Pulmonary Aspergillosis and Low HIES Score in a Family with STAT3 N-Terminal Domain Mutation

Abstract

Signal transducer and activator of transcription 3 (STAT3) plays a key role in leukocytic and non-leukocytic cells. Germ line mutations in STAT3, which are mainly found in the SH2, DNA binding and transactivation domains, can be loss- or gain-of-function (LOF and GOF). STAT3 N-terminal domain (NTD) mutations are rare, and their biological effects remain incompletely understood. We explored the significance of STAT3 NTD p.Trp37* variant in a patient with chronic pulmonary aspergillosis and a low Hyper-IgE syndrome (HIES) score. In cell culture models, the expression of full-length p.Trp37* allele showed shorter STAT3 protein expression suggesting a re-initiation (Met99 or Met143). STAT3 activity using luciferase reporter assay showed a twofold-increased activity of the STAT3 p.Trp37* STAT3 protein compared with WT STAT3 at basal level and upon IL-6 stimulation. In contrast, the activity of the short pTrp37* peptide (amino acids 1 to 37) was amorphic but without dominant negative (DN) effect on transcriptional activity or STAT3 Tyr705 phosphorylation. The proteins initiated at Met99 and Met143 were surprisingly hypermorphic. In carriers' peripheral blood mononuclear cells (PBMCs), both WT and mutated STAT3 mRNA were equally present and the global amount of STAT3 protein was not significantly reduced. In stimulated heterozygous carriers' PBMCs, however, STAT3 Tyr705 phosphorylation and Th17 were reduced but not completely abolished. This suggests a DN effect of an unknown product of the p.Trp37* allele. Transcriptomics analysis of PBMCs from the index revealed selectively distinct gene expression. We conclude that heterozygosity for the NTD p.Trp37* STAT3 mutation defines a novel allelic form of STAT3 deficiency, associated with a chronic pulmonary aspergillosis and minor signs of HIES.

Keywords: Mutation; Pulmonary aspergillosis; STAT3.

Fig. 1

The index presented with bloody sputum at age 22 when her chest CT was consistent with chronic pulmonary aspergillosis (A, B). Histology of the lung lesion was consistent with aspergillosis and displayed high numbers of eosinophils (C-E). Seven years later, hemoptysis recurred, and chest CT showed a new aspergillosis lesion (F, G) which was surgically removed (H). One year later, the CT scan appeared unremarkable (I). Soon after, several new aspergillosis lesions were found (J, K) leading to surgical removal of the largest lesions. Medications (vori, voriconazole; posaconazole; isavuconazole; Ambisome, liposomal amphotericin B; IgG, intravenous IgG substitution) are indicated. Blue solid arrows on the timeline indicate the surgical operations

Fig. 2

A Family segregation of the STAT3 p.Trp37* allele in the kindred. The index (II:5) is marked with a solid arrow. B A schematic of the filtering criteria and amounts of variants from whole exome analysis (C). Whole exome sequencing revealed a private heterozygous premature stop gained (p.Trp37*) in STAT3 gene. Possible re-initiation sites (methionine 99 and 143) are indicated with arrows. D p.Trp37* variant affects the splicing of exon 2 in STAT3. E STAT3 immunoblotting on STAT3 deficient A4 cells transfected with WT or p.Trp37* STAT3 variant. p.Trp37* STAT3 variant produced lower MW products compared to WT STAT3, indicating a re-initiation. F The STAT3 p.W37X mutation expression from RNA-Seq data. G Intracellular STAT3 immunostaining measured by flow cytometry on PBMC from heterozygous Trp37* variant carriers and controls without (left) and with stimulation with IL-6 (right) (H). Time course of phospho-STAT3 by intracellular immunostaining in PBMC from heterozygous Trp37* variant carriers, non-carrier relatives, and healthy controls upon IL-6 or IL-21 stimulation. I Time course of phospho-STAT1 by intracellular immunostaining in PBMC from Trp37* variant carriers, non-carrier relatives, and healthy controls upon interferon-alpha, IL-6 or IL-21

Fig. 3

Luciferase activity (fold change) in STAT3 deficient A4 cells in non-transfected (mock), empty vector (EV), wild type STAT3 (WT), whole STAT3 sequence with p.Trp37* (W37*_Full-length), STAT3 N-terminal peptide amino acids 1–37 (W37*_short), or dominant negative STAT3 (Arg382Trp) (A). Luciferase activity (fold change) in STAT3 positive HEK293T cells in non-transfected (mock), empty vector (EV), wild type STAT3 (WT), whole STAT3 sequence with p.Trp37* (W37*_Full-length), STAT3 N-terminal peptide amino acids 1–37 (W37*_short), or dominant negative STAT3 (Arg382Trp). The y-axis represents STAT3 transcriptional activity levels normalized against unstimulated activity in EV-transformed cells. Each dot represents a biological replicate obtained from technical replicates. B Luciferase activity (fold change) in HEK293T cells with endogenous STAT3 expressing an increasing amount of wild type (WT) or whole STAT3 sequence with p. Trp37X or dominant negative STAT3 (Arg382Trp). D HEK293 cells were transfected with GFP and MAC-tag-N vector containing STAT3 p.Trp37*_short using calcium phosphate. Two days after transfection, cells were stimulated with or without IL-6 for 30 min. Cells were collected for further Western blot detections of tyrosine phosphorylated STAT3 and total STAT3. Immunoblots from two independent experiments are shown

Fig. 4

Selected immunological expression profiles of index PBMCs. mRNA fold changes compared to healthy controls in unstimulated index STAT3 p.Trp37* and Arg278His (STAT3 GOF) PBMCs of seven IFN-regulated genes (A), type II IFN (B), inflammasome (C), NF-κB pathway (D), JAK/STAT pathway (E), and interleukins (F) related genes. Mean of three measurements together with SD is presented

Fig. 5

Transcriptomics analysis reveals distinct gene expression profiles in cases with STAT3 p.Trp37* mutation. A PCA plot of transcriptomes of IL-6 stimulated PBMCs from three STAT3 p.Trp37* variant positive cases and sex/age matched controls X-axis represents the contributor rate of the first component. Y-axis represents the contributor rate of the second component. Points represent each sample. Ellipses were drawn manually. B The number of differentially expressed genes from IL-6 stimulated PBMCs from three STAT3 p. Trp37X mutated cases and sex/age matched controls are shown. C STAT3 mRNA expression of the three family members with the STAT3 p.W37X mutation and their controls from RNA-Seq data obtained from IL-6 stimulated PBMCs. D KEGG pathway analysis was performed on up-regulated and down-regulated genes in IL-6 stimulated PBMCs from the indexed patient versus her control comparison. The pathways presented in the plot are significantly enriched; The colour indicates the q-value, the lower q-value indicates the more significant enrichment. Point size indicates DEG number. The larger the value of the rich factor, the more significant enrichment. E Heatmap showing the log fold change values of selected genes significantly changed in IL-6 stimulated PBMC from patient versus control (F). Quantitative RT-PCR detections of Foxp3, RORC and TBX21, key transcription factors for Treg, Th17 and Th1 cells respectively. G Quantitative RT-PCR mRNA expression using 5’UTR probe (upstream the W37* variant), and probe in the middle of the transcript (downstream the W37* variant) the p.W37X mutation site respectively, fragments of STAT3. EF1α was used as endogenous control in all the qPCR detections