De novo mutations promote inflammation in children with STAT3 gain-of-function syndrome by affecting IL-1β expression

De novo mutations promote inflammation in children with STAT3 gain-of-function syndrome by affecting IL-1β expression | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article De novo mutations promote inflammation in children with STAT3 gain-of-function syndrome by affecting IL-1β expression Jiyu Chen, Yan-Fang Li, Xue-Mei Jiang, Xin Bi, Mi-Feng Yang, Zhu Zhou, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4181172/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background STAT3 Gain-of-Function (GOF) syndrome characterized by early onset autoimmunity and primary immune regulatory disorder, the immunological mechanisms remain poorly understood. Employing whole-genome sequencing within familial trios, our study elucidated the pivotal role of de novo mutations in genetic diseases. Results We identified 37 high-risk pathogenic loci affecting 23 genes, notably including the novel STAT3c.508G>A mutation. Furthermore, significant downregulation of pathogenic genes in affected individuals, potentially associated with inflammatory responses regulated by PTPN14 via miR378c, was observed. Conclusion These findings not only contribute to our understanding of the pathogenesis but also highlight potential therapeutic strategies. Our study suggests that combined JAK inhibitors and IL-6R antagonists could offer promising avenues for mitigating the severity of these genetic disorders. STAT3 gain-of-function syndrome de novo mutation PTPN14 microRNA inflammation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Signal transducer and activator of transcription-3 (STAT3), a crucial gene involved in cell survival, proliferation, differentiation, and metabolism, plays a pivotal role in immune cell differentiation and inflammation control[ 1 – 4 ]. Intriguingly, mutations in the same STAT3 gene can give rise to different diseases and clinical phenotypes. Autosomal dominant inherited mutations in STAT3 can result in STAT3 Loss-of-Function (LOF) variants leading to conditions such as Hyper-IgE syndrome (HIES) or Job syndrome (OMIM 147060)[ 5 ]. Somatic mutations in STAT3 within somatic cells can lead to Gain-of-Function (GOF) variants, contributing to certain malignant tumors[ 6 , 7 ]. Meanwhile, germline mutations in reproductive cells can cause STAT3 GOF, associated with autoimmune disorders[ 8 , 9 ]. Traditional genetic approaches, such as linkage analysis and genome-wide association studies, have traditionally focused on inherited genetic variations[ 10 , 11 ]. Recent findings indicate that disruptive de novo mutations in one or two genes are major contributors to many rare genetic syndromes. Examples include Kabuki syndrome[ 12 ], Schinzel-Giedion syndrome[ 13 ], Bohring-Opitz syndrome[ 14 ], Baraitser-Winter syndrome[ 15 ], and Coffin-Siris syndrome[ 16 ]. However, the clinical presentation of STAT3 GOF syndrome is highly diverse, with distinct clinical features observed in different patients[ 17 ]. Therefore, the association between specific de novo mutations (DNMs) identified through whole-genome sequencing and STAT3 GOF syndrome holds practical significance for the diagnosis and intervention of this syndrome. In this study, we established joint analyses for multiple trios within a multifetal family to minimize the impact of non-pathogenic variants arising from similar environments. Leveraging Whole Genome Sequencing (WGS) data, we curated a dataset specific to non-inherited variations in the affected child. By comparing the immune cell gene expression profiles of healthy family members, we identified miRNA-mediated regulation of STAT3 , potentially leading to aberrant inflammatory responses associated with the autoimmune phenotype observed in the affected child. This research not only provides evidence for new pathogenic loci in STAT3 GOF syndrome but also significantly advances our understanding of the relationship between genetic variations and observed clinical manifestations. Materials and Methods Sample collection All sample collections in this study followed the ethical standards of the Kunming Children's Hospital Ethics Committee (Approval Number 2023-03-336-K01). With the informed consent of the pediatric patient and their family, blood specimens were collected from the patient, the patient's parents, and the patient's sibling, and stored temporarily in EDTA anticoagulant tubes. DNA and RNA extraction Collect a whole blood specimen of 300 µL, and extract DNA and total RNA following the instructions provided for TIANamp Genomic DNA Kit (DP304, TIANGEN) and RNAprep Pure Hi-Blood Kit (DP443, TIANGEN), respectively. Assess nucleic acid integrity using Agilent 5400, and quantitate the samples with NanoDrop 2000. Whole-genome sequencing (WGS) The DNA sample was used as input material for the DNA library preparations. Sequencing library was generated and index codes were added to each sample. Briefly, genomic DNA sample was fragmented by sonication to a size of 350 bp. Then DNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for Illumina sequencing, followed by further PCR amplification. After PCR products were purified. Subsequently, library quality was assessed and quantified by qPCR. The qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy, according to effective library concentration and data amount required. lncRNA and miRNA sequencing Total RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB E7420) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. rRNA is removed from the total RNA samples. After adenylation of 3’ ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 370 ~ 420 bp in length, the library fragments were purified with AMPure XP system (Beverly, USA). Then 3 µL USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) library quality was assessed on the Agilent 5400 system༈Agilent, USA༉ and quantified by QPCR (1.5 nM). The Qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy in Biolinker Technology (Kunming) Co., Ltd., according to effective library concentration and data amount required. Small RNA Sequencing libraries were generated using NEB Next® Multiplex Small RNA Library Prep Set for Illumina® (NEB E7300L). Briefly, 3’ and 5’ adaptors were ligated to 3’ and 5’ end of small RNA, respectively. Then the first strand cDNA was synthesized after hybridazition with reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between18 ~ 40 bp were ready for sequencing on Illumina sequencing with SE50. Subsequently, library quality was assessed on the Agilent 5400 system (Agilent, USA) and quantified by QPCR༈1.5nM༉. The Qualified libraries were pooled and sequenced on Illumina platforms with SE50 strategy in Biolinker Technology (Kunming) Co., Ltd., according to effective library concentration and data amount required. Alignment and post alignment processing After obtaining raw sequencing data, we employed the fastp tool[ 18 ] for data filtering and cleaning, removing adapter sequences and low-quality reads. The sequencing results for all three types of libraries were processed using parameters “-g -q 5 -u 50 -n 15”. The reference genome used for alignment was hg38(GCF_000001405)[ 19 , 20 ]. For WGS data, we utilized the BWA mem algorithm for alignment[ 21 ], employing parameters “-M -a”. Subsequently, the resulting BAM files underwent sorting and index construction using samtools[ 22 ]. For the lncRNA sequencing data, alignment was performed using hisat2 with default parameters[ 23 ]. Following alignment, the resultant BAM files were subjected to sorting and index establishment using samtools. For small RNA sequencing data, we employed Bowtie[ 24 ] for alignment against three different references: the reference genome, all miRNA hairpins, and all mature miRNA sequences[ 25 , 26 ]. The parameters used for alignment were -v 0 -m 5 -a -q --strata --best. Following alignment, the resulting SAM files were sorted using the samtools sort command and then converted to BAM format, with subsequent index construction. SNP/INDEL calling and filtering For the SNP/INDEL analysis of WGS data, we followed a Genome Analysis Toolkit (GATK) comprehensive workflow[ 27 ]. Initially, we employed GATK to mark and remove duplicate reads using the MarkDuplicates tool. The sequencing depth was calculated using the samtools depth command. Subsequently, BaseRecalibrator was applied for base quality score recalibration. Known SNP positions for recalibration were sourced from Homo_sapiens_assembly38.dbsnp138.vcf, while INDEL positions were obtained from Mills_and_1000G_gold_standard.indels.hg38.vcf. High-confidence SNP positions from the 1000 Genomes Project were extracted from 1000G_phase1.snps.high_confidence.hg38.vcf.gz. After the first round of Base Quality Score Recalibration (BQSR), the recalibrated BAM files were subjected to variant calling using HaplotypeCaller in GVCF (Genomic Variant Call Format) mode, calling variants per-sample. After merging all the GVCF files using GATK's CombineGVCFs, we performed joint genotyping using GenotypeGVCFs. Subsequently, we constructed a recalibration model and assessed variant quality scores using VariantRecalibrator (VQSR) and ApplyVQSR. This process enabled us to filter variants based on their quality. For SNP recalibration, we applied prior probability correction using sites from hapmap, omni, 1000G, and dbsnp. For INDEL recalibration, we used mills as a reference, incorporating parameters such as "-an QD -an MQ -an DP -an MQRankSum -an ReadPosRankSum -an FS -an SOR" for fine-tuning the recalibration model. DNM calling and filtering We conducted DNMs analysis using triodenovo for both the affected child and their sibling. The input files consisted of VQSR-calibrated VCF files. Subsequently, we filtered out any mutation sites identified in the sibling, retaining only those found exclusively in the affected child. The retained variants included single-nucleotide positions with only two alleles, a QUAL score of at least 30, and mutations where the child was heterozygous while the parents were homozygous reference. Furthermore, we specifically kept positions where the child was heterozygous, and the PL (Phred-scaled likelihoods) for heterozygous genotype was zero, while the minimum PL for the other two genotypes was 30 (i.e., the genotype likelihoods, defined as P(R|G), where R represents the aligned base, and G is the potential genotype, for the heterozygous mutation > 1000 times greater than the other two genotypes). DNM annotation and validation We performed mutation filtering using SnpSift[ 28 ] filter with the criteria "(( DP > 30 ) && ( GEN.DQ > 5 ) && ( GEN.DP > 10 ))". This filtering strategy retained variants with a depth (DP) greater than 30, a quality score (DQ) greater than 5 for each genotype, and a genotype depth (DP) greater than 10. Additionally, we utilized SnpSift annotate to annotate the variants with existing information from the NCBI ClinVar database[ 29 ]. This annotation step aimed to provide additional insights into the clinical significance and known associations of the identified variants. For newly discovered variants that were not annotated in the National Center for Biotechnology Information (NCBI) ClinVar database, we conducted PCR amplification of the target regions and subsequently validated the correctness of the variant positions through Sanger sequencing[ 30 ]. This experimental validation step aimed to confirm the presence of the identified variants and ensure the accuracy of the genomic alterations identified in our analysis. Differential gene analysis We quantified gene-level expression for each sample using htseq-count[ 31 ], utilizing a gene annotation file sourced from Genecode version 44[ 20 ]. Subsequently, we aggregated the expression values for all samples into an expression matrix. Differential gene expression analysis was conducted using DESeq2[ 32 , 33 ], with the following parameters: fold change threshold of 2, a p -value cutoff of 0.05, a minimum count threshold of 10, and the normalization method set to VST (variance stabilizing transformation). This approach allowed us to identify genes exhibiting significant differences in expression between conditions while considering fold changes, statistical significance, and data quality. miRNA target gene analysis We obtained the mature human miRNA sequences from the miRBase database[ 25 , 26 ] and concurrently extracted the 3'UTR sequences of differentially expressed genes. Utilizing the miranda algorithm with default parameters[ 34 – 36 ], we predicted the binding sites for miRNA-mRNA interactions. The analysis was comprehensive, outputting all predicted binding sites to provide a thorough examination of potential miRNA-mRNA interactions associated with the differentially expressed genes. Weighted correlation network analysis (WGCNA) WGCNA package of R software was used to reveal the correlation between genes[ 37 ]. Genes were filtered by MAD (median and median absolute deviation). We set the power of β based on the point of the plateau phase. The minimum number of module genes was set at 30. The hierarchical clustering dendrogram summarized the Gene modules with different colors. Heatmap and topological overlap matrix (TOM) plot were used to visualize. We calculated the correlation between modules and traits. The corresponding module gene information was extracted for further analysis. Cytoscape was to use to visualize gene networks[ 38 ]. Enrichment analysis We performed enrichment analysis on differentially expressed genes (p < 0.05) using the R packages DOSE[ 39 ], topGO[ 40 ], and clusterProfiler[ 41 ]. The analysis involved the exploration of enriched terms within biological ontologies. The specific parameters were set to output all terms. A significance threshold of p < 0.05 was considered for determining enriched results. We employed ggplot2[ 42 ] for visualizing the enrichment results. Plasmid construction and transfection Human microglial cells (HMC3) were transfected with STAT3 and STAT3 c.508G > A plasmids in 6-well and 12-well plates. After a 36-hour transfection, cells in the 6-well plate were treated with 500ng/ml LPS for 6 hours for protein extraction and subsequent western blot analysis. Meanwhile, cells in the 12-well plate, subjected to a 3-hour LPS treatment after transfection, were harvested for RNA extraction and RT-qPCR analysis to assess gene expression changes. Results Case history The proband was a 1-year-old boy, the third child of healthy non-consanguineous parents of Chinese ancestry (Fig. 1 A and Table S1 ). His growth parameters were abnormal, loss of subcutaneous fat, body weight of 6.3 kg (<-3SD), and height of 67 cm (<-3SD). He presented to a community hospital with recurrent fever and aggravated abdominal distension, all of 2 months duration. These digestive symptoms include increased abdominal distention, bloating, difficulty with defecation, appetite reduction, and an abdominal circumference of 46 cm, after 1 week of recurrent fever. Additionally, parents noticed dry, brown, and plate-like scales mainly on both ankles and the dorsa of both feet. A review of systems was negative for nausea, vomiting, loose stools, or a history of other joint pain or swelling. No family history was identified within 3 generations, including his two brothers, aged 4 and 6. Before admission, Cefperazone-Sulbactam was given against infection for 2 weeks, and meropenem + vancomycin for 1 week. However, no significant effects on the persistent fever were observed. On physical examination, palpable lymph nodes (1.0-1.5 cm) were detected in the occipital, axilla, and inguinal regions. Examination revealed abdominal distension and visible abdominal wall veins, without arrhythmia, edema of the eyelids and lower limbs, enlarged thyroid and clubbing fingers. The liver was palpable 3 cm under the costal margin, and hepatosplenomegaly was confirmed by Ultrasound. The patient was physical growth retardation. Notably, after 1 year of treatment follow-up, focal epileptic seizures manifested, characterized by stiffness and tremors in the left limbs. At times, these seizures were accompanied by bilateral eye deviation to the right and lasted for several seconds to 1 minute. Moreover, no interictal behavioral changes were observed in addition to the seizures. The laboratory findings indicated elevated inflammatory markers in the blood (WBC and differential leukocyte count, CRP, SSA, PCT, IL-6), decreased thyroid function, hypogammaglobulinemia, abnormal liver function, hypertriglyceridemia, and hypercalcemia (Table 1). When examined for hematological parameters, the patient showed elevated counts of leukocytes(18.71×109/L), lymphocytes (11.49×109/L), neutrophils (5.9×109/L), and basophil granulocyte (0.14×109/L). Blood platelet count (735.00×109/L), C-reactive protein (CRP) (33.77 mg/L), Procalcitonin (PCT) (0.69 ng/ml), and IL-6 (365.02 pg/ml) were also found to be increased. Blood biochemical examination revealed decreased thyroid function, with reduced levels of triiodothyronine (T3) (1.02nmol/L) and free triiodothyronine (FT3) (2.41pmol/L). Thyroid peroxidase antibodies (TPOAb) (16.95IU/ml) were elevated, while both thyroid-stimulating hormone (TSH) (2.18mIU/L) and thyroxine (T4) (13.65pmol/L) remained within normal ranges. Additionally, abnormal liver function was observed, with elevated alanine aminotransferase (ALT) (160 U/L), aspartate aminotransferase (AST) (140 U/L), and elevated blood triglycerides at 5.12 mmol/L. Simultaneously, in the fluid immunological examination, the patient was found to have hypogammaglobulinemia, with reduced levels of immunoglobulin G (IgG) and immunoglobulin M (IgM). The total IgE level was measured at 5.4 IU/ml, which falls within the normal range. Hypercalcemia was also observed, with a significant increase in blood calcium levels, elevated by 3.49 mmol/L. Bone scan showed no abnormalities. Surprisingly, the patient presented with recurrent fever; however, no definitive pathogens were identified. Blood culture, fecal culture, urine culture, Cryptococcus, and fungal cultures all yielded negative results. Both human cytomegalovirus (CMV) DNA and Epstein-Barr virus (EBV) DNA levels in the blood were below the detection limit. The bronchial lavage culture tested negative for bacteria as well as respiratory pathogens including influenza A, influenza B, and parainfluenza virus. Additionally, the interferon-γ release assay test showed a negative result. The patient’s immune-related antibodies were negative, which included anti-nuclear antibodies, anti-Sm antibodies, and antineutrophil cytoplasmic antibodies. Computerized tomography (CT) imaging revealed bilateral pneumonia with pulmonary interstitial involvement; bilateral cerebral deepening and widening with patchy high-density lesions in the right frontal lobe; esophageal dilatation observed along the entire length; abdominal CT revealed bilateral kidney enlargement. Head MRI indicated periventricular white matter and partial cortical FLAIR T2WI high signal changes, along with patchy T2WI high signal changes in the right frontal lobe. Abdominal ultrasound showed multiple enlarged lymph nodes in the neck, axilla, and groin regions; bilateral kidney enlargement; hepatosplenomegaly, with no focal lesions observed in the liver. The electroencephalogram (EEG) revealed a few sharp and slow wave discharges in the right hemisphere and a slow wave discharge focus in the right temporal central region. Trio sequencing reveal potential pathogenic mutations in patient Given the presence of a healthy sibling, we conducted an independent analysis of de novo mutations in both the affected patient and their healthy brother (Fig. 1 A). This analysis involved excluding any de novo mutations (DNMs) observed in the healthy sibling, allowing us to maximize the screening for potential pathogenic variations specific to the affected individual. By comparing the de novo mutations between the affected patient and their healthy brother, we aimed to identify and prioritize genetic alterations that could be associated with the observed medical condition. We proceeded to collect blood specimens from the entire family, including the patient, the patient's sibling, and both parents. Initially, we conducted high-quality Whole Genome Sequencing (WGS) on the four individuals within the family, including the affected child, the sibling, and both parents. The sequencing depth was approximately 25 \(\times\) , ensuring robust coverage, with 95% coverage across the genome. Moreover, the sequencing data met stringent quality standards, achieving a Q30 score of over 85%. Next, we conducted independent trio family analyses for both the affected patient and their genetically related brother (4 years old). The former is referred to as the "patient trio family" (p-trio), and the latter as the "patient’s healthy brother trio family" (h-trio). In the p-trio analysis, the specific mutation rate for the affected patient was calculated as 89.7% (Figures S1 A and S1B), indicating that the majority of detected DNMs were unique to the patient (calculated by dividing the total number of mutations found exclusively in the patient by the total number of mutations identified in the trio). Similarly, in the h-trio analysis, the specific mutation rate for the patient's brother was 90.36%, reinforcing that the majority of DNMs were specific to each individual (Fig. 1 B). Furthermore, we employed ClinVar database to identify potential pathogenic variants in the affected patient[ 29 ]. A total of 37 candidate pathogenic variants were identified in the patient, comprising 26 SNPs and 11 INDELs. These variants were associated with 23 different genes (Fig. 1 C). Interestingly, we identified a mutation, STAT3 c.508G > A, on the patient's STAT3 gene that has not been previously reported in the ClinVar database[ 29 ]. This mutation is speculated to be associated with a STAT3 GOF syndrome, aligning with the clinical presentation of the patient. Through Sanger sequencing, we confirmed that this mutation is indeed a de novo mutation specific to the affected child (Fig. 1 D), emphasizing its potential relevance to the observed clinical phenotype. STAT3 G508A is involved in regulating the expression of genes related to cell growth and differentiation Patients with STAT1 GOF syndrome mutations often exhibit diverse immune characteristics[ 43 – 45 ], commonly presenting with early-onset autoimmune manifestations[ 46 , 47 ]. Consequently, we conducted a detailed analysis of the blood transcriptomic profiles within the patient's family. Gene expression levels in the patient's blood were computed, and individuals within the family, including the healthy brother and parents, served as controls (Figures S2 A and S2B). Our findings revealed that the blood gene expression pattern in the patient closely resembled that of the father, exhibiting a higher similarity with the paternal profile compared to the brother (Figs. 2 A and S3). Compared to the healthy control group, the gene expression profile in the blood of the affected child revealed a predominance of downregulated genes (n = 74) and a minority of upregulated genes (n = 2) (Fig. 2 B and Table S2 ). Notably, five significantly downregulated genes ( p < 0.05) — phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta ( PIK3C2B ), SEC13 homolog, nuclear pore and COPII coat complex component pseudogene 1 ( SEC13P1 ), solute carrier family 16 member 3 ( SLC16A3 ), solute carrier family 2 member 3 pseudogene 4 ( SLC2A3P4 ), and protein tyrosine phosphatase non-receptor type 14 ( PTPN14 ) — play crucial roles in cellular growth and differentiation (Table S3). The downregulation of these genes, known to be involved in various aspects of cell development, provides a potential molecular basis for understanding the clinical phenotype of developmental delay observed in the affected child. Interestingly, these genes exhibited minimal expression in the affected child, while the healthy brother (the sibling without the condition) showed comparatively higher expression levels (Figs. 2 C- 2 G). DEGs shows abnormalities in neurological functions To explore the biological functions and signaling pathways influenced by differentially expressed genes, we conducted enrichment analysis on them including Molecular Function, Biological Process, Cell Component and KEGG pathway[ 48 – 50 ]. Phosphorylation is one of the significantly enriched functions identified in the enrichment analysis (GO:0035091, p = 0.013; GO:0032266, p = 0.003; GO:0016303, p = 0.018; GO:0035004, p = 0.021) (Fig. 3 A). Interestingly, we also found significant enrichment of differentially expressed genes in neurological functions, included regulation of dendritic spine development (GO:0061000, p = 0.022), neuronal differentiation, and synaptic development (GO:0014069, p = 0.021; GO:0032279, p = 0.023; GO:0099572, p = 0.025 and GO:0098984, p = 0.028) (Figs. 3 B and 3 C and Table S4). The patient also clinically presents with refractory epilepsy symptoms. Our analysis of KEGG signaling pathways is consistent with the aforementioned results, indicating that abnormalities in the nervous system are a characteristic feature of STAT3 G508A (Figure S4). IFN-γ is downregulated in the JAK-STAT pathway, while cyclin dependent kinase inhibitor 1A ( CDKN1A ) is upregulated. PIK3CA, AKT3, and mTOR are also affected (Fig. 3 D and Table S4). miR378c-mediated regulation of PTPN14 promotes the activation of inflammatory pathway Furthermore, to explore the regulatory mechanisms of differentially significant genes and their regulatory mechanisms, we conducted small RNA sequencing on corresponding samples[ 51 – 53 ], including the affected child, their sibling, and their parents. We quantified the expression levels of microRNAs (miRNAs) and performed Weighted Gene Co-expression Network Analysis (WGCNA)[ 37 , 54 ] (Figures S5A-S5D and Table S5). Our results revealed that miRNAs were divided into 14 modules (defined as densely interconnected clusters of genes) (Fig. 4 A and S6). The modules MElightgreen and MElightcyan exhibit the highest positive correlation, with correlation coefficients of 0.97 and 0.95, respectively ( p < 0.05). Conversely, the modules MEmagenta and MEred display the highest negative correlation, with correlation coefficients of -0.59 and − 0.54, respectively ( p -values not significant) (Fig. 4 B and S7). Interestingly, miRNAs not only regulate multiple target genes but also exhibit inverse regulatory relationships with mRNA expression[ 55 , 56 ]. The differentially expressed gene PTPN14 is associated with the activation of the PI3K/AKT/mTOR pathway. In our analysis, it is regulated by miR-548j-3p, miR-11401, miR-424-5p, miR-378c, and miR-452-5p, and exhibits inverse regulatory relationships with miR-424-5p, miR-378c, and miR-452-5p (Fig. 4 C and Table S6). Among them, miR-378c exhibits the highest expression level. Next, we analyzed the differential expression of miRNAs (|log 2 (Fold Change)| >= 2) involved in the regulation of different modules (Table S7). After excluding cases where expression is 0, we obtained 242 differentially expressed miRNAs. In the MElightgreen module, 31 miRNAs exhibited differential expression, annotated to 1 target gene kalirin RhoGEF kinase ( KALRN ). In the MEmagenta module, 14 miRNAs showed differential expression, regulating target genes including Sodium voltage-gated channel alpha subunit 2 ( SCN2A ), PTPN14 , glutaredoxin 5 ( GLRX5 ), and prohibitin 1 pseudogene 12 ( PHB1P12 ). STAT3 c.508G > A Enhances the Activity of Immune-Related Protein RT-qPCR was primarily utilized to evaluate the expression of four pro-inflammatory genes. The results indicate that, compared to the wild-type STAT3 plasmid, transfection with the STAT3 G508A mutant plasmid significantly increases the expression of several pro-inflammatory genes in response to LPS stimulation (Figs. 5 A- 5 D and S8). This suggests that the STAT3 c.508G > A mutation promotes inflammation. The Western blot primarily assessed the expression of STAT3-HA, p-STAT3, and the inflammatory factor IL-1beta (Fig. 5 E). The results indicate that the expression of STAT3-HA tagged protein confirms successful plasmid transfection into cells. Furthermore, transfection with the STAT3 c.508G > A plasmid significantly enhances the expression of p-STAT3 protein (Fig. 5 F), indicating that the STAT3 c.508G > A increases the activity of the STAT3 enzyme, thereby promoting inflammation. This effect is supported by the elevated expression of the active form of IL-1beta in the presence of overexpressed STAT3 and STAT3 c.508G > A mutant plasmids (Fig. 5 G), compared to the control group transfected with the Vector plasmid. Additionally, when comparing the overexpression of STAT3 c.508G > A mutant plasmid to that of the wild-type STAT3 plasmid, there is a further increase in the expression of IL-1beta protein. This suggests that the STAT3 c.508G > A has an enhanced pro-inflammatory effect. These results indicate that the STAT3 c.508G > A enhances STAT3 protein activity, thereby promoting immune cell inflammatory responses. JAK inhibitor and IL-6R antagonist helps to block inflammation and treat STAT3 -GOF syndrome Prior to the genetic test results, treatment with cephalosporins, glycopeptides, and carbapenems for anti-infective therapy was ineffective. Blood inflammatory markers, including white blood cell count, CRP, and ferritin, remained elevated. The patient received intravenous methylprednisolone (2mg/kg*d) for 5 days, resulting in the normalization of body temperature. However, temperature fluctuations persisted upon dose reduction, and inflammatory and immune markers remained elevated. After obtaining informed consent from the patient's family, we initiated treatment with IL-6R antagonist (tocilizumab) at a dose of 8mg/kg, administered intravenously every two weeks, in combination with JAK inhibitors (tofacitinib) at a dose of 0.4mg/kg*d orally for 3 months. The patient's inflammatory markers normalized, recurrent infections significantly reduced, abdominal distension and pulmonary imaging improved, and serum interleukin-6 levels gradually returned to normal. Due to the presence of hypogammaglobulinemia, the patient received regular intravenous immunoglobulin (IVIG) infusions, leading to an initial improvement in weight and height. After six months of treatment, we gradually extended the interval of tocilizumab to 3–4 weeks (Table 1). This demonstrates that our clinical combination targeted therapy for STAT3 -GOF has achieved good results, successfully controlling inflammation and immune responses in the patient. Discussion In this study, we identified a novel pathogenic mutation (c.508G > A, p.D170N) associated with STAT3 GOF syndrome[ 57 ], resulting in increased STAT3 phosphorylation and transcriptional activity. We proposed a mechanism where miR378c promotes the activation of inflammatory pathways by targeting PTPN14. Combining clinical data, we support the efficacy of JAK inhibitors and IL-6R antagonists in effectively suppressing inflammation and improving clinical symptoms in STAT3 -GOF patients. Therefore, early identification of newly emerging pathogenic mutations through whole-genome familial analysis and targeted treatment interventions can improve the prognosis of patients with STAT3 GOF syndrome. STAT3 GOF syndrome exhibits significant heterogeneity, where different mutation types and inheritance patterns may lead to varied clinical phenotypes and prognoses[ 17 , 58 ]. We found a rare case of STAT3 GOF syndrome with neurological involvement. Early manifestations in STAT3 GOF children may include gastrointestinal and endocrine system disorders, such as intestinal diseases, hypothyroidism, and growth hormone deficiency. As the disease progresses, additional clinical features may include non-malignant lymphoproliferation, interstitial pneumonia, and inflammatory arthritis. However, unlike previously reported cases of STAT3 GOF syndrome, the child we tracked exhibits a rare neurological clinical phenotype, and seizures were difficult to prevent with JAK inhibitors and IL-6R antagonists. This observation suggests potential genetic influences on the immune response and highlights distinctive transcriptional regulatory patterns associated with the STAT13 GOF mutation within the familial context. Additionally, there was no reversal of periventricular white matter and right frontal lobe brain lesions. To date, we have not elucidated the molecular mechanisms underlying the neurological abnormalities, but we speculate that they may be associated with STAT3 haploinsufficiency and immunoheterogeneity. This differs significantly from a previously reported case of STAT3 GOF syndrome primarily presenting with bilateral facial muscle weakness due to myositis and muscle atrophy[ 59 ]. STAT3 , whether excessively activated or inactivated, leads to human inflammatory or immune-related diseases. The precise underlying mechanisms remain incompletely elucidated. STAT3 orchestrates pro-inflammatory signal transduction by activating genes associated with inflammation, such as IL-6, IL-17, and TNF-α[ 60 ]. This corresponds with our research findings that STAT3 G508A mutation has significant pro-inflammatory effects. Experimental results from transfecting human astrocytes with plasmids also revealed elevated expression of pro-inflammatory factors IL-6, IL-18, IL-1β, and TNF-α. Specifically, STAT3 can bind to specific DNA sequences in the promoters of pro-inflammatory genes, augmenting their transcription and expression[ 61 ]. These genes encode cytokines or enzymes that foster inflammatory responses by stimulating immune cells or generating prostaglandins. Furthermore, STAT3 induces the participation of helper T cells in inflammatory reactions. For instance, Th17 cells express pro-inflammatory cytokines like IL-17, IL-21, and IL-22, thereby enhancing the functional efficacy of Th17 cells and promoting inflammation[ 62 ]. Current research primarily focuses on STAT3 GOF syndrome leading to increased Th17 differentiation and reduced function of Treg cells[ 9 ]. A previous study suggested that STAT3 gene mutations result in heightened activity, suppressing STAT5 phosphorylation responses and impairing Treg cell function[ 8 , 9 ]. Additionally, a study using STAT3 mutant mice revealed phenomena such as T cell proliferation, activation, and Th1 polarization, while the quantity, phenotype, and function of Tregs remained essentially normal[ 63 ]. Similarly, an international cohort study on STAT3 found that the abundance of Tregs and Th17 cells does not predict the severity of clinical phenotypes in patients[ 59 ]. Our research harnessed transcriptomic data from patients with STAT3 GOF syndrome to analyze miRNA-mediated regulation of downstream JAK-STAT targets. Notably, the differentially expressed gene PTPN14, which exhibits a close association with the highly expressed miR-378c, holds potential significance in the context of downstream inflammatory signaling pathways. However, further evidence is required to definitively establish this connection. Perhaps delving into the downstream mechanisms activated by STAT3 mutations could unlock the secrets behind the observed phenotypic variations. Moreover, comprehensive longitudinal studies are imperative for unraveling the pathogenesis of STAT3 GOF syndrome in the future. Given the heterogeneity of symptoms in STAT3 -GOF syndrome, we attempted to associate its phenotype with genotype. A group of significantly downregulated genes in the STAT3 G508A mutant group, including PIK3C2B, SEC13P1 , SLC16A3 , SLC2A3P4 , and PTPN14 , may exacerbate aberrant signal transduction in STAT3 -GOF syndrome. Targeting the functions of these genes could explore the development of related therapeutic targets and drugs. For example, inhibitors targeting the PIK3C2B , functional restorers for the SEC13P1 gene, and modulators for the SLC16A3 , SLC2A3P4 , and PTPN14 genes could potentially serve as novel drugs for the treatment of STAT3 -GOF syndrome, thereby saving patients' lives. Declarations Supplementary Information The online version contains supplementary material available. DNA and RNA sequencing data have been deposited at The Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/) and are publicly available as of the date of publication. Accession numbers is PRJCA025000. Authors contributions Conceptualization: Bo Zhao, Ji-Yu Chen.Formal analysis: Ji-Yu Chen, Bo Zhao, Zhu Zhou. Methodology: Yan-Fang Li, Mi-Feng Yang.Resources: Xue-Mei Jiang, Xin Bi. Software: Ji-Yu Chen. Supervision: Bo Zhao, Ji-Yu Chen. Validation: Xin Bi. Project administration: Yan-Fang Li. Visualization: Xue-Mei Jiang . Writing – original draft: Ji-Yu Chen. Writing – review & editing: Yan-Fang Li, Zhu Zhou, Bo Zhao. Funding The work was supported by the Kunming Health Science and Technology Talent Project-10 Projects of China (2023-SW（GUIDE）-07); Yunnan Clinical Medical Research Center Chronic Kidney Disease Clinical Medical Research Center (202102AA100060); "Spring City Plan" High-Level Talent Training - Spring City Famous Medical Special Project (C202112012). Availability of data and materials The data are available from the corresponding author upon reasonable request. Competing interests The authors declare no competing interests. Ethics approval and consent to participate The study was approved by the Ethics Committee at the Kunming Children’s Hospital, Yunnan, China (2023-03-336-K01). Written informed consent has been obtained from the legal guardian of the parent for publication of this case details and accompanying images. Consent for publication Not applicable. References Akira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T: Molecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway . Cell 1994, 77 (1):63-71. O'Shea JJ, Holland SM, Staudt LM: JAKs and STATs in immunity, immunodeficiency, and cancer . New england journal of medicine 2013, 368 (2):161-170. Yuan ZL, Guan YJ, Wang L, Wei W, Kane AB, Chin YE: Central role of the threonine residue within the p+1 loop of receptor tyrosine kinase in STAT3 constitutive phosphorylation in metastatic cancer cells . 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Schmitt EG, Toth KA, Risma SI, Kolicheski A, Saucier N, Berríos RJF, Greenberg ZJ, Leiding JW, Bleesing JJ, Thatayatikom A et al : A human STAT3 gain-of-function variant confers T cell dysregulation without predominant Treg dysfunction in mice . JCI Insight 2022, 7 (21):null. Table 1 Table 1. After one year of continuous treatment, the prognosis of the patient is summarized. PNE pneumonia, ILD interstitial pneumonia, BEK bilaterally enlarged kidneys. CEF Cefoperazone, ACY acyclovir, MER meropenem, VAN vancomycin, IVIG human immunoglobulin for intravenous injection, MP methylprednisolone, TCZ tocilizumab, JAK jak inhibitor (Tofacitinib Citrate Sustained-release Tablets), Biw biweekly, TIW Once every 3 weeks, FIW Once every four weeks, LEV levetiracetam, LAC lacosamide, MO month-old. PNE pneumonia, ILD interstitial pneumonia, BEK bilaterally enlarged kidneys. CEF Cefoperazone, ACY acyclovir, MER meropenem, VAN vancomycin, IVIG human immunoglobulin for intravenous injection, MP methylprednisolone, TCZ tocilizumab, JAK jak inhibitor (Tofacitinib Citrate Sustained-release Tablets), Biw biweekly, TIW Once every 3 weeks, FIW Once every four weeks, LEV levetiracetam, LAC lacosamide, MO month-old. Additional Declarations No competing interests reported. Supplementary Files SupplementaryFigues1.docx SupplementaryTables2.xlsx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4181172","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":290653745,"identity":"656336a8-7271-44a0-8415-f7b75c18fed2","order_by":0,"name":"Jiyu Chen","email":"","orcid":"","institution":"Kunming Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Jiyu","middleName":"","lastName":"Chen","suffix":""},{"id":290653746,"identity":"6efafb55-2ab8-4717-a15b-d560c0ba7125","order_by":1,"name":"Yan-Fang Li","email":"","orcid":"","institution":"Kunming Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Yan-Fang","middleName":"","lastName":"Li","suffix":""},{"id":290653747,"identity":"f6cd98a5-a596-46dc-abc7-dbaaa7bc4ad2","order_by":2,"name":"Xue-Mei Jiang","email":"","orcid":"","institution":"Kunming Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xue-Mei","middleName":"","lastName":"Jiang","suffix":""},{"id":290653748,"identity":"4b9718f4-4c75-4759-8e1d-75cf71b612cd","order_by":3,"name":"Xin Bi","email":"","orcid":"","institution":"Kunming Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Bi","suffix":""},{"id":290653749,"identity":"283e18b1-e28f-4182-8e82-a69814fafe4b","order_by":4,"name":"Mi-Feng Yang","email":"","orcid":"","institution":"Kunming Children's Hospital","correspondingAuthor":false,"prefix":"","firstName":"Mi-Feng","middleName":"","lastName":"Yang","suffix":""},{"id":290653750,"identity":"cad23b49-72b9-4629-a8ac-16a8fb7f6c2f","order_by":5,"name":"Zhu Zhou","email":"","orcid":"","institution":"Yunnan Clinical Medical Research Center of Chronic Kidney Disease","correspondingAuthor":false,"prefix":"","firstName":"Zhu","middleName":"","lastName":"Zhou","suffix":""},{"id":290653751,"identity":"c4baae25-5e34-418a-a635-288f6cbd9815","order_by":6,"name":"Bo Zhao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAv0lEQVRIiWNgGAWjYDACCTYIzc/MfPgBCVoSGBgk29nSDEjTYnCeR0GCKB3ys9tSN3z8cThx82EeBgOGGptogloY5xw7dnNGwuHEbYd5DzxgOJaW20BIC7NEetttHrAWvgQDxobDhLWwgbT8AWrZ3MxjIEGUFh6JtGO3GYBaNjATq0VC5ljazZ60dOMZh4GBnECMX4AhZnbjh421bH//4cMPPtTYENYCBc2OYJUJRCoHgTp7EhSPglEwCkbBSAMAC8ZDIm+aRigAAAAASUVORK5CYII=","orcid":"","institution":"Kunming Children's Hospital","correspondingAuthor":true,"prefix":"","firstName":"Bo","middleName":"","lastName":"Zhao","suffix":""}],"badges":[],"createdAt":"2024-03-28 09:35:46","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4181172/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4181172/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54931986,"identity":"86046b4a-406e-445a-8ef9-5276beca8fe8","added_by":"auto","created_at":"2024-04-18 18:52:37","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":584606,"visible":true,"origin":"","legend":"\u003cp\u003eConfirmation of patient's de novo mutation (DNMs). \u003cstrong\u003e(A)\u003c/strong\u003e Three-generation pedigree of aptient’s family. Squares represent males, circles represent females, three generations are indicated by Greek letters, and individuals with potential therapeutic relevance are marked in black. The red dashed boxes represent the samples subjected to high-throughput sequencing in this study.\u003cstrong\u003e (B)\u003c/strong\u003e SNP and INDEL statistics for the patient and patient's brother. DNMs, de novo mutations; specific-DNMs, Individual-specific de novo mutations. CLNHGVS, variants annotated through ClinVar database. \u003cstrong\u003e(C)\u003c/strong\u003e potential pathogenic de novo mutations and associated genes in this patient. The horizontal bars represent chromosomes, with a window size of 1×10\u003csup\u003e6\u003c/sup\u003ebp. Within each window, the density of DNMs is depicted using a heatmap. Red gene indicates novel pathogenic DNMs discovered in this study.\u003cstrong\u003e (D) \u003c/strong\u003eSanger sequencing was employed to validate the \u003cem\u003eSTAT3\u003c/em\u003e mutation site.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/8eaa1d963a3aa5602b72d5e9.png"},{"id":54931985,"identity":"dce6dcc7-08a3-4321-8dba-e7482b2f953f","added_by":"auto","created_at":"2024-04-18 18:52:37","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":325428,"visible":true,"origin":"","legend":"\u003cp\u003eDifferential gene expression profiles. \u003cstrong\u003e(A)\u003c/strong\u003e The expression heatmap of the top 100 differentially expressed genes. \u003cstrong\u003e(B)\u003c/strong\u003e The volcano plot of differentially expressed genes. The dashed line represents the threshold range for fold change greater than 2. Genes from the JAK-STAT signaling pathway are highlighted in the plot. \u003cstrong\u003e(C-G) \u003c/strong\u003eThe expression levels of genes \u003cem\u003ePIK3C2B\u003c/em\u003e (C), \u003cem\u003eSLC16A3\u003c/em\u003e(D), \u003cem\u003ePTPN14\u003c/em\u003e (E), \u003cem\u003eSEC13P1\u003c/em\u003e (F) and \u003cem\u003eSLC2A3P4\u003c/em\u003e (G) from the JAK-STAT signaling pathway in different samples.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/c64f819c73b0f93b27e492e0.png"},{"id":54931989,"identity":"6de2909c-1a6e-46f3-ac7f-3e6160d3f3b3","added_by":"auto","created_at":"2024-04-18 18:52:37","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1462162,"visible":true,"origin":"","legend":"\u003cp\u003eResults of differential gene enrichment analysis. \u003cstrong\u003e(A-C) \u003c/strong\u003eGene Ontology enrichment analysis results, included Molecular Function (A), Biological Process (B) and Cell Component.\u003c/p\u003e\n\u003cp\u003eThe size of the dots represents the number of genes enriched, while the color gradient from blue to red indicates adjusted p-values from large to small.\u003cstrong\u003e (D) \u003c/strong\u003eTranscriptome differential expression of genes in the JAK-STAT3 signaling pathway. Differential gene expression has been shown in heatmap and the linkage relationship between different genes is indicated by arrows. The color gradient within each rectangle represents the log2(FC) values of the gene, ranging from red to blue indicating larger to smaller values, respectively.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/2a5c23f9a8dbff9e02f871e4.png"},{"id":54932455,"identity":"19383c34-f1e4-4af8-89df-ea7d287e643e","added_by":"auto","created_at":"2024-04-18 19:00:37","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":800150,"visible":true,"origin":"","legend":"\u003cp\u003eWeighted correlation network analysis (WGCNA) results. \u003cstrong\u003e(A)\u003c/strong\u003e Gene dendrogram and model colors. \u003cstrong\u003e(B)\u003c/strong\u003e Heatmap plot with correlation values of the adjacencies in the eigengene network including the trait weight. Significance values in parentheses. \u003cstrong\u003e(C) \u003c/strong\u003eRegulatory networks of differentially expressed microRNA (miRNAs). miRNA target genes are connected by lines. \u003cstrong\u003e(D)\u003c/strong\u003eNetwork connections in light green module.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/166431618cd149e88ec683f9.png"},{"id":54931990,"identity":"4e696bee-6667-4ee3-ad29-ec6769829909","added_by":"auto","created_at":"2024-04-18 18:52:38","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":607671,"visible":true,"origin":"","legend":"\u003cp\u003eExperimental validation of \u003cem\u003eSTAT3c\u003c/em\u003e.508G\u0026gt;A function. \u003cstrong\u003e(A-D)\u003c/strong\u003eExpression of pro-inflammatory factors IL-6 (A), IL-18 (B), IL-1β (C) and TNF-α (D) in STAT3c.508G\u0026gt;A transfected plasmids. \u003cstrong\u003e(E) \u003c/strong\u003eSTAT3-HA, p-STAT3, and inflammatory factor IL-1β protein expression. \u003cstrong\u003e(F-G)\u003c/strong\u003e p-STAT3 (F) and IL-1β (G) relative protein level. The bars of different colors represent different treatment conditions: blue for empty vector, red for transfection with wild-type STAT3, and green for transfection with mutant STAT3. * indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.05, ** indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.01, *** indicates \u003cem\u003ep\u003c/em\u003e \u0026lt; 0.001.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/6b674fe5956e6454ab2d8cc9.png"},{"id":58300847,"identity":"d1774ce9-38a3-4473-b704-6394cfc9c417","added_by":"auto","created_at":"2024-06-13 16:05:57","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5928311,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/faacdb40-67aa-4fe4-9098-88baa7659d8e.pdf"},{"id":54931988,"identity":"4139a823-265b-484f-924e-46b301faa561","added_by":"auto","created_at":"2024-04-18 18:52:37","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":1495673,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryFigues1.docx","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/9e2c4e494809a86546defc48.docx"},{"id":54932456,"identity":"57d59405-5534-46fb-ba58-f8f1db7c92c6","added_by":"auto","created_at":"2024-04-18 19:00:38","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":1967924,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementaryTables2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-4181172/v1/6fd7dea6f25694ec0df0f15d.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"De novo mutations promote inflammation in children with STAT3 gain-of-function syndrome by affecting IL-1β expression","fulltext":[{"header":"Introduction","content":"\u003cp\u003eSignal transducer and activator of transcription-3 (STAT3), a crucial gene involved in cell survival, proliferation, differentiation, and metabolism, plays a pivotal role in immune cell differentiation and inflammation control[\u003cspan additionalcitationids=\"CR2 CR3\" citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Intriguingly, mutations in the same STAT3 gene can give rise to different diseases and clinical phenotypes. Autosomal dominant inherited mutations in \u003cem\u003eSTAT3\u003c/em\u003e can result in \u003cem\u003eSTAT3\u003c/em\u003e Loss-of-Function (LOF) variants leading to conditions such as Hyper-IgE syndrome (HIES) or Job syndrome (OMIM 147060)[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Somatic mutations in STAT3 within somatic cells can lead to Gain-of-Function (GOF) variants, contributing to certain malignant tumors[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Meanwhile, germline mutations in reproductive cells can cause \u003cem\u003eSTAT3\u003c/em\u003e GOF, associated with autoimmune disorders[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eTraditional genetic approaches, such as linkage analysis and genome-wide association studies, have traditionally focused on inherited genetic variations[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Recent findings indicate that disruptive de novo mutations in one or two genes are major contributors to many rare genetic syndromes. Examples include Kabuki syndrome[\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e], Schinzel-Giedion syndrome[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], Bohring-Opitz syndrome[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], Baraitser-Winter syndrome[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], and Coffin-Siris syndrome[\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. However, the clinical presentation of \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome is highly diverse, with distinct clinical features observed in different patients[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Therefore, the association between specific de novo mutations (DNMs) identified through whole-genome sequencing and \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome holds practical significance for the diagnosis and intervention of this syndrome.\u003c/p\u003e \u003cp\u003eIn this study, we established joint analyses for multiple trios within a multifetal family to minimize the impact of non-pathogenic variants arising from similar environments. Leveraging Whole Genome Sequencing (WGS) data, we curated a dataset specific to non-inherited variations in the affected child. By comparing the immune cell gene expression profiles of healthy family members, we identified miRNA-mediated regulation of \u003cem\u003eSTAT3\u003c/em\u003e, potentially leading to aberrant inflammatory responses associated with the autoimmune phenotype observed in the affected child. This research not only provides evidence for new pathogenic loci in \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome but also significantly advances our understanding of the relationship between genetic variations and observed clinical manifestations.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eSample collection\u003c/h2\u003e \u003cp\u003e All sample collections in this study followed the ethical standards of the Kunming Children's Hospital Ethics Committee (Approval Number 2023-03-336-K01). With the informed consent of the pediatric patient and their family, blood specimens were collected from the patient, the patient's parents, and the patient's sibling, and stored temporarily in EDTA anticoagulant tubes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eDNA and RNA extraction\u003c/h2\u003e \u003cp\u003eCollect a whole blood specimen of 300 \u0026micro;L, and extract DNA and total RNA following the instructions provided for TIANamp Genomic DNA Kit (DP304, TIANGEN) and RNAprep\u003c/p\u003e \u003cp\u003ePure Hi-Blood Kit (DP443, TIANGEN), respectively. Assess nucleic acid integrity using Agilent 5400, and quantitate the samples with NanoDrop 2000.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eWhole-genome sequencing (WGS)\u003c/h2\u003e \u003cp\u003eThe DNA sample was used as input material for the DNA library preparations.\u003c/p\u003e \u003cp\u003eSequencing library was generated and index codes were added to each sample.\u003c/p\u003e \u003cp\u003eBriefly, genomic DNA sample was fragmented by sonication to a size of 350 bp. Then\u003c/p\u003e \u003cp\u003eDNA fragments were end-polished, A-tailed, and ligated with the full-length adapter for\u003c/p\u003e \u003cp\u003eIllumina sequencing, followed by further PCR amplification. After PCR products were\u003c/p\u003e \u003cp\u003epurified. Subsequently, library quality was assessed and quantified by qPCR. The qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy, according to effective library concentration and data amount required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003elncRNA and miRNA sequencing\u003c/h2\u003e \u003cp\u003eTotal RNA was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB E7420) following manufacturer\u0026rsquo;s recommendations and index codes were added to attribute sequences to each sample. rRNA is removed from the total RNA samples. After adenylation of 3\u0026rsquo; ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. To select cDNA fragments of preferentially 370\u0026thinsp;~\u0026thinsp;420 bp in length, the library fragments were purified with AMPure XP system (Beverly, USA). Then 3 \u0026micro;L USER Enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37\u0026deg;C for 15 min followed by 5 min at 95\u0026deg;C before PCR. Then PCR was performed with Phusion High-Fidelity DNA polymerase, Universal PCR primers and Index (X) Primer. At last, PCR products were purified (AMPure XP system) library quality was assessed on the Agilent 5400 system༈Agilent, USA༉ and quantified by QPCR (1.5 nM). The Qualified libraries were pooled and sequenced on Illumina platforms with PE150 strategy in Biolinker Technology (Kunming) Co., Ltd., according to effective library concentration and data amount required.\u003c/p\u003e \u003cp\u003eSmall RNA Sequencing libraries were generated using NEB Next\u0026reg; Multiplex Small RNA Library Prep Set for Illumina\u0026reg; (NEB E7300L). Briefly, 3\u0026rsquo; and 5\u0026rsquo; adaptors were ligated to 3\u0026rsquo; and 5\u0026rsquo; end of small RNA, respectively. Then the first strand cDNA was synthesized after hybridazition with reverse transcription primer. The double-stranded cDNA library was generated through PCR enrichment. After purification and size selection, libraries with insertions between18\u0026thinsp;~\u0026thinsp;40 bp were ready for sequencing on Illumina sequencing with SE50. Subsequently, library quality was assessed on the Agilent 5400 system (Agilent, USA) and quantified by QPCR༈1.5nM༉. The Qualified libraries were pooled and sequenced on Illumina platforms with SE50 strategy in Biolinker Technology (Kunming) Co., Ltd., according to effective library concentration and data amount required.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eAlignment and post alignment processing\u003c/h2\u003e \u003cp\u003eAfter obtaining raw sequencing data, we employed the fastp tool[\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e] for data filtering and cleaning, removing adapter sequences and low-quality reads. The sequencing results for all three types of libraries were processed using parameters \u0026ldquo;-g -q 5 -u 50 -n 15\u0026rdquo;.\u003c/p\u003e \u003cp\u003eThe reference genome used for alignment was hg38(GCF_000001405)[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. For WGS data, we utilized the BWA mem algorithm for alignment[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], employing parameters \u0026ldquo;-M -a\u0026rdquo;. Subsequently, the resulting BAM files underwent sorting and index construction using samtools[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. For the lncRNA sequencing data, alignment was performed using hisat2 with default parameters[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e]. Following alignment, the resultant BAM files were subjected to sorting and index establishment using samtools. For small RNA sequencing data, we employed Bowtie[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e] for alignment against three different references: the reference genome, all miRNA hairpins, and all mature miRNA sequences[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The parameters used for alignment were -v 0 -m 5 -a -q --strata --best. Following alignment, the resulting SAM files were sorted using the samtools sort command and then converted to BAM format, with subsequent index construction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eSNP/INDEL calling and filtering\u003c/h2\u003e \u003cp\u003eFor the SNP/INDEL analysis of WGS data, we followed a Genome Analysis Toolkit (GATK) comprehensive workflow[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Initially, we employed GATK to mark and remove duplicate reads using the MarkDuplicates tool. The sequencing depth was calculated using the samtools depth command. Subsequently, BaseRecalibrator was applied for base quality score recalibration. Known SNP positions for recalibration were sourced from Homo_sapiens_assembly38.dbsnp138.vcf, while INDEL positions were obtained from Mills_and_1000G_gold_standard.indels.hg38.vcf. High-confidence SNP positions from the 1000 Genomes Project were extracted from 1000G_phase1.snps.high_confidence.hg38.vcf.gz.\u003c/p\u003e \u003cp\u003eAfter the first round of Base Quality Score Recalibration (BQSR), the recalibrated BAM files were subjected to variant calling using HaplotypeCaller in GVCF (Genomic Variant Call Format) mode, calling variants per-sample.\u003c/p\u003e \u003cp\u003eAfter merging all the GVCF files using GATK's CombineGVCFs, we performed joint genotyping using GenotypeGVCFs. Subsequently, we constructed a recalibration model and assessed variant quality scores using VariantRecalibrator (VQSR) and ApplyVQSR. This process enabled us to filter variants based on their quality.\u003c/p\u003e \u003cp\u003eFor SNP recalibration, we applied prior probability correction using sites from hapmap, omni, 1000G, and dbsnp. For INDEL recalibration, we used mills as a reference, incorporating parameters such as \"-an QD -an MQ -an DP -an MQRankSum -an ReadPosRankSum -an FS -an SOR\" for fine-tuning the recalibration model.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eDNM calling and filtering\u003c/h2\u003e \u003cp\u003eWe conducted DNMs analysis using triodenovo for both the affected child and their sibling. The input files consisted of VQSR-calibrated VCF files. Subsequently, we filtered out any mutation sites identified in the sibling, retaining only those found exclusively in the affected child.\u003c/p\u003e \u003cp\u003e The retained variants included single-nucleotide positions with only two alleles, a QUAL score of at least 30, and mutations where the child was heterozygous while the parents were homozygous reference. Furthermore, we specifically kept positions where the child was heterozygous, and the PL (Phred-scaled likelihoods) for heterozygous genotype was zero, while the minimum PL for the other two genotypes was 30 (i.e., the genotype likelihoods, defined as P(R|G), where R represents the aligned base, and G is the potential genotype, for the heterozygous mutation\u0026thinsp;\u0026gt;\u0026thinsp;1000 times greater than the other two genotypes).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eDNM annotation and validation\u003c/h2\u003e \u003cp\u003eWe performed mutation filtering using SnpSift[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e] filter with the criteria \"(( DP\u0026thinsp;\u0026gt;\u0026thinsp;30 ) \u0026amp;\u0026amp; ( GEN.DQ\u0026thinsp;\u0026gt;\u0026thinsp;5 ) \u0026amp;\u0026amp; ( GEN.DP\u0026thinsp;\u0026gt;\u0026thinsp;10 ))\". This filtering strategy retained variants with a depth (DP) greater than 30, a quality score (DQ) greater than 5 for each genotype, and a genotype depth (DP) greater than 10.\u003c/p\u003e \u003cp\u003eAdditionally, we utilized SnpSift annotate to annotate the variants with existing information from the NCBI ClinVar database[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This annotation step aimed to provide additional insights into the clinical significance and known associations of the identified variants.\u003c/p\u003e \u003cp\u003eFor newly discovered variants that were not annotated in the National Center for Biotechnology Information (NCBI) ClinVar database, we conducted PCR amplification of the target regions and subsequently validated the correctness of the variant positions through Sanger sequencing[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e]. This experimental validation step aimed to confirm the presence of the identified variants and ensure the accuracy of the genomic alterations identified in our analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003eDifferential gene analysis\u003c/h2\u003e \u003cp\u003eWe quantified gene-level expression for each sample using htseq-count[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e], utilizing a gene annotation file sourced from Genecode version 44[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Subsequently, we aggregated the expression values for all samples into an expression matrix. Differential gene expression analysis was conducted using DESeq2[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], with the following parameters: fold change threshold of 2, a \u003cem\u003ep\u003c/em\u003e-value cutoff of 0.05, a minimum count threshold of 10, and the normalization method set to VST (variance stabilizing transformation). This approach allowed us to identify genes exhibiting significant differences in expression between conditions while considering fold changes, statistical significance, and data quality.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003emiRNA target gene analysis\u003c/h2\u003e \u003cp\u003eWe obtained the mature human miRNA sequences from the miRBase database[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e] and concurrently extracted the 3'UTR sequences of differentially expressed genes. Utilizing the miranda algorithm with default parameters[\u003cspan additionalcitationids=\"CR35\" citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e], we predicted the binding sites for miRNA-mRNA interactions. The analysis was comprehensive, outputting all predicted binding sites to provide a thorough examination of potential miRNA-mRNA interactions associated with the differentially expressed genes.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eWeighted correlation network analysis (WGCNA)\u003c/h2\u003e \u003cp\u003eWGCNA package of R software was used to reveal the correlation between genes[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Genes were filtered by MAD (median and median absolute deviation). We set the power of β based on the point of the plateau phase. The minimum number of module genes was set at 30. The hierarchical clustering dendrogram summarized the Gene modules with different colors. Heatmap and topological overlap matrix (TOM) plot were used to visualize. We calculated the correlation between modules and traits. The corresponding module gene information was extracted for further analysis. Cytoscape was to use to visualize gene networks[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003eEnrichment analysis\u003c/h2\u003e \u003cp\u003eWe performed enrichment analysis on differentially expressed genes (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) using the R packages DOSE[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], topGO[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e], and clusterProfiler[\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. The analysis involved the exploration of enriched terms within biological ontologies. The specific parameters were set to output all terms. A significance threshold of \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered for determining enriched results. We employed ggplot2[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e] for visualizing the enrichment results.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003ePlasmid construction and transfection\u003c/h2\u003e \u003cp\u003eHuman microglial cells (HMC3) were transfected with \u003cem\u003eSTAT3\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003ec.508G\u0026thinsp;\u0026gt;\u0026thinsp;A plasmids in 6-well and 12-well plates. After a 36-hour transfection, cells in the 6-well plate were treated with 500ng/ml LPS for 6 hours for protein extraction and subsequent western blot analysis.\u003c/p\u003e \u003cp\u003eMeanwhile, cells in the 12-well plate, subjected to a 3-hour LPS treatment after transfection, were harvested for RNA extraction and RT-qPCR analysis to assess gene expression changes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec17\" class=\"Section2\"\u003e \u003ch2\u003eCase history\u003c/h2\u003e \u003cp\u003eThe proband was a 1-year-old boy, the third child of healthy non-consanguineous parents of Chinese ancestry (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA and Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). His growth parameters were abnormal, loss of subcutaneous fat, body weight of 6.3 kg (\u0026lt;-3SD), and height of 67 cm (\u0026lt;-3SD). He presented to a community hospital with recurrent fever and aggravated abdominal distension, all of 2 months duration. These digestive symptoms include increased abdominal distention, bloating, difficulty with defecation, appetite reduction, and an abdominal circumference of 46 cm, after 1 week of recurrent fever. Additionally, parents noticed dry, brown, and plate-like scales mainly on both ankles and the dorsa of both feet. A review of systems was negative for nausea, vomiting, loose stools, or a history of other joint pain or swelling. No family history was identified within 3 generations, including his two brothers, aged 4 and 6. Before admission, Cefperazone-Sulbactam was given against infection for 2 weeks, and meropenem\u0026thinsp;+\u0026thinsp;vancomycin for 1 week. However, no significant effects on the persistent fever were observed.\u003c/p\u003e \u003cp\u003eOn physical examination, palpable lymph nodes (1.0-1.5 cm) were detected in the occipital, axilla, and inguinal regions. Examination revealed abdominal distension and visible abdominal wall veins, without arrhythmia, edema of the eyelids and lower limbs, enlarged thyroid and clubbing fingers. The liver was palpable 3 cm under the costal margin, and hepatosplenomegaly was confirmed by Ultrasound. The patient was physical growth retardation. Notably, after 1 year of treatment follow-up, focal epileptic seizures manifested, characterized by stiffness and tremors in the left limbs. At times, these seizures were accompanied by bilateral eye deviation to the right and lasted for several seconds to 1 minute. Moreover, no interictal behavioral changes were observed in addition to the seizures.\u003c/p\u003e \u003cp\u003eThe laboratory findings indicated elevated inflammatory markers in the blood (WBC and differential leukocyte count, CRP, SSA, PCT, IL-6), decreased thyroid function, hypogammaglobulinemia, abnormal liver function, hypertriglyceridemia, and hypercalcemia (Table\u0026nbsp;1). When examined for hematological parameters, the patient showed elevated counts of leukocytes(18.71\u0026times;109/L), lymphocytes (11.49\u0026times;109/L), neutrophils (5.9\u0026times;109/L), and basophil granulocyte (0.14\u0026times;109/L). Blood platelet count (735.00\u0026times;109/L), C-reactive protein (CRP) (33.77 mg/L), Procalcitonin (PCT) (0.69 ng/ml), and IL-6 (365.02 pg/ml) were also found to be increased. Blood biochemical examination revealed decreased thyroid function, with reduced levels of triiodothyronine (T3) (1.02nmol/L) and free triiodothyronine (FT3) (2.41pmol/L). Thyroid peroxidase antibodies (TPOAb) (16.95IU/ml) were elevated, while both thyroid-stimulating hormone (TSH) (2.18mIU/L) and thyroxine (T4) (13.65pmol/L) remained within normal ranges. Additionally, abnormal liver function was observed, with elevated alanine aminotransferase (ALT) (160 U/L), aspartate aminotransferase (AST) (140 U/L), and elevated blood triglycerides at 5.12 mmol/L. Simultaneously, in the fluid immunological examination, the patient was found to have hypogammaglobulinemia, with reduced levels of immunoglobulin G (IgG) and immunoglobulin M (IgM). The total IgE level was measured at 5.4 IU/ml, which falls within the normal range. Hypercalcemia was also observed, with a significant increase in blood calcium levels, elevated by 3.49 mmol/L. Bone scan showed no abnormalities.\u003c/p\u003e \u003cp\u003eSurprisingly, the patient presented with recurrent fever; however, no definitive pathogens were identified. Blood culture, fecal culture, urine culture, Cryptococcus, and fungal cultures all yielded negative results. Both human cytomegalovirus (CMV) DNA and Epstein-Barr virus (EBV) DNA levels in the blood were below the detection limit. The bronchial lavage culture tested negative for bacteria as well as respiratory pathogens including influenza A, influenza B, and parainfluenza virus. Additionally, the interferon-γ release assay test showed a negative result. The patient\u0026rsquo;s immune-related antibodies were negative, which included anti-nuclear antibodies, anti-Sm antibodies, and antineutrophil cytoplasmic antibodies. Computerized tomography (CT) imaging revealed bilateral pneumonia with pulmonary interstitial involvement; bilateral cerebral deepening and widening with patchy high-density lesions in the right frontal lobe; esophageal dilatation observed along the entire length; abdominal CT revealed bilateral kidney enlargement. Head MRI indicated periventricular white matter and partial cortical FLAIR T2WI high signal changes, along with patchy T2WI high signal changes in the right frontal lobe. Abdominal ultrasound showed multiple enlarged lymph nodes in the neck, axilla, and groin regions; bilateral kidney enlargement; hepatosplenomegaly, with no focal lesions observed in the liver. The electroencephalogram (EEG) revealed a few sharp and slow wave discharges in the right hemisphere and a slow wave discharge focus in the right temporal central region.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003eTrio sequencing reveal potential pathogenic mutations in patient\u003c/h2\u003e \u003cp\u003eGiven the presence of a healthy sibling, we conducted an independent analysis of de novo mutations in both the affected patient and their healthy brother (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). This analysis involved excluding any de novo mutations (DNMs) observed in the healthy sibling, allowing us to maximize the screening for potential pathogenic variations specific to the affected individual. By comparing the de novo mutations between the affected patient and their healthy brother, we aimed to identify and prioritize genetic alterations that could be associated with the observed medical condition. We proceeded to collect blood specimens from the entire family, including the patient, the patient's sibling, and both parents. Initially, we conducted high-quality Whole Genome Sequencing (WGS) on the four individuals within the family, including the affected child, the sibling, and both parents. The sequencing depth was approximately 25\u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\times\\)\u003c/span\u003e\u003c/span\u003e, ensuring robust coverage, with 95% coverage across the genome. Moreover, the sequencing data met stringent quality standards, achieving a Q30 score of over 85%.\u003c/p\u003e \u003cp\u003eNext, we conducted independent trio family analyses for both the affected patient and their genetically related brother (4 years old). The former is referred to as the \"patient trio family\" (p-trio), and the latter as the \"patient\u0026rsquo;s healthy brother trio family\" (h-trio). In the p-trio analysis, the specific mutation rate for the affected patient was calculated as 89.7% (Figures \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003eA and S1B), indicating that the majority of detected DNMs were unique to the patient (calculated by dividing the total number of mutations found exclusively in the patient by the total number of mutations identified in the trio). Similarly, in the h-trio analysis, the specific mutation rate for the patient's brother was 90.36%, reinforcing that the majority of DNMs were specific to each individual (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Furthermore, we employed ClinVar database to identify potential pathogenic variants in the affected patient[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. A total of 37 candidate pathogenic variants were identified in the patient, comprising 26 SNPs and 11 INDELs. These variants were associated with 23 different genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC).\u003c/p\u003e \u003cp\u003eInterestingly, we identified a mutation, \u003cem\u003eSTAT3\u003c/em\u003ec.508G\u0026thinsp;\u0026gt;\u0026thinsp;A, on the patient's STAT3 gene that has not been previously reported in the ClinVar database[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This mutation is speculated to be associated with a \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome, aligning with the clinical presentation of the patient. Through Sanger sequencing, we confirmed that this mutation is indeed a de novo mutation specific to the affected child (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD), emphasizing its potential relevance to the observed clinical phenotype.\u003c/p\u003e \u003cp\u003e \u003cb\u003eSTAT3\u003c/b\u003e \u003cb\u003eG508A is involved in regulating the expression of genes related to cell growth and differentiation\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePatients with \u003cem\u003eSTAT1\u003c/em\u003e GOF syndrome mutations often exhibit diverse immune characteristics[\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], commonly presenting with early-onset autoimmune manifestations[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Consequently, we conducted a detailed analysis of the blood transcriptomic profiles within the patient's family. Gene expression levels in the patient's blood were computed, and individuals within the family, including the healthy brother and parents, served as controls (Figures \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003eA and S2B). Our findings revealed that the blood gene expression pattern in the patient closely resembled that of the father, exhibiting a higher similarity with the paternal profile compared to the brother (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA and S3).\u003c/p\u003e \u003cp\u003eCompared to the healthy control group, the gene expression profile in the blood of the affected child revealed a predominance of downregulated genes (n\u0026thinsp;=\u0026thinsp;74) and a minority of upregulated genes (n\u0026thinsp;=\u0026thinsp;2) (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB and Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Notably, five significantly downregulated genes (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) \u0026mdash; phosphatidylinositol-4-phosphate 3-kinase catalytic subunit type 2 beta (\u003cem\u003ePIK3C2B\u003c/em\u003e), SEC13 homolog, nuclear pore and COPII coat complex component pseudogene 1 (\u003cem\u003eSEC13P1\u003c/em\u003e), solute carrier family 16 member 3 (\u003cem\u003eSLC16A3\u003c/em\u003e), solute carrier family 2 member 3 pseudogene 4 (\u003cem\u003eSLC2A3P4\u003c/em\u003e), and protein tyrosine phosphatase non-receptor type 14 (\u003cem\u003ePTPN14\u003c/em\u003e) \u0026mdash; play crucial roles in cellular growth and differentiation (Table S3). The downregulation of these genes, known to be involved in various aspects of cell development, provides a potential molecular basis for understanding the clinical phenotype of developmental delay observed in the affected child. Interestingly, these genes exhibited minimal expression in the affected child, while the healthy brother (the sibling without the condition) showed comparatively higher expression levels (Figs.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC-\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eG).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003eDEGs shows abnormalities in neurological functions\u003c/h2\u003e \u003cp\u003eTo explore the biological functions and signaling pathways influenced by differentially expressed genes, we conducted enrichment analysis on them including Molecular Function, Biological Process, Cell Component and KEGG pathway[\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Phosphorylation is one of the significantly enriched functions identified in the enrichment analysis (GO:0035091, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.013; GO:0032266, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.003; GO:0016303, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.018; GO:0035004, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Interestingly, we also found significant enrichment of differentially expressed genes in neurological functions, included regulation of dendritic spine development (GO:0061000, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.022), neuronal differentiation, and synaptic development (GO:0014069, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.021; GO:0032279, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.023; GO:0099572, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.025 and GO:0098984, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;=\u0026thinsp;0.028) (Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC and Table S4). The patient also clinically presents with refractory epilepsy symptoms.\u003c/p\u003e \u003cp\u003eOur analysis of KEGG signaling pathways is consistent with the aforementioned results, indicating that abnormalities in the nervous system are a characteristic feature of \u003cem\u003eSTAT3\u003c/em\u003e G508A (Figure S4). IFN-γ is downregulated in the JAK-STAT pathway, while cyclin dependent kinase inhibitor 1A (\u003cem\u003eCDKN1A\u003c/em\u003e) is upregulated. PIK3CA, AKT3, and mTOR are also affected (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD and Table S4).\u003c/p\u003e \u003cp\u003e \u003cb\u003emiR378c-mediated regulation of\u003c/b\u003e \u003cb\u003ePTPN14\u003c/b\u003e \u003cb\u003epromotes the activation of inflammatory pathway\u003c/b\u003e\u003c/p\u003e \u003cp\u003eFurthermore, to explore the regulatory mechanisms of differentially significant genes and their regulatory mechanisms, we conducted small RNA sequencing on corresponding samples[\u003cspan additionalcitationids=\"CR52\" citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e], including the affected child, their sibling, and their parents. We quantified the expression levels of microRNAs (miRNAs) and performed Weighted Gene Co-expression Network Analysis (WGCNA)[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e] (Figures S5A-S5D and Table S5). Our results revealed that miRNAs were divided into 14 modules (defined as densely interconnected clusters of genes) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA and S6). The modules MElightgreen and MElightcyan exhibit the highest positive correlation, with correlation coefficients of 0.97 and 0.95, respectively (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Conversely, the modules MEmagenta and MEred display the highest negative correlation, with correlation coefficients of -0.59 and \u0026minus;\u0026thinsp;0.54, respectively (\u003cem\u003ep\u003c/em\u003e-values not significant) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB and S7).\u003c/p\u003e \u003cp\u003eInterestingly, miRNAs not only regulate multiple target genes but also exhibit inverse regulatory relationships with mRNA expression[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. The differentially expressed gene \u003cem\u003ePTPN14\u003c/em\u003e is associated with the activation of the PI3K/AKT/mTOR pathway. In our analysis, it is regulated by miR-548j-3p, miR-11401, miR-424-5p, miR-378c, and miR-452-5p, and exhibits inverse regulatory relationships with miR-424-5p, miR-378c, and miR-452-5p (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC and Table S6). Among them, miR-378c exhibits the highest expression level.\u003c/p\u003e \u003cp\u003eNext, we analyzed the differential expression of miRNAs (|log\u003csub\u003e2\u003c/sub\u003e(Fold Change)| \u0026gt;= 2) involved in the regulation of different modules (Table S7). After excluding cases where expression is 0, we obtained 242 differentially expressed miRNAs. In the MElightgreen module, 31 miRNAs exhibited differential expression, annotated to 1 target gene kalirin RhoGEF kinase (\u003cem\u003eKALRN\u003c/em\u003e). In the MEmagenta module, 14 miRNAs showed differential expression, regulating target genes including Sodium voltage-gated channel alpha subunit 2 (\u003cem\u003eSCN2A\u003c/em\u003e), \u003cem\u003ePTPN14\u003c/em\u003e, glutaredoxin 5 (\u003cem\u003eGLRX5\u003c/em\u003e), and prohibitin 1 pseudogene 12 (\u003cem\u003ePHB1P12\u003c/em\u003e).\u003c/p\u003e \u003cp\u003e \u003cb\u003eSTAT3\u003c/b\u003e \u003cb\u003ec.508G\u0026thinsp;\u0026gt;\u0026thinsp;A Enhances the Activity of Immune-Related Protein\u003c/b\u003e \u003c/p\u003e \u003cp\u003eRT-qPCR was primarily utilized to evaluate the expression of four pro-inflammatory genes. The results indicate that, compared to the wild-type STAT3 plasmid, transfection with the \u003cem\u003eSTAT3\u003c/em\u003e G508A mutant plasmid significantly increases the expression of several pro-inflammatory genes in response to LPS stimulation (Figs.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA-\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD and S8). This suggests that the \u003cem\u003eSTAT3\u003c/em\u003ec.508G\u0026thinsp;\u0026gt;\u0026thinsp;A mutation promotes inflammation.\u003c/p\u003e \u003cp\u003eThe Western blot primarily assessed the expression of STAT3-HA, p-STAT3, and the inflammatory factor IL-1beta (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eE). The results indicate that the expression of STAT3-HA tagged protein confirms successful plasmid transfection into cells. Furthermore, transfection with the \u003cem\u003eSTAT3\u003c/em\u003ec.508G\u0026thinsp;\u0026gt;\u0026thinsp;A plasmid significantly enhances the expression of p-STAT3 protein (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eF), indicating that the \u003cem\u003eSTAT3\u003c/em\u003ec.508G\u0026thinsp;\u0026gt;\u0026thinsp;A increases the activity of the STAT3 enzyme, thereby promoting inflammation. This effect is supported by the elevated expression of the active form of IL-1beta in the presence of overexpressed \u003cem\u003eSTAT3\u003c/em\u003e and \u003cem\u003eSTAT3\u003c/em\u003e c.508G\u0026thinsp;\u0026gt;\u0026thinsp;A mutant plasmids (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eG), compared to the control group transfected with the Vector plasmid. Additionally, when comparing the overexpression of \u003cem\u003eSTAT3\u003c/em\u003e c.508G\u0026thinsp;\u0026gt;\u0026thinsp;A mutant plasmid to that of the wild-type \u003cem\u003eSTAT3\u003c/em\u003e plasmid, there is a further increase in the expression of IL-1beta protein. This suggests that the \u003cem\u003eSTAT3\u003c/em\u003e c.508G\u0026thinsp;\u0026gt;\u0026thinsp;A has an enhanced pro-inflammatory effect. These results indicate that the \u003cem\u003eSTAT3\u003c/em\u003e c.508G\u0026thinsp;\u0026gt;\u0026thinsp;A enhances STAT3 protein activity, thereby promoting immune cell inflammatory responses.\u003c/p\u003e \u003cp\u003e \u003cb\u003eJAK inhibitor and IL-6R antagonist helps to block inflammation and treat\u003c/b\u003e \u003cb\u003eSTAT3\u003c/b\u003e\u003cb\u003e-GOF syndrome\u003c/b\u003e\u003c/p\u003e \u003cp\u003ePrior to the genetic test results, treatment with cephalosporins, glycopeptides, and carbapenems for anti-infective therapy was ineffective. Blood inflammatory markers, including white blood cell count, CRP, and ferritin, remained elevated. The patient received intravenous methylprednisolone (2mg/kg*d) for 5 days, resulting in the normalization of body temperature. However, temperature fluctuations persisted upon dose reduction, and inflammatory and immune markers remained elevated.\u003c/p\u003e \u003cp\u003eAfter obtaining informed consent from the patient's family, we initiated treatment with IL-6R antagonist (tocilizumab) at a dose of 8mg/kg, administered intravenously every two weeks, in combination with JAK inhibitors (tofacitinib) at a dose of 0.4mg/kg*d orally for 3 months. The patient's inflammatory markers normalized, recurrent infections significantly reduced, abdominal distension and pulmonary imaging improved, and serum interleukin-6 levels gradually returned to normal. Due to the presence of hypogammaglobulinemia, the patient received regular intravenous immunoglobulin (IVIG) infusions, leading to an initial improvement in weight and height. After six months of treatment, we gradually extended the interval of tocilizumab to 3\u0026ndash;4 weeks (Table\u0026nbsp;1). This demonstrates that our clinical combination targeted therapy for \u003cem\u003eSTAT3\u003c/em\u003e-GOF has achieved good results, successfully controlling inflammation and immune responses in the patient.\u003c/p\u003e \u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn this study, we identified a novel pathogenic mutation (c.508G\u0026thinsp;\u0026gt;\u0026thinsp;A, p.D170N) associated with \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e], resulting in increased \u003cem\u003eSTAT3\u003c/em\u003e phosphorylation and transcriptional activity. We proposed a mechanism where miR378c promotes the activation of inflammatory pathways by targeting PTPN14. Combining clinical data, we support the efficacy of JAK inhibitors and IL-6R antagonists in effectively suppressing inflammation and improving clinical symptoms in \u003cem\u003eSTAT3\u003c/em\u003e-GOF patients. Therefore, early identification of newly emerging pathogenic mutations through whole-genome familial analysis and targeted treatment interventions can improve the prognosis of patients with \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome.\u003c/p\u003e \u003cp\u003e \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome exhibits significant heterogeneity, where different mutation types and inheritance patterns may lead to varied clinical phenotypes and prognoses[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. We found a rare case of \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome with neurological involvement. Early manifestations in \u003cem\u003eSTAT3\u003c/em\u003e GOF children may include gastrointestinal and endocrine system disorders, such as intestinal diseases, hypothyroidism, and growth hormone deficiency. As the disease progresses, additional clinical features may include non-malignant lymphoproliferation, interstitial pneumonia, and inflammatory arthritis. However, unlike previously reported cases of \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome, the child we tracked exhibits a rare neurological clinical phenotype, and seizures were difficult to prevent with JAK inhibitors and IL-6R antagonists. This observation suggests potential genetic influences on the immune response and highlights distinctive transcriptional regulatory patterns associated with the \u003cem\u003eSTAT13\u003c/em\u003e GOF mutation within the familial context. Additionally, there was no reversal of periventricular white matter and right frontal lobe brain lesions. To date, we have not elucidated the molecular mechanisms underlying the neurological abnormalities, but we speculate that they may be associated with \u003cem\u003eSTAT3\u003c/em\u003e haploinsufficiency and immunoheterogeneity. This differs significantly from a previously reported case of \u003cem\u003eSTAT3\u003c/em\u003e GOF syndrome primarily presenting with bilateral facial muscle weakness due to myositis and muscle atrophy[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cem\u003eSTAT3\u003c/em\u003e, whether excessively activated or inactivated, leads to human inflammatory or immune-related diseases. The precise underlying mechanisms remain incompletely elucidated. \u003cem\u003eSTAT3\u003c/em\u003e orchestrates pro-inflammatory signal transduction by activating genes associated with inflammation, such as IL-6, IL-17, and TNF-α[\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e]. This corresponds with our research findings that STAT3 G508A mutation has significant pro-inflammatory effects. Experimental results from transfecting human astrocytes with plasmids also revealed elevated expression of pro-inflammatory factors IL-6, IL-18, IL-1β, and TNF-α. Specifically, \u003cem\u003eSTAT3\u003c/em\u003e can bind to specific DNA sequences in the promoters of pro-inflammatory genes, augmenting their transcription and expression[\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. These genes encode cytokines or enzymes that foster inflammatory responses by stimulating immune cells or generating prostaglandins. Furthermore, \u003cem\u003eSTAT3\u003c/em\u003e induces the participation of helper T cells in inflammatory reactions. For instance, Th17 cells express pro-inflammatory cytokines like IL-17, IL-21, and IL-22, thereby enhancing the functional efficacy of Th17 cells and promoting inflammation[\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. Current research primarily focuses on STAT3 GOF syndrome leading to increased Th17 differentiation and reduced function of Treg cells[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. A previous study suggested that \u003cem\u003eSTAT3\u003c/em\u003e gene mutations result in heightened activity, suppressing STAT5 phosphorylation responses and impairing Treg cell function[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. Additionally, a study using \u003cem\u003eSTAT3\u003c/em\u003e mutant mice revealed phenomena such as T cell proliferation, activation, and Th1 polarization, while the quantity, phenotype, and function of Tregs remained essentially normal[\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Similarly, an international cohort study on \u003cem\u003eSTAT3\u003c/em\u003e found that the abundance of Tregs and Th17 cells does not predict the severity of clinical phenotypes in patients[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e]. Our research harnessed transcriptomic data from patients with STAT3 GOF syndrome to analyze miRNA-mediated regulation of downstream JAK-STAT targets. Notably, the differentially expressed gene PTPN14, which exhibits a close association with the highly expressed miR-378c, holds potential significance in the context of downstream inflammatory signaling pathways. However, further evidence is required to definitively establish this connection. Perhaps delving into the downstream mechanisms activated by STAT3 mutations could unlock the secrets behind the observed phenotypic variations. Moreover, comprehensive longitudinal studies are imperative for unraveling the pathogenesis of STAT3 GOF syndrome in the future.\u003c/p\u003e \u003cp\u003eGiven the heterogeneity of symptoms in \u003cem\u003eSTAT3\u003c/em\u003e-GOF syndrome, we attempted to associate its phenotype with genotype. A group of significantly downregulated genes in the STAT3 G508A mutant group, including \u003cem\u003ePIK3C2B, SEC13P1\u003c/em\u003e, \u003cem\u003eSLC16A3\u003c/em\u003e, \u003cem\u003eSLC2A3P4\u003c/em\u003e, and \u003cem\u003ePTPN14\u003c/em\u003e, may exacerbate aberrant signal transduction in \u003cem\u003eSTAT3\u003c/em\u003e-GOF syndrome. Targeting the functions of these genes could explore the development of related therapeutic targets and drugs. For example, inhibitors targeting the \u003cem\u003ePIK3C2B\u003c/em\u003e, functional restorers for the \u003cem\u003eSEC13P1\u003c/em\u003e gene, and modulators for the \u003cem\u003eSLC16A3\u003c/em\u003e, \u003cem\u003eSLC2A3P4\u003c/em\u003e, and \u003cem\u003ePTPN14\u003c/em\u003e genes could potentially serve as novel drugs for the treatment of \u003cem\u003eSTAT3\u003c/em\u003e-GOF syndrome, thereby saving patients' lives.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eSupplementary Information\u003c/p\u003e\n\u003cp\u003eThe online version contains supplementary material available.\u003c/p\u003e\n\u003cp\u003eDNA and RNA sequencing data have been deposited at The Genome Sequence Archive (GSA, https://ngdc.cncb.ac.cn/gsa/) and are publicly available as of the date of publication. Accession numbers is PRJCA025000.\u003c/p\u003e\n\u003cp\u003eAuthors contributions\u003c/p\u003e\n\u003cp\u003eConceptualization: Bo Zhao, Ji-Yu Chen.Formal analysis: Ji-Yu Chen, Bo Zhao, Zhu Zhou. Methodology: Yan-Fang Li, Mi-Feng Yang.Resources: Xue-Mei Jiang, Xin Bi. Software: Ji-Yu Chen. Supervision: Bo Zhao, Ji-Yu Chen. Validation: Xin Bi. Project administration: Yan-Fang Li. Visualization: Xue-Mei Jiang . Writing \u0026ndash; original draft: Ji-Yu Chen. Writing \u0026ndash; review \u0026amp; editing: Yan-Fang Li, Zhu Zhou, Bo Zhao.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eFunding\u003c/p\u003e\n\u003cp\u003eThe work was supported by the Kunming Health Science and Technology Talent Project-10 Projects of China (2023-SW（GUIDE）-07); Yunnan Clinical Medical Research Center Chronic Kidney Disease Clinical Medical Research Center (202102AA100060); \u0026quot;Spring City Plan\u0026quot; High-Level Talent Training - Spring City Famous Medical Special Project (C202112012).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAvailability of data and materials\u003c/p\u003e\n\u003cp\u003eThe data are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003eCompeting interests\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003eEthics approval and consent to participate\u003c/p\u003e\n\u003cp\u003eThe study was approved by the Ethics Committee at the Kunming Children\u0026rsquo;s Hospital, Yunnan, China (2023-03-336-K01). Written informed consent has been obtained from the legal guardian of the parent for publication of this case details and accompanying images.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConsent for publication\u003c/p\u003e\n\u003cp\u003eNot applicable. \u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAkira S, Nishio Y, Inoue M, Wang XJ, Wei S, Matsusaka T, Yoshida K, Sudo T, Naruto M, Kishimoto T: \u003cstrong\u003eMolecular cloning of APRF, a novel IFN-stimulated gene factor 3 p91-related transcription factor involved in the gp130-mediated signaling pathway\u003c/strong\u003e. \u003cem\u003eCell \u003c/em\u003e1994, \u003cstrong\u003e77\u003c/strong\u003e(1):63-71. \u003c/li\u003e\n\u003cli\u003eO\u0026apos;Shea JJ, Holland SM, Staudt LM: \u003cstrong\u003eJAKs and STATs in immunity, immunodeficiency, and cancer\u003c/strong\u003e. \u003cem\u003eNew england journal of medicine \u003c/em\u003e2013, \u003cstrong\u003e368\u003c/strong\u003e(2):161-170. 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PNE pneumonia, ILD interstitial pneumonia, BEK bilaterally enlarged kidneys. CEF Cefoperazone, ACY acyclovir, MER meropenem, VAN vancomycin, IVIG human immunoglobulin for intravenous injection, MP methylprednisolone, TCZ tocilizumab, JAK jak inhibitor (Tofacitinib Citrate Sustained-release Tablets), Biw biweekly, TIW Once every 3 weeks, FIW Once every four weeks, LEV levetiracetam, LAC lacosamide, MO month-old.\u003c/p\u003e\n\u003cp\u003e\u003cimg 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6UQ1nXerJyDQ882bN/Sp4Qi3k6FtvnrYoforqaXwWICnn41H7g/N1OufoDyogoGH9fDg220+tcXpA70xKdttV4jz2fOwKzpzed9whs6MncmXpzIjNkHHuNpEZjoeFpcA3Krl1NidXDoNwZmYMAVNrHH98ccfdQO9Suzb9LHdm3K4lqNB7IOzc4XqCfakA5252gdnvHsLOdCCccVwCC55HMOhsfeHMBIKqaE11mFv344Snz7zeFNNmRoNegPFjtD0VrhGUjBO4QG+zT81HCV7/0zKD5jQGutw+Z+XjwlCcfpj/LcfU5BVcjf19XBIwQghhDBECkYIIYQhUjBCCCEMkYIRQghhiBSMEMKtIH/YcnkOomB8+vo1S/90xL8hoVl/SPfly5dF/tKxn8U/42Nq5Ndf//bcGRHokz/MCOFo4Ik2h/B0+9SHPTiIgkGFIGWTrN1R/+a60rc8f/78ktvMFEyEEWdBhvorb/QcKTv4h+EpGCEcDTzjpBQecAsGIABPutlGaLYB4TwO6xMG0DShI/cF4/Rv4y/31/GcFSxUKaq5mB396dzT1zg5MTRTMEI4JniiK7HwyPvg99nAPECWsH84l4MoGIXbZqZ2I9UDewmtsRd9wWiqb3FqunG2UjBCOCZmBaPSC8LsBVlT93A+B1Ew2CfSNLvIjp6Whakw1I5K6feG4TVLU32FU8J5sgN3mTcFI4Rjoh58CwYyV398rCsZIE/9Zg6iYLhhwBayYVxRqgG38PL/hgEapBi09lfQeAtPPFWlgbP9Qwg3C5MMPxFyJZPA9HD/898wUjBGOKxfSV3EUn8lFUIIYW9uRsEIIYRw7aRghBBCGCIFI4QQwhApGCGEEIZIwQghhDBECkYIIYQhblHBeD3RGiGEEHbkFhWM+lJeCCGEPbhdv5KiYFA2WiOEEMIu3LqCcfn3i4QQwu0kBSOEEMIQ+ZVUCCGEIfKP3iGEEIa4RQUjf1YbQgiX4Xb9SiqEEMLepGCEEEIYIgUjhBDCEN8UDP/PwhBCCDeRlspX45sJfgshhHBjaal8NVIwQgjhSGipfDVSMEII4UhoqXw1thSMX3755c6dO1yRX716hfz+/XtvLcuLFy+4MsXjr/z666/eKiUaePbsGZ0/fvzo3UHoz0Aos4AdLSOzRi17aw/wU+MnJyeYoqke+iU01S4w/Pvvvy9ZI2/evCnlrixucMba9mWNWZayuYgdBnpsgLFnbdYBBqe4DFjArJab6uuDuQhY1lWeDp5HZrEJzOLsdPNR3fUB38wsULOJ0Dx58oTYcnXtXEkIXBkI9Cnn7bAZNgvL4ESz2fum/XtIsLjRGr/9hlf01zGsYVk9SeZcT1oqX41NBQP/WLMFA7+V+4JBU7x1//59lPRUOVha3La7d++29qQhQFxt9mFFRg8MaaoxiK+x7gfOLHOlm3u8K2weS6hDhoYlVATOPRnjEFJia9lGVsAma6ko7cTiBmesbV/WmGUpm4vYYSAgMOThw4fIM5sYnDruA4cTtMahRWYWrlpWieYyU8woy1yJA1dXJzad8dxUeHkwW/P2EyHQRGD5JmvyALJrpz+pYzwO9K+Mb6KfjT1ryowhyDqJjJ94goB7HIC6YrOmmNFS+Wps/5UUqV/vieCsDNAkvbIqzrHN//iP/+CKjH4wxKy/9kYImTMKt6yx9Kxug8YLQowRqDPKpERfpXdRMm912An2mIEMr8pk07v9EtTsBKaw6VnRLM5jkCBD67QLixucsbZ9WWOWpWwuYoeBgIARjhDyzCZKruKQcbDAgeS4YgSXuGL8dL7JMq5yi1ONvg24NFjGpsaZC8HMCASHK3pWh9AGLA1rIXSzibjimB0KOpiUASeNP0qayK3TBRjA1phgLBrpm6xaf3SGJneZAoFJ7Xk6voNubspFbrRUvhr7FAw8RvDKLQLkwmj++OOPFg/0CoNUaAgEIVMWZ/c0c7LdQjbPu4M4nGsZZyLOBAKmcNtD43Km+zvAEExxdbgOlwD9EtTshKYYjqvKCD57HLXWaRcWNzhjbfuyxixL2VzEDgN5iHg0OPB1wHqb3EKQNmYYhmABwUePpk+Beh8TnpF6MC8Plo0A/kPvPzLwGd3S1QYsCvaZSKGfqPRquMWqdc+dIuC6rWznDbic1pioZULfpBvpFIFdtol99tqpLwo+HXC1Nc7QUvlqXPYTBrdYqguj+fPPP3NF3nXjqzMh68NB1NAgGFlTPMpZUdkK/RmFUBNhSmtMxyo06OGY7u8ANoFdZ5sxpQXOXHnbL+F0wI54mBBwkomQnQjQcLXbOIsbnLG2fVljlqVsLmIHI9Aa59nk6q09KGsaodkXDJUX5az90LKyv79FYxOqeerBXo/JZngezV2ziWbJhPXytNKn9Mj/8i//Mv4Ik0+qDwIDZzE8G9LSMKOzn3o2/SsAsrfoYwZDX8qztFS+GgsXDJsI0PfcSoWsDwd7zNXPE4QSg+RiZXPxOPRnFGOxRhPBh4HpPBZoANld2YPyXLM1Edd+Caddd6Qs4xtP2g8//FBVjXU50U4sbnDG2vZljVmWsrmIHYxAa5yxicxJKzb81HkuZY2xNvuCgTWcRPbuImCNqjk5e+ots/j5SWjSwZ40iZLyUmBT4exE5nSeUK7cqrsIKnlsicb4I0xP+gtNLBQsnKvdZrCt5iIhVmr0ofYXr9y4c2mpfDW2F4wQQgg3gpbKVyMFI4QQjoSWylcjBSOEEI6ElspXIwUjhBCOhJbKVyMFI4QQjoSWylcjBSOEEI6ElspXIwUjhBCOhJbKVyMFI4TbyKtvXzJ4lq0djpj379/X9x5uFi2Vr0YKRgi3ke+nLyH7FbCP0/vcLA9cVaJBTxNhwzfFbgq1ClfkYtGwxun+N9R39wqG1FcvD5mWylcjBSOE2wg58cmTJ/wczZW86euMXkyvR0QQ9I+nr0DbbCNvIJRGF+u3plmLX8N2+a1TR18bqCh0w0JrHzYtla9GCkYItxGSpj9xkzcRrAcmR2SzqgVj6v7PV2vcRKgKfpLg6kJqyeeuy1viZwtjdfi0VL4aKRgh3EasBwh+wqjsiUyKRDimgkFRtGD42iiEfsmnPb7l7G+fqv+B01L5aqRghHAbIf2RK19MVDakeCDU722OpmD8+vXdo1QCF7K5YHjrJtJS+WqkYIQQwjfwGetV/krqPFIwQgjhSGipfDVSMEII4UhoqXw1UjBCCOFIaKl8NVIwQgjhSGipfDVSMEII4UhoqXw1UjBCCOFIaKl8NVIwQghHzqtXrx5/5c2bNx8/fpy+f/LCN0rd3G9dnKWl8tVIwQghnH61rUkbIck26UZBSbAqUCoePnxI/fhleu0g9QPlzf3WxVlaKl+NFIwQbikkSvMmwrNnz/76178iWDm8heBdu9Ekw9IB1FcHhEOmLxgWCYRSwo14E+0ILZWvRgpGCLeRN2/e8HGBn6x9lxRp9Oeff/adtfVuV2/xI7mvYCLJ3r17l9yKHg01Bjv0t8YcMjjJ0lgCPusta3FR1UHhptNS+WqkYIRwSyFjmkmREagN5s1Ko1QUqohKf43jLXtSNpC9Hjh46yqEhVMwEGql+YQxSApGCLcRftYmb75//54PEDTJp1UwqAHmU/pUwSClzgoGJYSKciNSLd66CsF/lobzfkgiCPk3jEFSMEK4jZBAyZjgJwzKxk8//WRW/fXru13Jp+RWf09lqegLhr+hsrTcaPpactNpqXw1UjBCCBdibWiNb6FU+BN6OBxaKl+NFIwQwoX4J1Kt8S18Cjn8f+6+bbRUvhopGCGEcCS0VL4aKRghhHAktFS+Gt8UjBBCCOEiUjBCCCEMkYIRQghhiC0F49OnT3fu3OGK/PTpU+QHDx54a1lOTk4UXr9+/fLly3fv3tn8/fffmVdUfvnyhT7e3ZWa5Vwj2Gc6BH348OGD+q1gDctQw5Fr+GwJTXr6lD52GMT+vQWnA+bC4Z0MasEhs5hDzVJT7IQBqRgakFnAawuYQufReGsQ+juwd/Js8Pt1bUWbMFs4RnYNhQHEmdae2Mmf2gVgRTQrhvqD8XZ7953aY0VydiBB+/z5c2tsox8+M7XHMb4kFcDnz5/jCaCBXY9iD4tiFcrYwTiCs0jfdN6pb4NkiwVlouqO080Ic2t2BpSvjE0Fw5VbMMBSwRXZDt4FVo7enixMZS1sMz6i9+7dQ3bDECriBZrT/fz9d2LHdE07zGyWs0ZQchf/ObV6jieDjwGBYqCHAztv375F+ejRI+8WLkGZPnUsttI7L2hYAlebrkU31GyGbrU7uHQ25ntEuAfjeoKdmotZavnAdM7ClYVA78AIGHQWQqFmFny2EmHwHArDLTa9M1joj/0IfYQL1riTP/S0cw0sN5ARLrNNWGClGME455wrSoS///3vXGux6O2DTHjBzgp24+AZ9q0wyqcMuV+OuJzxY7wfGHeLwfOJgGOcIk4US6sMsB+MZbMIDjJr8bGd7RTpUcF5lX0McaA6exqB06hLfdCQVw3UuWz/lRRr692iaSzAZ56mfVwVGlNh33MDBIhuxgiZqGGkgii1hfTsAzpOP8u5RnSeVVTK2Gk/MEh/sEkELloC4MDs7mZ656VypTg1NunWVBvRPZaMgPGzMWculGi421S7Mwty797pQ/Dhg3erT995hHJvNrCCz9NIlFia+hEYwsINSFNNSprjhwH6CDfVtE00x/2hp52NJDJBIxnRRMYflFzFIeNgAQ857Tqpn2jUMxFXTx23uDIpTzR3f/zxR6ZDY5QEI5rdTD1lyGejOlna4RjvCmZZL/O29nRyyhmXQB8Elj/d3weWwCyATIiYAoFdYyJwdhMm0I3+TIpsH+KMA0aAUHCl6QFApvPsDKC8SnYrGJ4YFoMA3333nWtDNtBQBXCnH8oMh0YYNTs0NAlQa+yeWYp+YC+zqDq+7g07rSetxzYYVRkQGIh80RL22+kyjh1PUkETg/g8eNBxTN/8UehszHUPa7iqZlcMYz2cTGFKAmbBVQRXhJ6p6aByHIxjoZ7Jog8+8k5LcMlcZxHedcuYXQeIsBpsstKd/KEnDxFrITh1PrHDkpW5hSBtzDAMcUUYYbOYAofLMno0pDPigIY+TlcDlcFbYHMDDKxVlKYfuOsx3hWMsyL3RXCeDeLKpD6e3KUPTTvsgYtiLRjEjoFiH9GDTwEJE9ljT5P+NO2DA0SJgcYKIxYSeno4kfszcDrlFbJDwWA9Lq+gCaywLxgEqILS781mDCtb5VnpY+FpVhY770E/cCYDC7R067Z7ZofN4LOnDSMMcdSGJcyS0SAYV2C6Sr7AdJ5vpmNSlZsph/HqbMy5pYY4DBqc4XqNA+CeBgXnp3g/5dM6st0YsmtYcM9VVGROQ/9t8MdjIj7nCGVTyuAgfYTVEARscsA4Zjq5FSbtnS8fcBJTyDMnd6KsaQQ/PfD9LHU2DLU97VBT64lDNkNP8ClTU3MBU+96jPeDIwfKOq8MvV5hD1wUa0FgUZqaGSQ3KvQO9H2QsUDY2QINshEoK2J1Bux/ZYwWDEKJIBVWDhmwKpRc+ybstOsGy6zBQJrIFx0gO+9BP1CZHa3luBn6wNTOPgKjWDj9uY4sYT//a5R+Kmv/1NcpHw1mIoa7Rkyd67DLQc9uOmQnGO4PbkB4yRHKlRQEDVd/oGOuQecLww7uIEZma7FPH/mt4IORwSzN8raP+Qh9hGmWnZ38oWffGVkfWJr/EjAFtVHHeJCyxliuyOZx9GhwGJgIWY1yDVQGBIJmBzWbqXl72fg4qQan+6vDXOUM1FHcNZg9LoolGE8DxX4hCLdIj1Pf00qMRrkHTzyB/V0c8/eByHUGvHVlbC8YIYSjh9RDkkLoE3oIM1IwQrjt8DMvP8/6EYFqsd/HynAbSMEIIYQwRApGCCGEIVIwQgghDJGCEUIIYYgUjBBCCEOkYNwMpj/gPv3r+NYOYX1+n76l9OXrV1nDHvDMfjrzPkGaftcE+fSbRF9fFXX4pGDcADhwN+U8hRsEycuvXFgS6g9qaSJzVU92e9u9gqmEsIH62Y6f8wgjV5tE8sPXN3wYWPXWFeVDJgXjBsDBen7mTXYhXAbSll9p5kqqevToEcmLvPaPf/wDjS+iQE9qA38KpniQ+xCaiXAell7iybVKhdFDILZcLRhVRW4QKRg3AI6ap82XDYRwechc/FRL5nowvSQUASXXv/3tbxwzbvFJQj1XoaJQYHy7VLgIAmVUufLYWhUIpiGtONOtCgYCMffWgZOCcQPgEeVHFYT8cBeWgoLhoarCgMyVgkGaM5395S9/MbUBd9HkBA7SF4MSqBn+5GdUvXrXpvIhk4JxA+Aw1c+DTRXC5aBOmMKoAZWtuFIwOGwI6JER7El1Qaa/w8MgVTD4fFbllkgSc3+/R7S9EmfvHjIpGCGEISwerRFuJSkYIYTtvNvl/woMx0oKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCECkYIdxqzn4Xr9fkixehJwUjhNvI6evJJiwJCJ+mt9X6vjyU6l++fKkQAqRghHAbef36NYXh3bt3XKkTyr6I8N69e2/fvvVlFerbmHDrScEI4Tbi64wQKAklK6BBRqAJp71DmEjBCOE2Ym1AoDzwecKXlp9M/41PCka4iBSMEG4jfcHw3y2oFv63P33BoJbkDbWhSMEI4bZT//T9/Ot/khHCuaRghHDb8f+64INF/je9sJkUjBBCCEOkYIQQQhgiBSOEEMIQKRghhBCGSMEIIYQwRApGCOEc3r17N3uL1JcvXz5//twaE3TIi0NuFSkYIdxGSP3gd/eAYoBsPaAMALLfybCJQG14+/YtggMdkq+C3ypSMEK4jZDonz9//uHDB64UAGW/9X3nzh3u/vjjj9SDk5MT5OLly5d0Q0nxYEgKxm0jBSOE2wiJnnSPQA3gymcINBQMlL4axA4PHjxAqFvALWoGGl9ti4Am3BJSMEK4jZDozf58UODjAk1qRv/yQTsoA3dpgh8y+FDii6dSMG4VKRgh3EZI9BQDKoRFgrKB4MeIvmD4qyeLCjWjhkA+YdxCUjBCuI1YD1ojhDFSMEK4jVAt/JuoEMZJwQghhDBECkYIIYQhvikYv4UQQrixtFS+GikYIYRwJLRUvhopGCGEcCS0VL4aWwrGL7/8cufOHa7Ijx8/Rn727Jm3luXFixcK33//PTJXm8DsTGqHX3/9FQE+fvzo3UEYgv/SVL/99ubNG/Q1Fzb7eXcFUwpnlwB4/urVKwXuwvgSGFjWkLGAgPN7e7u4wRlr25c1ZmFTPGxaAyy3c/P4MUrs12ncwCK+MYqxNVxNc+XxY6yp3A/MLh69zVz9jFthHw3mkydP8AdOn8zd00sPS6skiR2MY9ZZYOtiKzKwR0ZqqXw1NhUMV27BgPv3779//55mu/3bb94FAsFdBLrZBwaX6iN69+5dZIY7ij2rqGGc68nJCf64l3sfMmzWXuKnMpaREWg6165sXQIOP3z4sPS7LoGehBfLygq4yqSYmrrsxuIGZ6xtX9aYhd3RDhlEjWAQDVdPCEfRbhexiG+MYmwNV6MDgD+l3wPMXsEe9Vz9jOfCvDx9ysyuGzyqxPOS6UUYy8NuSuGckBaYot+1Sgtn6UMERMaBDMGgA7lruGjKadevtFS+Gtt/JWUZUO4XA67HCoGeJgtGQ5i4i9KobYbF060CKoTVsYSDXUTAvruInolqy3eCWepoUiewjEazGISZG4NsXgKwivIfdl0CA+nMKWQiZExhAeeJs6HelcUNzljbvqwxC7uGNYTZbmIW+whMBP3mngt9Lu8bo6bZToeXphzzrvIeMPYK9qjn6mecwXQ89X2GJZjGFiWPJAJ99k4vwtKYBZBZnVPUrm0+ObhhZJDxARzIFT2OAbHCCLeQUfpzatFS+WrsVjCAmvHzzz+jhD/84Q84zfqRDTRUUZlVl81UQAEjdYCwQFB6wV1nJ6b7O1AWBFkjXP/t3/5NuXdjVy5agvSz77oEBjKcI8IQZQQsIPD4tU67sLjBGWvblzVm4YllH7HT7yYJjkdUGcGJmFfNuSziG50Z6/DSlGPeVd4Dza69Rz1XP+MMJmK6Pl8TTKZ2x/1BYe/0Urg0zgkGsYNxmqR1BBN963cBDscNfXC7vaLnrk01ygrSUvlqjBYM0p+ezT4xAWvrCwbdzJUq7bmVWjZhmsXUZxUlG1lZeBamEbDc+6NBBPbgX/7lXzAI7Oss0Y9TLp1dAtRm77EEzxACoWAUMqecK5i/7DbO4gZnrG1f1piFE2Li6HeH3XTXKCc+xhh3Ny9iEd/ohp0ypaYcQ7/3WYUyu2D0NnP1M54LQau46YYy9HqFPXCZnBMEf/6gOW7Q4XjCEDAjORw9dxFoEjEPKjE8HfaVlspXY4dPGJQKZEqCerBCzAqGTXBtgxgRQsMsp3Ga/qXIh5MrGCCLP3Jt7ThOIRjhih2c7PW9vCuOPXcJUJu9xxI8QwhEgAP0ww8/WOqAKYi58jiLG5yxtn1ZYxYMMhDcHbevJlIDbq6ac1nENyxgB0gKnqiffvoJa8r6tjeLeLgTVz/jVghj7SzgD1HdL70ULpMVmcqdgqt3t1JREgd6Rc9dm9jnStDINqf9vtJS+WpsLxghhBAOCooHdRcob0010VL5aqRghBDCDcPf6FAt+KjRVBMtla9GCkYIIRwJLZWvRgpGCCEcCS2Vr0YKRgghHAktla9GCkYIIRwJLZWvRgpGCCEcCS2Vr0YKRgghHAktla9GCkYIISzGx48fX7169X56qcTjif4rltya/SHssrRUvhopGCGEsBhv3ry5c+fOyfSOLL+YLRQSSsXsi9yL01L5aqRghBDCYvDZgsJAVeDDxJMnT55Nr5lByUcNhP5dfGvQUvlqpGCEEMLyUCQA4eHDh37gQH7x9T35K9FS+WqkYIQQwvK8efPm4/RCWT5kUDB8g3UKRgghhDkUhifT/+L3/fff+5ZAhMffvh93cVoqX40UjBBCOBJaKl+NFIwQQjgSWipfjRSMEEI4EloqX41vCkYIIYSdaKn6MGg+rUYKRgghhCFSMEIIIQyRghFCCGGILQXj06dPd+7c4Wrz9evXT58+VV6Wk5MTBaZ4+fLlu3fvbMLvv//OXfjy5QtNrvTx1q7ULG/fvp3NAkxUGgSayoNonIGESHa1sJly/sOHD8izIOwaFjpjBFMl98PZceLjjFhGgPHlOAQL2j8b7YqSNvfb05lj/SznhmgEbGIE+sXuYU075ZsYZ2Mywh6HcByM14pqInzbI2iDXP2MW2E7PIfPnz/HH0ADbF/rsTssja1Xxo6H3FngGhe7CJsKhiuvgvH582dk1uxd8K7KBw8eINDTbjAYGh+te/fuIbthCBVxcEYsc7bowNb2Pgxy7iyYwtvp/unBffTokT5zZTlVJrfSGxc0GOfa2pejt4/DBof8WM4DyvGwsLOAQBxYpqtGJjLT/RZzpvAMMBE+jJ91eho97Jwb7d7VvfcU35gFx87OYohc3dR3FJZsQtcCXBTwzdTU+KMGD7GAwDFTsxXDzkAErmhcL03Qvk3vclUYoT/k/X6xWGxOXRbm6mc8F+Z1i4HZdYO4sVPsGp5csoYxli1mU5A5ojy2TFHHm1nG9+gA2f4rKcsAAvsKtXKwSWjsQ5NgoTEZoTRqm/HEaxaZPcNI/5ghA8btybX3YZB+lkoKdWqBiZA9KMj9ra30xmWPVLWBmfMYR9ZVYTkwHhbiyXD6GwdBySwILJ8pEAwI3Qg+d3c96O7UudFGzxTYdGn29NZOMBCzcHYWrrWicVw7YEfNuQHfiktDmK2LR4NbrbEN1+LUbAE4nCZX1suVDmRhNPosbfxG6IYFUpsxxA6xwghTQOu0KFc/4wymYzf7Y0xscQMBJeFFoA+CZ2k/WBqzALInx030Llu265k8KEYLhmtmX7myYJTw3XffEVybBhrqh4idfkg3oBphVIUV2Z/I2EL3ACr6u+JAD2svCDIa5dmtEcorngf8V16Qcp5TiMDVo0+g1IyHhZ4+EjWEsNdDW3FQYHNZ0a6Loj/G8bAi2YdUAR+cCMad72EWjDD27CymgF2ffF3iWoulOQv4CPTEK4bM1oVvaDzbW3EtWkCgidAv1oel9FxhcJs0wkpxUhmBRwxh/DPQTlz9jDOYiOn64BM0puaKJ5wlNNylD0077IFLYxcwiB2M0+RzBgLKXQ/koTFaMDiapyVigoh4CyUQYpRcbRIXU49Ke26FUVzZKgNq0BEqT9Gsee28Bw7EFBMhsJ19CuinKAfGKa9Ywho/MWkf4zpfHlrFgUM5OG8tXJs0Z+fYmDuXfaCErbBrnASnOBttszwCx+Mye4oRH3LG9rP8v//3/7Tfb+ggPucI5Y9BQNjpSLC0mR3W7vLH7dhTC7UWmmUBb3s9117YTG+EIcikTq6gWbstyNXPeC48I/WY6IYy9HqFPXCZnEMEdtwpLmPwoNjhV1JAQPuVWyFmBcMmeI4H0SzPGEeHgTSRCTpKmghosDz13X87ayACZvEW2VmAZZbPCP1JGqGM7zF2hLJfIUIu56E6bIVIYoSxwJ7yWZCxwPnWoLfowy6QMQkUsll4BPr7gxvQ5DqLNrL2L7OnBFk7Wu5n4WqIzNHj0N/IaEdv0VTAB9E3MAdhZ3a27bYZOmPHeRFoIrhMrtiE0jMR0zFFZb3NaBwBZ/hR409/+pN1EQgCppQX5Opn3Aph1CXBH0I6HsNzcZmsyI9NTuEmHgHbC0YI4aCozBvCFZOCEcINg2pRn8xCuEpSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCECkYN4Z3a76KLoSwICdfvyD19rz3nJ7Lly9f6FZ//1YWDooUjJsBpcK3mLV2uAXUzwcKXBXIKeBhQFMpJn9uewiQ96kQPK3I7I55//n0HdVpA0/3iz71LLtr6D98+HDnzh0KzMwCVzTurM1rJAXjZsAByte1bhtsOpnC9EHe4QAUJCAyy9Ovr3ejjzL6HJLrhb0gufvVbvbI9yOwZewLNYA9ejS9FZtbXNlWP4LQ37JBt94CHVAyij402V+u10gKxg2Ag8Wh8cw1VbgFsN1kE5IFScdX0gJ5pE6COYXmn//8Z9OQP1hMo8N1UlvjTinMlDTVKCsUanjwrSig7N3rIgXjBjCdq9P/ccS304TbAz9R+kOl6QP6Hx1U0qRg+KsPfzg97ReuFbeGbTLF+0Gh9mtWMPyAiNBTGsZSLbDDSbj2zU3BuDHUOQu3BzbdSuCvmwDhbMGgSU6hJxoS0+nIcK1Uukdgg9idUvbb57Zyt/oXpbFasK3+vOh5uC5SMEII4XrwXzgoBuNlgHrTpOsgBSOEEK4HPmr4CePL2BvvYbznGqRghBBCGCIFI4QQwhApGCGEEIZIwQghhDBECkYIIYQhUjBCuBnkCxY3iPoz2dfTN2lG/hb2y9eXDyIw5OXLl/7R7UGRghHCgULugE9fXy600x9fhuuCPWKnfHUge2epoABQ74WtpI/f3bMPGvT18sH+m33oETwACDavkRSMEA4UEsfz6SWDXMkgpA+zRjhkTO6zb26zgygHXz7oEOygtNIwij7a8e51kYIRwoHS/6Rpc1KHG0BfMMj1ZHyE2sfa1upWglgtKBVgRQHl1uOaSMEI4UBJwbi5VAHgI0L9U0Tt46xgWB5Oe0zQtDzY9N8z/KB57R8xUzBCOFBmBePRo0eVRMKB45bxweLBgwenZWH6uKCy31Y6UAaoB94Smuz16ZhJabVg+CG8rDoFI4QQroc9Xj54vaRghBDC9cBHDT9hfLkhf/+WghFCCGGIFIwQQghDfFMwfgshhHBjaal8NVIwQgjhSGipfDVSMEII4UhoqXw1thSMX3755c6dO1yRHz9+jAzv37/37iJ8/Pjx2bNnL168+PXXX5tqguarV68QuDI1zDrsBPY1Ak31228nJydM7SwYpw/gj3d3AjvQe1gzIr9584a733//vbd2BQ9rLLKzYPNwDM5Y276sMctSNhexUwPXYKmVjnP1M26lHtInT57gD0w5YM8kIGUTkNGwQISaot2b7laTgFRPYjJZ2kQFcEZL5auxqWDgE2muCgaC+sK7wILv37+PQE/KicrBc0CAtE9A1QAb9vDhQy1gXOXlIcT4XLLbybz47Cx4MrJbMyg8jMLn3tWSS08fj8WuEAfCa5SQFbDJWvY72YsbnLG2fVljlqVsLmKHY0lnDiqC2QE7aGiCNm16l6vCCEutdJyrn/FcmLeecWbXDeJGHiAh4Al3cc8Oe4NlBUyRHxCwTE5TCcxFNqg4AE3zg264xQq1rWhsMhA/a2zRUvlqbP+VlGUALANsebsxFQziwgLsQ5NloHHZKLllzw2wTx6XCjFgByNckdETwep2GTBVRszyCMzy008/YR/2O7vlWy2BhXM4cBvYXa4oCZEr2hVG4SoGmQUZO9jH5nTA9qxAyxqcsbZ9WWOWpWwuYseBHCoETiYwlsNGkyv2udKBRxINxrlKG78Rul3BHvVc/YwzmI6nvq+pxBY3EFASXgT6IODndH9/KhWU0MN6nY4rEWAfccCdpT+z4xWBohtXfxGChp7KHgn604GrNqWl8tUYLRit8dtvnM4//vGPp6Xjzp0//OEPeE+IkQ001A8RJWyGSLF+YlGRJV4EgrEEhaZGUNrcmzIoyFpG+PHHH9kYZGbxx4GdKM9L4JHwXLIuZiEsCLDfEnQV37SAjICfCLq9K4sbnLG2fVljlqVsLmLHgR4qBJoINNUjc656PVeYZZCL0Mjae9Rz9TPOYCKmI1O19hQ0puaKJzyzaCwqNO2wN7UdJRRmPGVc4srUaIDg0ERA32+rTWWVBhCZM6BSWipfjdGCQQbX3b4MnNaHMwWDbnTmrkp7boA+blWFg91CZiOZ6z//8z8NIt2M4N5gtveHVXAyENAzi4+ZGzPd3wGGWB5qCTTVWPmUWYgz7kodDpxkCmSCwxXQcLXbOIsbnLG2fVljlqVsLmJHIwxHRqCJQLM33uu59sJmFvFwJ65+xnPhkTRBgW4oQ69X2JuyQEqsKVD+9a9/5WrGg5qRbiQiE4Ve9dtqU9nq0geTa9FS+Wrs8AmD9I2s0zIViHnBsAkueCsYd6Cxq9peIeMWSuLSp/s96D13FuOOfWQElGj2mIUhDGR4VaDaVLeTKZTroOwEY90Cht+9e/eHH36owsMJ0/+dWNzgjLXtyxqzLGVzETsa8dwi0ETwXHneoPQ8PpjljFUO2swiHu7E1c+4FcKoS4I/hHQ8hhtw14DFYhCzaDBLEEikyMKMLBz4aZI0Yk/ot5urTYbTgc4oaXK18zRPo6Xy1dheMEIIB0Vl3nCr8HctFNdZkehpqXw1UjBCuGFQLS75aTvcRNh3P2HwqaWpztBS+WqkYIQQwpHQUvlqpGCEEMKR0FL5aqRghBDCkdBS+WqkYIQQwpHQUvlqpGCEEMKR0FL5aqRghBDC6vR/C/tq+LWS/R/RPvn6XTSUflPk4/Tm1v6PploqX40UjBAOlMopClwVyBrgVzHQmEQgf257yLh3JHeE8W/S0M3v7r3p3niNEgFT3NJs0VL5aqRghHCg+MMjIPBjJjmi4IfNk5MT8gU/aXI1d/hN4MFMFK4YNohy7jYNvmcPqmB4GJRRch58scrU65+0VL4aKRghHCikA6oC+YWfLkkxlgpSBlczhemD5k8//UT6QKgfQsOhwWaxm24c6f5srj8XujGQUmF5YH85DAh37971Z4XW7ystla9GCkYIhwufGAChUkP/C42+YPibbjJLfit1mFgwfL3HSMGgHrDXdKMzA910hmMHwR8LuCoULZWvRgpGCIcL6cBK4K+bAAHlrGDQ9NdWaGa/1A4HAltDOWcH2S+TvnqKAXrlHjrQDSwSTTv9DPHzzz9XneBWv+Mtla9GCkYIIVwnlf0vT0vlq5GCEUIIR0JL5auRghFCCEdCS+WrkYIRQghHQkvlq5GCEUIIR0JL5auRghFCCEdCS+WrkYIRQghHQkvlq5GCEUIIR0JL5auRghHCzSDfyLvpfPz4cfaVC5ovXrzw69+L0FL5aqRghHCgvJ/45ZdffOGHr59r98JNg3r/5MmT/jvbryYQqBluce21Pxwgozk9BMOve2mpfDVSMEI4UPjxkxRzcnLC1RdFjCeOcGiwdxSA2Us+2GI0vk/w4cOH1A+u7Dg/HLjjaLiOfxW8pfLVSMEI4UAhTZA1EMwy41kjHCx9wbBUKLDR7q8daNaLh+vWCC2Vr0YKRggHSgrG8dEXjBcvXvirp3MLxv/4H//D905SVFIwQghbmBWMhw8f5t+9bzpu5avp1eXv379/8uQJVQHOFgya9QbiFIwQQgib8BdW9b9ojNBS+WqkYIQQwiGy6794Q0vlq5GCEUIIR0JL5auRghFCCEdCS+WrkYIRQghHQkvlq5GCEUIIR0JL5auRghFCCEdCS+WrkYIRQgir82L6Fp708q+//uobpRahpfLVSMEIIYTVeTy9CsyvXtYXMH+Z2OkPZzfTUvlqpGCEEMLq+I0K8evcXPf4psVmWipfjRSMEEJYHYsEPHv2DJkPGdaJnV4VtZWWylcjBSOEEFanCob/KwYFw3/JSMEIIYTwDRQJPltQLagQFg8KBtRrzBehpfLVSMEIIYQjoaXy1UjBCCGEI6Gl8tVIwQghhCOhpfLVSMEIIYQjoaXy1fimYIQQQggXkYIRQghhiBSMEEIIQ2wpGJ8+fbpz5w5X5JcvXyI/ffrUW8tycnKi8Pr1a+QPHz7YBGSmtgOzS/Uf5MuXLwyB33//van++7/fvn2L5Xfv3iE7C7N7a1cYC71x5tLV1r4EeFiOITsLDu/t7eIGZ6xtX9hTYl572u9mwa2ZZjPnnhMoO/1p3MAiEVjEyEWsavxcrn7GrbCPPqTPnz/HH5g2/4Rj0HrsDkvjhChjB+OYdRaoxaKkJ8Lnz5/pD5eZ9MrYVDBcuQUDHjx4gJImK7SDd4FAcBeBbtxVOXgOfOzv3buHTBB57BEePXo03TwF41xxBuNq6GOsx3G4+6fGw4HAWfn73/+unuO7q2XAHwb2xqGXLwmRJLwuH1kB+3sfssUNzljbvrB9WvZpr92s88mmcJBwwOYIZ88J0Cw76vvTeC6LRGARIxexqvFzufoZz4V5ORjKzK4bnB9OjqXikjWMsZwWDyHnhMzGFEzkXc9qfzLRMClK4sAoKJc0oobrVUbpIrb/SsoywMpZT1N9BQ2BYCX2oUkI0JhzUbrgzRAFulVAgeEEsTWmDYBKBFz7u+MwkaZsmuUR0Pz5z3823bCK6jCOW45Qq8BJDgQ2NXtJcMkT5hI8PVgmUIZ6VxY3OGNt+9KHvd9Nppvunz5ye2yoPvejkHFbzXTnn6fxIuhz+QgsYuQiVjV+Llc/4wym45xwKlr724JhfqMPgmdpP1gaswAyq3OKygyenP5k1i0ENHRgLDIx4erPsshVa6+XHQoGAuA3C1b+7rvvCK5NAw21sJ1WWFEDRtHEoE0iiIbQu4seNW/tBMcUCzVR2VHAc6YAd3EnymYJzOW5xOB+3vboIc7rHjICpwqBx6912oXFDc5Y274QYQKOZa7OgrIEQUbTGmPMzgkyh7/szE7jRejGJSOwiJGLWNX4uVz9jDOYiOkqsQC7zNSeIvYdDXfpQ9MOe+DSOCcYxA7GafI5AwFlHRuU9ESok4bg2FLSJC+pqVvXy2jBANfQl4GpQMwLBt38eUGlPbeicVKAebaiQ9PNo2l8CTrXXWGfPBBOBBjkZCBgv+alm8qdwDGHl/EyaK5RuTcVDdbOFMiccq5g/rLbOIsbnLG2feF01Z5is9/N6f4p6D02g5w9Jz7z+M/J/7//9//OTuNFLBKBVcN4NXvUc/UznguPpAkKdEMZer3CHrhMzgkCR9EpzhpESQcE1s6VU4dQIbI/zT/+8Y/WGAz2rl4XowUDgTUg89ioByvErGDYBMMxiAGqqNFE9uHkCmhMBPbcFZaAb9jhShOBK6aYSw1XZGY3X+wES8YUNitnYUTjnoZLgh23ALP8qPKnP/2pqhox0f+dWNzgjLXti3sKPuf9brq/QB+UyiNoc3ZO1GsHDTCXp/EiFonAqmFc1fi5XP2MW2EfdUnwh83lma3KsQcukxVRDmk6BVfvFijpiUA9YEaWr0Z/7E/zb3/7GzJeWVxPR14r2wtGCCGEa4EfRqk9CPv9LLs4KRghhHCgUCT4nMGHDH8xde2kYIQQQhgiBSOEEMIQKRghhBCGSMEIIYQwRApGCCGEIVIwwk3lywUvCgzh0Hj39WWLBef26VeQOcwvu6+C0b/d+/o96AMhBSPcVHiQPp15UWAI145fnrBCcD4R6ht5s8pRRxdBPdf+276lPxBSMMINhqeRRxFaO4Tr5sP0Avy3b9/eu3fPd0BQJHyjEnirakBfMBR66HxQHy8gBSPcYCgYPJ/nPmwhXAv1+YBjSW3wg8XJ9CYofrJB2ZeBDQWDPoxqjYMhBSPcVCgVFAyEFIxwONQ7PB49ekTB8BvaFgwEaoaC1NGls6O4opz9YupwSMEINxU/4PMocm2qEK4bcz2F4cGDB2R/ZWpAXyeKKhiMQqYnlUMZgSsc1OeMFIwQQlgecn2TjogUjBBCWJ5D+/fqRUjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBCWpP/C3YcPHxBeD7zu7F33XkKEw/yr3BSMEEJYDN8Zpfz582dfQ/D27dt6hS3Cly9f6ovfCN5iFNVFPR1UItsfwWZfV66eFIwQQlgMEvrz5899wyB1go8XfNqwhNy7dw/lo0ePaHKLK3cR6GwfBjrcqoOSUfahbCCgpzPN66oZKRghhLAYZHZAsDCQ5ZG5kuItACj9GEFTjXLpKQkWDAsDzSoSVhGa1BUHXjEpGCGEsBj1EmXSOjKJHtliYHk4WzDoj1D6+oRB4bFUnFaMr2/YrG7XQgpGCCEsBtmcjM9HCjI7TWQEC4PXyvg06/dXFgyuDAQLBn0ePHjABwtqBkqa104KRgghXA/+bxmUhAOpB1tJwQghhOvBjyN8wvC3WIdPCkYIIYQhUjBCCCEMkYIRQghhiBSMEEIIQ6RghBBCGCIFI4QQFubdBMLnz5+fP39eyt+nV3r0f0c7+4Pa8ZcVXgspGCGEsDBkfJM+2f/l9KoPlQhUi6dPn36Z4Na9e/emEaf41W6Etxe/rFAl2LxiUjBCCGFhqmDw8aLKABoqwYMHDygANLlyy69/i9/o9lvfNM++rJDPH5hC6MvMVZKCEUIIC0NOr/zOhwOSPuUBjdkfWr+v7wsR9P6GiiufIbyF0o8XNKtzCVdMCkYIISyGGZ8sT7Xg4wVX0v1piZio1F+/YupTP535JIFgT2/1ozDopxMKD9erJwUjhBAWg/zOpwoS+t///vf6527ws4Wpn6TvZw7kKhhWGobQTeXZguHHDuw/ePAAzdWTghFCCDcDKgdlBvpSdJWkYIQQws3Afz+nWuSvpEIIIRw0KRghhBCGSMEIIYQwRApGCCGEIVIwQgghDJGCEUIIi1FvGAQEX0EoftPiRpOCEUIIi/F6+o53fUHv8+fPCFyhvqN3c0nBCCGExaBgPH/+nA8WXCkb1o/rfWPggqRghBDCYtSbPHztoPXDW9f1AqgFScEIIYTFqIJRnzAQvlzrGwMXJAUjhBAWgwrx9OnTkwkLxu+//07NQLiuNwYuSApGCCGEIVIwQgghDJGCEUIIYYgUjBBCCEN8UzB+CyGEcGNpqXw1UjBCCOFIaKl8NVIwQgjhSGipfDW2FIxffvnlzp07XL///nsEefXqVbu9HC9evFA4OTl59uzZbIpff/1VDQI94ePHj94a5/FETQSzubDJMpUXgbhhv58RzrqxFTwsx5AJAsKbN2/29nZxgzPWti9rzMIZYGvYNew01W+/YXCm2coivq2xwGJV4+dCbAkjONe5T/oVw177PD558gSvAA3skV4KVsS6lLGDccw6C5wb3nNPHXYcwvCmGqCl8tXYVDBcORWCxKcG4f79+8rgXWBV6O35/v17lYMnz2N09+5dZDcMgf3DznT/9BA/fPhQa+7lHsfamtcaE2fnwg0WMt3cHyaqXdcaD0Y9FWfdGIEhhJexygoYx+H9TvbiBmesbV/WmKW3o6YOG6elzuRWFvFtESMXsarxc+FB8NFginOf9KuBlfZPqAvHHzzZO730MJZ85aJ48MlsTFHHycqkXPTxVwO9zBAMOpCebhBNmbo0Wipfje2/krIMKNcJEzaeVbES+9Bk5WjMjyhHjgKLp5vRqSNVEQQiglxbSGcmqi0fBJfYKgZW7p7NhQz9Ju0KjmGz9g/BR6J3/qwbIzAc3ziFxEpvmQvjGNnJTrG4wRlr25f1ZsFgnYRzz+RWFvFtvQXCqsbPxScCmG6/qF4SFtg/ocAuOztKnkoE+uyRXnpcHSCzWKeo47ShQPanDpAZTn/1mMUxYHdQ4iEySn/ULloqX40dCgZXCgYCC0YJf/jDH3DapoGGKiqz6rIZI1WnZ3aMkNEoMxGbQShtDsIoQOAJUdPP9fPPP2uw37BdwRqOOYtNfZ45P3NjBF3liPiwISMwF8JOdorFDc5Y276sNIvPZ6UVm1jmbGNZ5VYW8W2lBcqqxs+FuZiIK/nOGVGWcAUwEQusJxTYWRbr/rLRaLhLH5p22ANXZKLHDsZpktYRTPSt37fMTh0YFoPGLTXICDTVKCtIS+WrsUPB4IHR3WIqEPOCwQL8CUWlPbfispmI3UIg0LPYOXX97DML01aIuwcCD9X0c/3rv/4rBoF9veSPVwwvC5wPrkztRMozN0bwCCJgECeROeVcAQ1Xu42zuMEZa9uXNWZhd9ia/uyh8RjPzuRmFvFt1TBezR71YNbD73TnPulXQP+E6oky9HqFPTCwrAuBpTnFZoPnnjpLC2cPOw7HDjICzQomu8a1aKl8NXYoGHhZARUWCbOCYRNc2yAVUAQGYgeZoKusSFn8CdbMk61ggVHuIk0t93NJubEIzAJuLQ5zIGZuDEJntwA7lLQffvihKhA2e/8HWdzgjLXtyxqzMIonkGPgSWCnMO6WgX1GWMS3VcN4NXvUg1kjqXEijA9rTDQOPhgEIQK4h5O7ppceA8tiTeVO4XG6CIIwO3UqdYaMqh47GEegiX2u9PG3PkVL5auxvWCEEEI4KCgeVHqgojTVREvlq5GCEUIINwx/o0O14KNGU020VL4aKRghhHAktFS+GikYIYRwJLRUvhopGCGEcCS0VL4aKRghhHAktFS+GikYIYRwJLRUvhopGCGEsC5+Das1Jl58fe8hIH+cvrv3/fffP/n6ta1278y33zbTUvlqpGCEEMKSvJ9eFGiFIPsj1NckZ5WDelCCeq79VytKP0hL5auRghFCCIvxZnot4MnJyd27d/2SNkXCF+uBt6oG9AVDoYfOO328gJbKVyMFI4QQFqM+H1ADqA1+sHjx4gUCnzNQ9mVgQ8Ggj+8I2YmWylcjBSOEEBbDf4RAePjwIQXDdwhaMBDqd1NSdYLOjuKKcvaLqXFaKl+NFIwQQlgMcz2F4f79+2R/ZWpAXyeKKhiMQqYnlUO5Xke40+eMlspXIwUjhBCWh1zfpCukpfLVSMEIIYTl2fXfqxehpfLVSMEIIYQjoaXy1UjBCCGEI6Gl8tVIwQghhCOhpfLVSMEIIYQjoaXy1UjBCCGEI6Gl8tVIwQghhCOhpfLVSMEIIYQl6b9w9+bNG4Tvv//eWxt41b2XEGG/v8ptqXw1UjBCCGExfGeU8vv37589e4ZwcnJSr7BF+PjxY33xG8FbjKK6qKeDSmT7I9js68pZWipfjRSMEEJYDBL6kydPfMMgdYKPF3zasITcvXsX5cOHD2lyiyt3EehsHwY63KqDklH2oWwgoKczzYtqRkvlq5GCEUIIi0FmBwQLA1kemSsp3gKA0o8RNNUol56SYMGwMNCsImEVoUldceCMlspXIwUjhBAW482bN/4GibSOTKJHthhYHs4WDPojlL4+YVB4LBWnFWP61RZUt3NpqXw1UjBCCGExyOZkfD5SkNlpIiNYGLxWxqdZv7+yYHBlIFgw6HP//n0+WFAzUNLcSkvlq5GCEUII14P/WwYlYbAebKWl8tVIwQghhOvBjyN8wvC3WJenpfLVSMEIIYQjoaXy1UjBCCGEI6Gl8tVIwQghhCOhpfLVSMEIIYQjoaXy1UjBCCGEI6Gl8tVIwQghhIV5NYHw/v37J0+elPLX6ZUe/d/Rzv6gdvxlhefSUvlqpGCEEMLCkPFN+mT/Z9OrPlQiUC0eP378cYJbd+/enUac4le7EU4uflmhSrA5o6Xy1UjBCCGEhamCwceLKgNoqAT379+nANDkyi2//i1+o9tvfdM8+7JCPn9gCqEvMz0tla9GCkYIISwMOb3yOx8OSPqUBzRmf2j9vr4vRND7GyqufIbwFko/XtCsziXMaKl8NVIwQghhMcz4ZHmqBR8vuJLuT0vERKX++hVTn/rpzCcJBHt6qx+FQT+dUHi4nqWl8tVIwQghhMUgv/OpgoT+v//3/65/7gY/W5j6Sfp+5kCugmGlYQjdVJ4tGH7swP79+/fRnKWl8tVIwQghhJsBlYMyA30p6mmpfDVSMEII4Wbgv59TLfJXUiGEEC5FS+WrkYIRQghHQkvlq5GCEUIIR0JL5auRghFCCEdCS+WrkYIRQghHQkvlq5GCEUIIi1FvGAQEX0EoftNiVVoqX40UjBBCWIzvp+941xf03r9/j8AV6jt669FS+WqkYIQQwmJQMJ48ecIHC66UDevH5jcGLkhL5auRghFCCItRb/LwtYPWD29d9AKoBWmpfDVSMEIIYTGqYNQnDISPG98YuCAtla9GCkYIISwGFeLx48cvJiwYv/76KzUD4aI3Bi5IS+WrkYIRQghHQkvlq5GCEUIIR0JL5auRghFCCEdCS+WrkYIRQghHQkvlq/FNwQghhBAuIgUjhBDCECkYIYQQhthSMO5MvHz5Evnp06clL8iXL1+wCb///ntT/fd/v337Fs27d++QP3z4gPz69WtvXQYM9rNg8+TkBPvI6JEBf7y7E9MKvlkCZrG2iNuCtRLYC7AJzoUD485rQZs4yVijLXsY7GGUw43tWYeBWDkjAj3ps99csz2FsrwfumpkpD8ng8wiAGf93ApD6vzUcAwucqjONX4F1Fznnjo0i6xukDqZz58/xytAA/sdRSmbgIyGFSHUFO3edLeaxsGeO520K2ZTwXBHP3/+TJ349OnTgwcPUCKjsQN3T+vJnTssmLsIdLM/DG48tcEAYU0NQTTQhPjvf/87xpHp05+tPcDsvXv38LCaTI3w6NEjrs7C3T12yyVwyDQCBMHlcKvCtTdYxhrO26xZigqRK9oKy6zdqWhX/GFXgzMwbpy1c9ZhLBN2feAuC3SN3h1ntqeAnbK8B31kZHZOBplFgCYPSO/nCP2o3iCBYplTl/051zhXjivGPbSsnWs1L0/t19lTxyzGirNxySd9M8zOFMrM6MLxh2yDS7jBXQJih71xLYApzw+W+yPEXCyzIg80XbhuEHNuKbgRgKZvXj3bfyWFixwsVmII+nPPZqNkAZYKmqwfjctGOXLOPDpQm0R83VE0f/7zn7mFjP3qsB+6V84Lrlr5ucJ+jyIDHVWnhCXgNs1L+iweGo0jcOymgJ2GpYdJjfxWXDWLRcA4pvATTbv9lXGD54Jlj8dZhwl4bWgFrYRxzu4pTXzeO+x9ZJpqQn1rDGMEEFjvzM8RGGJlxY7DCSZhxJmZe3tw1jiuukz0Lpa7eI7S5/Hy1H6dPXXmAQTuole5LESPI81Erf1twcA3BPogXH69m081IXU6rqyaIOAAVxZOf2bHK4JPN674TDc09FQ+1+bVsKVg4KXFANf1koLxl7/85fQTxJ073333Hd4TYmQDDVVR+tKyAaJDmOqMgkeqBGwSKbjMMWIsW1KWC5qsixWxMTRxgy3x1ji1fyUwEQ4jcO0P6GXQOE+aBrHcr6WO4AhsFiCwajcOU8Rfpexk8CzmSly9yGFkN5SJmNpd9tYgZ/eU7cNaWd6DKTAtMmoEmyynj89WKgI2ez8HcQiLIjLKCJxPhJl7ezAzzk9m2ERgI7ziOVczVBtzOTBb+0UkMYvQnzo0OKM/apaF6VhOv4lsEKvmyqTsFxru0oemHfYGmzOhIAilxCWuHhVgO2gioDcIyHajqVzKa2FTwSB2VAKeQGQ8pgAgWBum+6cbDHbjapPFOKTvuQFOjFtVUXBfEdg2ggvI+6XyAuPAEupJK8vsxN/+9jfLVe3TTjBEU7WE8pZb2FR5STRebpsZpzun8k5u454xZ7/w02Pau7qrwRkYx7J+nuswVKi9yxC3YJxpS7/ZUw4MGpooNbsrfWTUTO6fmtppK/sIyE7DpYYQGdaFzNK4AhqudtuPmfH6KI/nPLZslv5zhWnEZTndra/7de6pM1z17KwESwNl/OnD2OsV9qYsEL2aAuU//vEPrp4xqBnpZrpD1isio8zVpjJjFa6FTQWDNZD0hTOElwj96UGGWcGwCS54K8SII0uwGEvTU8tcDFfDFZk+FeW9wY6bxyxYw6bbgIyAEhn/7TyOwWG4B90llPGpywJoyuNiQGgyF7Pfu3cPJdT52wxBMObYmcVhP4Mz2DKSghbOOmyfeiQIGkru9ul1HIzUnqopy3vQR4Ymwiw+dttKHwE1Z/3cSg1hXnbkT3/6U2VSU7nyfsyMI2MQ33AY454B7rJ2M/tSOO8sqsZkwSd9EGY3CFJHcb9j31P77koNLGZZICUTWZiRVQNBJub2BLyiZ9mxyXA60FnltbD93zCOEjcjhBBuClZuyjkVRc3Vc0sLxpX9CBNCCIvA5ww/YVxj+rqlBSOEEMKupGCEEEIYIgUjhBDCECkYIYQQhkjBCCGEMEQKxg3AP1T3j7WbKhw809/Zf/MSw4Pi3cYXDh6s24cPT+vn6ZuPg98rItT25OmuP/ev+GMHEDB7CF8GSMG4AXBQ/HrRNX5hJ+xEffHqYKlDRbaq1FZyaehD+lOD4JCwAfL72+kFJP/4xz8qdFwtJAizYsxDXU93/cmsTzqm0CBg4fn0hXzk6yUF48bASUrBuCnwqPsn8/54eIBYMEheCAXeouHqSePqQuiJXJ21EM6F+LDvXOuFQ4++vsPx//yf/0Mkfb+LxQMIrE0jDyoR3AXg7oE8/ikYNwOPS/3cFw4cnnBAIDuoOTTIaOSpBw8enFaA6RUd/FxsnvKwcRc9PWmSAStblRDOhbgZOq7EjWeWq68A4UigN+bctb+hplrQB8EDg5L+lmq7wSFEPgXjBuATm2pxg/hw5iWGh4Y5q3IQpwvIaBQMfEZPkyzGLZRq7HkIaeuQIapgeA0mkeQKdqgAGl6aHBU/dkB9nuMWeqqLB8meCtdICsYNgKPGeeK4HMKJCSP45Ps7nKY6MCqj4ScgmN38xYgnDRmQSXl19nIIN2OsfvrpJ8JLrd3wDkeUBJzO3OLjhUqrSwXZg6Sskg5skJqrJwUjhBDCECkYIYQQhkjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBCW5PXr1ycnJ+Pvln7Xvdp29r087DTpMEjBCCGExSDdm/HJ9V+mN9R++vRJwaqA7JfAYRpxWmCoLii9q94Oh/a9+hSMEEJYjOfPn1MAfOEHNeDRo0fUD66+fxANt9BwrdfGIPh2Fq50sH7QGcE3ixwOKRghhLAYlgoFs78yV5qWAWUFQKCJYEWhWe+P8uW1h0MKRgghLMbJ1/9B79yC8T//5//0nyUoKmcLRn3CQPAltSkYIYRwtHz+/Jl0T1WAswWDJh8juIWmLxg0+yGUHIwgPHjwwD4HQgpGCCFcHf7C6u3btwo3ixSMEEK4Omb/4n2zSMEIIYQwRApGCCGEIb4pGL+FEEK4sbRUvhopGCGEcCS0VL4aKRghhHAktFS+GlsKxp2JZ8+eIT9+/Bj5/v373lqKjx8/Yv/Fixe//vprU/3228nJCcpXr14h//LLL3bw1mXAYD/L999/j1muyG/evGEW5V1hCdhhOEbUMBHhEmZEbwd62mEnsFaO1RKwuZ+3sLjBGWvbl3NPDlOgcSO47nFy3LV+FLJKZGzS3LqVi0Rg1TBizUUBxpt2I+XDfqy6nP2onX3y5An+ABrY7zmVsgnIaFggQk3R7k13q+kW2JOYTJb2oaXy1dhUMPCeB+P9+/fUCbK2pYIrsh24OxWUOywYvd3sD4PngABpkICqIYgGGg3WMI5MCRk81heB2bt375bzCHrIXP/5n//pLGzVHrNgR7MaKTh2LIFrGWcV3toJ7FfY+7m2pq2LWNzgjLXty7knxwg/fPiQq2FnQ+02Aj1xuDW+oh1R3rqVi0Rg1TCeXSnRA2WeO6cDlDMf9mPV5YzDvJWRmV03WCOniBOFJ9ydRWYPPCeAKY8Klj2WwlyczD6kNMFbdHYLFGpf0PTNs7RUvhrbfyWFi2xza0yfOfBYmZ0mLjRR0o0m60fjsvueGzClIlSIia87irWffvqJ8CFj/5K7qHvYae2v4MC//du/XX4WVlFLEGz207EuI7MruERAOG1M4RIIrAfuQAzOWNu+nD05whSWEKYG5JFzKI7lqJSfjGUhOA9qYOtWMu/lI7CIkYvAGsvkCjQxyxTCpASBKU7XPE3Xbkw+OHwPGH4Fp2IDTMfG9dmWk+OKUBINBPog4Od0f3/qTM4Op7Bep+NKBIi2FYuw0J/Z8YpA0Y0rPrtT9FQ+16a0VL4aWwoGXloMbPq5gZgiwB/+8Ae8t2mgoX6IKGEzRIr1E4uKQp1LhB9//JErMhqF/WAsE5XlAoc9rAj4APvNwjOA//1ZRMMJaI3uiOyBbnOMdA8ZgaODwHlqnXZhcYMz1rYvZ0+OMAsaDiTxd+rx559RgFB+so9uK3awhjCylYtEYNUwYoQoYRZoVgwJGrBqZK7AeuljZ0bZbQ8cvvap2AATMZ37KyyKqbniiUvmLn1o2mFvKp4lFJ5bZePJ1GjAg4qAvt8Xm8qlPJeWyldjU8EgdlQC8ylQAGbPCU2wG1ebLMYhKu25Afq4VRUF9xWBbSO4HF9kQqlyPzAOLKE/mth3h8CksN8s+M/CtVBgqkLnI6e8Bz5dCISCVSCzCq6Ahqvdxlnc4Iy17cvZk8MWuAs4sN9nU3ZNm2yomrLJJmJqcCsXicCqYWR4v5CKIQJrNwhEw3kJNUL5sx+rLmccdhCUdUMZer3C3pQFgllToPzrX//K1fBCzUg30x2yXrk72rGpzFiFc2mpfDU2FQzWQNKXP/7xj03qSggdgMOEkmvfhP44boBYOFCzPuREhOEo1QBHqqK8N3VkMch01A8mAvaJubi73yyM5ehriibGudZcBOTu3bverdDtRJnCN0z98MMPVdX0XHmcxQ3OWNu+MAWmoE4O07GDzE6okU/PzfT5w+dwBGxigVEYoYmAHSzQRD++lYtEYNUwYtk1CsvBIGtEwLhBYDrjaRjLn/1YdTn7waL6FeEPKzUITbUvWFZgsQbTM0MQKu0AM7JwoDZzuuwJtTv04WqT4XSgs8pzaal8Nbb/G0YIIYRrh6LCleJKRVFzlpbKVyMFI4QQbgB8zvATBp9amuoMLZWvRgpGCCEcCS2Vr0YKRgghHAktla9GCkYIIRwJLZWvRgpGCCEcCS2Vr0YKRgghHAktla9GCkYI4Z/4PYzWCPsy+8vXN1+/397aq9FS+WqkYIRwS6EwVG0o+f30/flfvkJTPUplO9iH6zQ6/BNi8uzZs7t377b2FFI0CCfTi0mmSLcwEkP7qASbe9NS+WqkYIRwG/Gbw/zY65VmQRZTIOv5dhD6q0Qm9zEEwWtqxgwCQjHov4ztF8jREECaRJXQPXz4kCa3uBJkomrAHbI3LZWvRgpGCLcRUpVJnwR3//59shWY1Pyxl1uAYO6rguHYJ19fwVI/I4ceAyUEyt9QceUzhLcqzjSrcwl701L5aqRghHAbIXPxgzAlgZ9tK0+hNJH5M69K76LpCwZNBH+vctopfEuf+okVEUMwtt7qCwbV1zDysYPrZWipfDVSMEK4jVgq/JUUkLMAwURWnzmQ0SN4NdmR3RBspmCci4ECP1v00fNqbG36sYPtIOxoLkNL5auRghFCCNcJlYO6C9SVptqXlspXIwUjhBCuk/fTn1FRLfJXUiGEEK6IlspXIwUjhBCOhJbKVyMFI4QQjoSWylcjBSOEEI6ElspXIwUjhBCOhJbKVyMFI4QQjoSWylcjBSOEEBbj/fv3T6b/eXvwb2R9XwiC36UvpYJfq1QeoaXy1UjBCCGExTg5ObFa/PWvf6V4oLEefJxeSohQxUAef30fl1/2ppsyV0oFmtNOw7RUvhopGCGEsBikeFM/nxj85vbDhw+pGX5WoDbQ5GrxAAsGzXpNi0oEy8ZOtFS+GikYIYSwGGT/7ycQ/KjB1Y8dVAX0vqeLu/a3YFAt6IPg+wctOdSb6jZIS+WrkYIRQgiLQYqf6sVpSeBTQtUMsEN9bvB3UzQ/fvzoxw6wSNgHPdXFX1IN0lL5aqRghBDCYpDuq2DwkcL/E4ky4Avhe/y3cWpDvf8crC5VVCwhyiO0VL4aKRghhHAktFS+GikYIYRwJLRUvhopGCGEcCS0VL4aKRghhHAktFS+Gt8UjBBCCOEiUjBCCCEMkYIRQghhiC0F487Ey5cvbb5+/frp06fKS/Hlyxfsw++//95U00QnJycfPnxA5oqMxlt7g+eAqdaeYNJ3794hMAs+XH6Wnk+fPmFzNuO5bozgELzVAlTEzp1oM1pgyLkGgX0xGgh0g/7uZhyCS+4gVJyFpjbp+fnzZ3oCcrs9xsyx/swUs3lHMBSYsjmLz2CoZxHQt11PF1PXEGSXWcyau8LwtqqnTwdDdNaHPajQnX2uMY4Gdj0Je8NcRuD58+fMvogDZROQ0bj7NUW7d0ES0AdlhvB0qHSPcGyPJ2VBNhUM1olzeEzNoKnAOr0L3J0KyqnywYMHCDxOdoPBx+Pt27c+VFhTQ3RQIjx69IireoKI8dPbe8HYs/4Qd6ZAj+C68GTw4bkIJqqcpU3WUjbPdWMrnpJ79+619qThMNW5OTvRZs66MTMIzKhZBPaUW+Oe09PN0gJjjfN08xT1xsp52fQ6AIOwWCwwFmuzMyMYn827lYs2iFn0czDUfQTKJmcYP6f7QzCKx0o7ZZCrSYRw7WRtRnlVYK0MMoXTAcqZD/tB9OoYY9/tJpIuB/pToWYNevvuDgJrZH/ZIJzk7iwye+BaAFOeTCz3h5O5PEJM3Z8l/HEsbhAiPUGJ4PGrPaq6cpVs/5UUvnJqEfAeKhBgk/22VNBkVWhcP8o6Chtg/awcZpuEEbZQGePII9YuQmt42O8NM9Lkqg9o3Bjv7grucTJqO8+1ea4bW+GgYLyPPJYxq7yH82fd6A0ChxWckSs96V9LG8TzjVBxVo8drOlA9YF+gYMwHLNl2XUpAxPtuqFnIyPGZ9dQ96sDLO90hpmCXSDLuEwmxZoelty67g4WWCZXoMm6mEKYlCkwjhIQ2o1LF4w6xjwpWEbGpndnp0LlssyeUMABV4SSeRHog0AEpvv74zJ7oYeQOh3z4pIb4S0EhxgHZZSEi5NgN/QINnc6UZdnS8HAIYsB4Drr5IqLp58g7tz57rvvWJVNAw31M1EJm2FvODSe0aaacEbD4XZeZhexo6kq8lhjOczikcX+9HTs/7svTLH3zmJTUyXAWTfG8egAx6WP1bkTbWbmxswgt4gDgjOyuXSY9dkK/RnOxs3iDMjOyy0iVusqYRxmwUgNxDKyS5PxmMgUmPkG1drL2ojZioBNDhhxUB6EKZiIBXos//3f/91JsYy1rQ5sxlhhRDsVQ1YKTIHMFXCbPnZmlN32xokw5Rnjaoiw3J8KhMVhiv4JBZxhUq644ZK5Sx99uwwVzxIK1ltKYstW0nQXwH3BGRxDNu8h8MmMzg7ESfqTYBfZkZ3YVDBwi2ThKcfv0xIxUWtDCXbjapMlOUSlPTdQp7OCSEA9Q0z0t7/9jXghE5eadw8w4ix4qIYzwYzsCnGvGS9/WFm7yweWxrW3edaNcSo+GKkp5OxEm5m5MTOIzFzQn1EoYSsYx7IhncXZuzrsniqXcpxaBfanDWxnpn+Edj02s8hIH5/BUPcRAILAEOVxai1Myhr//Oc/m8h81i7zOMAsMv0uY9wg4DNNejIjwiy2++FEFcCyWQdg5tjisJW1m65OGXq9wt6UBYJZU6D8xz/+wdXwSq2X2TkwNNllgmNkagvsw1UBLu/kHmwqGCx1KhCnmPpZUu8lHYBbduibUAvbDDHioBAjxtJE8Ogw3MiiV67Hbw+IuLPolQ+eejXOQp9+Ly8Js4A26zT0buxERZ6x2FHGlFcYd37mxlmD4owcWYJD//GUR39/cCufK87aR0bgLqcFsxhnSPkwCP0ZhR2uszNTq6h5B6F/H5nytnxDA/TZHOo+Amw9xVKZM6DNEWpe5qJ4Izu7dsgp46bOMosMTuIz6/KgGgSmMKpO2sdhb7CjoGWbLqQmMttcAczVr8iQGoSm2pdapifTdWGWNdZhAPsInqi0SDTtFKi//OUvDLTJrctkwkuy/d8wQgghBEjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgXjxuDfqofiy/QdHcJyjX+WvgG/VTD+5ZUDwe9htEbYnVkAp29WNN52LybhYMzOxtkH/Hn3LhkGetrPdrtKUjBuAGbG/uWDAXhy/NYVz5Waw4GsAQjX+3hvhhxU+atkMhTnjcAKTfUole1gH67T6PBP+EGByLRG9w0+QF/ngUNr9KrA9D2BcsJT7/f16PloeqUYMlUHpi7XQArGDYCDwlM6O0+hHrkDjAy+8ajj2OynyMMB90hV5C+vNAvymgI/o+C/4VWJTBZjCIJXtyAURKkvGCR6mtBXBYJGGBHQI/jJw1sFerqppBtm1V8vKRg3htl5Cj5jJLsDjAwuWSoOdtdIUvhG0icr+Ro7QMOV9EQHbgGCSzBnKTO2KqKdQ1EBlIqtwSR0HAyu9kFPDKsGTyNOYVOsNNyiv8Fv966VFIwbQ3+eAvi7EYQDjAxl7NzfMxwOuEcAyVOkpHISJYmJ9ESSQq/Su2jMbshmPYTaglAYwNY4cwCIGIHtf4la/fueRLg2An3/CwaU9XutqycF48YwO3mBJ4dnD/zZ7aDgCScp8GBf47O9GUsF7iEA3gKCeap+LkZGj+DVQ+ivSmymYMwgLPXeSQrwvXv3lMGfIQg79cDOPa3TV5r26z99u0fsF7euMeYpGCGEEIZIwQghhDBECkYIIYQhUjBCCCEM8U3B8H/KCyGEcBNpqXw18gkjhBDCECkYIYQQhkjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCECkYIYQQhkjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCECkYIYQQhkjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCECkYIYQQhkjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCECkYIYQQhkjBCCGEMEQKRgghhCFSMEIIIQyRghFCCGGIFIwQQghDpGCEEEIYIgUjhBDCAP/93/8fgKLxEOfykd8AAAAASUVORK5CYII=\"\u003e\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;PNE pneumonia, ILD interstitial pneumonia, BEK bilaterally enlarged kidneys. CEF Cefoperazone, ACY acyclovir, MER meropenem, VAN vancomycin, IVIG human immunoglobulin for intravenous injection, MP methylprednisolone, TCZ tocilizumab, JAK jak inhibitor (Tofacitinib Citrate Sustained-release Tablets), Biw biweekly, TIW Once every 3 weeks, FIW Once every four weeks, LEV levetiracetam, LAC lacosamide, MO month-old.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"STAT3 gain-of-function syndrome, de novo mutation, PTPN14, microRNA, inflammation","lastPublishedDoi":"10.21203/rs.3.rs-4181172/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4181172/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground\u003c/p\u003e\n\u003cp\u003eSTAT3 Gain-of-Function (GOF) syndrome characterized by early onset autoimmunity and primary immune regulatory disorder, the immunological mechanisms remain poorly understood.\u003c/p\u003e\n\u003cp\u003eEmploying whole-genome sequencing within familial trios, our study elucidated the pivotal role of de novo mutations in genetic diseases.\u003c/p\u003e\n\u003cp\u003eResults\u003c/p\u003e\n\u003cp\u003eWe identified 37 high-risk pathogenic loci affecting 23 genes, notably including the novel STAT3c.508G\u0026gt;A mutation. Furthermore, significant downregulation of pathogenic genes in affected individuals, potentially associated with inflammatory responses regulated by \u003cem\u003ePTPN14\u003c/em\u003e via miR378c, was observed.\u003c/p\u003e\n\u003cp\u003eConclusion\u003c/p\u003e\n\u003cp\u003eThese findings not only contribute to our understanding of the pathogenesis but also highlight potential therapeutic strategies. Our study suggests that combined JAK inhibitors and IL-6R antagonists could offer promising avenues for mitigating the severity of these genetic disorders.\u003c/p\u003e","manuscriptTitle":"De novo mutations promote inflammation in children with STAT3 gain-of-function syndrome by affecting IL-1β expression","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-18 18:52:32","doi":"10.21203/rs.3.rs-4181172/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e99888ec-c7fd-4f75-af96-f4a0303c6333","owner":[],"postedDate":"April 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-06-13T15:57:47+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-18 18:52:32","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4181172","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4181172","identity":"rs-4181172","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}