygous mutation in STAT1 leading to a gain of function. (A) Sanger sequencing revealed a heterozygous c.504T>A mutation in exon 7 resulting in a missense mutation in the coiled-coil domain (p.D168E). (B) Increased phosphorylation of STAT1 following in vitro stimulation of patient's EBV-LCL with IFNalpha and IFNgamma.
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F1
STAT1 GOF mutation was considered and genetic analysis of STAT1 exons 7-14 was performed on DNA of the patient. Sanger sequencing revealed a heterozygous variant in exon 7 (c.504T>A) resulting in a missense mutation in the coiled-coil domain (p.D168E) (Figure 1A). Thirty minutes after stimulation with either IFNalpha or IFNgamma, the patient's cells showed increased levels of pSTAT1 confirming a GOF phenotype as a result of the D168E missense mutation (Figure 1B).
Immunological effects of immunotherapy with G-CSF. (A) Longitudinal measurements of blood leukocyte and neutrophil cell counts over a 3 year interval. (B) Frequencies of Th17 and Tfh cell subsets within total CD4 T cells prior to, during (2 timepoints; 2015) and after discontinuation of G-CSF therapy.
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F2
Extensive follow-up of total leukocyte and neutrophil count showed a general increase in numbers during therapy (Figure 2A). T-cell immunophenotyping, and reporting of relative frequencies of Th17 and Tfh cells. Th17 cell frequencies were within the normal range on only 1 occasion under G-CSF therapy, whereas Tfh cell frequencies were not below the normal range (Figure 2B).
B-cell defects in STAT1 GOF. (A) Somatic hypermutation levels in IgG subclass transcripts of the patient and controls. (B) Relative distributions of IgG subclasses of unique transcripts. (C) STAT3 phosphorylation in EBV-LCL upon stimulation with IL-21. (D) Nuclear localization of pSTAT3 in EBV-LCL following stimulation with IL-21 as determined with the AMNIS ImageStream. Example images are shown on the left, and the collated events as overlays for nuclear localization scores on the right. (E) CD25 (IL2Ralpha) expression on EBV-LCL following stimulation with IL-21. Overlays are shown for one experiment to illustrate on the left and the combined data from 4 independent experiments on the right. Statistics: Paired t-test.
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F3
However, IgG3 transcripts of the patient contained negligible SHM, in contrast to IgG1 and IgG2 (Figure 3A). Further analysis of the IgG transcripts demonstrated a predominant usage of IgG3 compared to IgG2 (Figure 3B). Indeed, in EBV-immortalized B cells from the patient, IL-21 stimulation normally induced STAT3 phosphorylation (Figure 3C). Moreover, nuclear localization studies with imaging flowcytometry revealed normal nuclear localization of pSTAT3 after IL-21 stimulation as well (Figure 3D). IL-21, EBV-LCL from a healthy control upregulated CD25 surface expression (Figure 3E). In contrast, EBV-LCL from the patient had lower levels of CD25 expression.