Introduction: As a valuable genetic resource, native birds can contribute to the sustainable development of animal production. Tibetan chickens, known for their special flavor, are one of the important local poultry breeds in the Qinghai–Tibet Plateau. However, Tibetan chickens have a slow growth rate and poor carcass traits compared with broilers. Although most of the research on Tibetan chickens focused on their hypoxic adaptation, there were fewer studies related to skeletal muscle development.Methods: Here, we performed the transcriptional sequencing of leg muscles from Tibetan chicken embryos at E (embryonic)10, E14, and E18.Results: In total, 1,600, 4,610, and 2,166 DE  mRNAs, 210, 573, and 234 DE lncRNAs (long non-coding RNAs), and 52, 137, and 33 DE miRNAs (microRNAs) were detected between E10 and E14, E10 and E18, and E14 and E18, respectively. Functional prediction showed several DE mRNAs and the target mRNAs of DE lncRNAs and DE miRNAs were significantly enriched in sarcomere organization, actin cytoskeleton organization, myofibril, muscle fiber development, and other terms and pathways related to muscle growth and development. Finally, a lncRNA–miRNA–mRNA ceRNA (competing endogenous RNA) network associated with muscle growth and development, which contained 6 DE lncRNAs, 13 DE miRNAs, and 50 DE mRNAs, was constructed based on the screened DE RNAs by Gene Ontology (GO) enrichment. These DE RNAs may play a critical regulatory role in the skeletal muscle development of chickens.Discussion: The results provide a genomic resource for mRNAs, lncRNAs, and miRNAs potentially involved in the skeletal muscle development of chickens, which lay the foundation for further studies of the molecular mechanisms underlying skeletal muscle growth and development in Tibetan chickens. The Tibetan chicken is a unique Chinese native breed that is mainly distributed in the Qinghai–Tibet Plateau. It has excellent meat quality and flavor due to the rich content of essential amino acids and flavor amino acids. Tibetan chickens exhibit characteristics such as high adaptability, resistance to extensive management, and strong disease resistance, but with the challenges of slow growth rate and low meat yield . The yieYY1 (YY1 transcription factor) gene in the process of chicken skeletal muscle development at the embryonic stage (MEF2C (myocyte enhancer factor 2C) (IGF2 (insulin-like growth factor 2) expression  were shown to be involved in skeletal muscle growth and development in multiple species, including pigs, sheep, chickens, and cattle . miRNAs ic stage . lncRNA ic stage  that hasic stage . In receic stage . lncRNA-ic stage . lncRNAsctor 2C) , and thictor 2C) . lncRNA-pression . To explFor poultry, skeletal muscle development during the embryonic period plays a fateful role in the potential of post-incubation muscle development. The primary and secondary muscle fibers were formed in approximately 7–12 days, independent myotube formation and differentiation were initiated mainly from 12 to 18 days, and muscle fiber hypertrophy was observed during the post 1/4 of embryo incubation . TherefoIn this experiment, we collected eggs from Tibetan chickens (TCs) obtained from Mao Xian Jiuding Original Ecological Livestock and Poultry Breeding Co., Ltd. . The eggs were incubated at a humidity of 55% and a temperature of 37.8°C. At the target time, we dissected the chicken embryos and determined gender by observing the gonadal features of the fetus. All leg muscle samples from nine Tibetan chickens were collected from healthy male embryos at three developmental stages , with three samples in each stage. All collected tissues were immediately frozen in liquid nitrogen and subsequently stored at −80°C for RNA extraction and sequencing. All treatment and sample collection procedures were approved by the IACUC  of Southwest Minzu University  with approval number 2020MDLS44.® N60 , and the integrity and RNA integrity number (RIN) values of the isolated RNAs were evaluated using the Agilent Bioanalyzer 2100 system . The RIN values of all nine samples were greater than 8, and all could be used for subsequent analysis. The isolated RNAs that passed these tests were divided into three parts. One part was used for quantitative real-time PCR (RT-qPCR) by reverse transcription into cDNA strands using the PrimeScriptTM RT Reagent Kit with gDNA Eraser  , and the other two parts were used for long RNA-seq (mRNA and lncRNA sequencing) and small RNA-seq (miRNA sequencing).Total RNA was isolated from nine leg muscle samples at three differential development stages using the TRIzol reagent . The quality of the RNA samples was detected by 1% agarose gel electrophoresis. The purity and concentration of the RNA samples were assessed using the NanoPhotometer® Ultra™ Directional RNA Library Prep Kit for Illumina , nine cDNA libraries were constructed from RNAs obtained in the previous step, and three biological replicates per developmental stage were prepared. Finally, the cDNA libraries were sequenced on Illumina HiSeq 2500 , and 125-bp paired-end reads were generated. For small RNA-seq, approximately 3 μg of total RNA per sample (nine samples) was used for preparing the libraries following the manufacturer’s recommendations in the SMARTer smRNA-Seq Kit for Illumina . Subsequently, ligation of the RNA 3′- and 5′-adapters, reverse transcription of the synthetic first strand, PCR amplification, and PAGE gel purification were performed. The cDNA libraries for small RNA-seq were constructed. Finally, the libraries were also sequenced on Illumina HiSeq 2500, and 50-bp single-end reads were generated.For long RNA-seq, 9 μg of total RNA in each sample was used to remove ribosomal RNA (rRNA) using the Ribo-Zero™ rRNA Removal Kit . The total RNA of nine samples  from which rRNA was removed was used as the input material. According to the NEBNexthttps://github.com/jstjohn/SeqPrep) and Sickle (https://github.com/najoshi/sickle) software tools. Specific operations include removing the reads containing adapter, removing the reads containing poly-N (N rate >10%), and removing the low-quality reads  < 20 at the end of the sequence (3′-end); if there are still bases with Q < 10 in the remaining sequence, the whole sequence will be eliminated; otherwise, it will be retained). For small RNA-seq, in addition to the aforementioned quality control operations, reads shorter than 18 nt and longer than 32 nt were filtered out. After the data quality control, the quality assessment was carried out, and the Q20, Q30, and GC content of clean data were calculated. All further analyses were carried out based on the high-quality, clean data.Large volumes of raw data stored in the FASTQ format were obtained after sequencing. In order to ensure the reliability of the subsequent bioinformatics analysis, the raw data were first quality-controlled. Clean data were obtained from raw data using SeqPrep  using HISAT2 (http://ccb.jhu.edu/software/hisat2/index.shtml) (http://ccb.jhu.edu/software/tophat/index.shtml). The mapped reads were assembled using Cufflinks (http://cole-trapnell-lab.github.io/cufflinks/) and StringTie (http://ccb.jhu.edu/software/stringtie/) software applications  were mapped to the chicken reference genome (the resource of the reference genome: x.shtml)  and Bowtx.shtml) , respectications .http://www.noncode.org/index.php), Ensembl, and NCBI databases. Based on these databases, known lncRNAs were identified. For the identification of novel lncRNAs, the transcripts with class code “x (exonic overlap with the reference on the opposite strand),” “i ,” “j ,” “u ,” and “o (generic exonic overlap with a reference transcript)” were obtained using gffcompare software. On this basis, the transcripts whose length ≥200 bp, exon number ≥2, and ORF length ≤300 bp were screened as candidate lncRNAs. The encoding potential of candidate lncRNAs was screened using the analysis methods of coding potential calculator (CPC), coding-non-coding index (CNCI), coding potential assessment tool (CPAT), and Pfam protein domain analysis tools . The expression of lncRNAs was measured by TPM (transcripts per million reads). Based on the expression details, a correlation analysis between biological replicate samples for long RNA-seq was performed using the Pearson correlation algorithm.Quantitative analysis of lncRNA expression levels was performed using RSEM software (http://www.mirbase.org/). Simultaneously, ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), small nuclear RNAs (snRNAs), small nucleolar RNAs (snoRNAs), and other ncRNAs and repeats were removed, and unannotated reads were obtained. According to the particular hairpin structure of pre-miRNAs, novel miRNAs were predicted using miRDeep2 software (https://www.mdc-berlin.de/content/mirdeep2-documentation)  and mature miRNAs in the miRbase database (ntation) . First, Padj [corrected p-value by BH (fdr correction with Benjamini/Hochberg)] < 0.05, Padj < 0.001, and Padj < 0.05, respectively, and |log2(fold change)| ≥ 1 | ≥ 1 .The lncRNA target genes of cis-acting were predicted by satisfying the requirement that the lncRNA affects the expression of surrounding genes (within 100 kb). When the expression of lncRNAs is significantly positively or negatively correlated with the expression of some genes, the lncRNA target genes of trans-acting were predicted by the correlation analysis of expression between lncRNAs and protein-encoding genes. The prediction of miRNA target genes was carried out using miRanda, TargetScan, and RNAhybrid software applications .http://www.geneontology.org/) analyses were carried out for DE mRNAs, the target genes of DE lncRNAs, and DE miRNAs using GOATOOLS software. Furthermore, KEGG (http://www.genome.jp/kegg/) pathway enrichment analyses were performed for DE mRNAs, the target genes of DE lncRNAs, and DE miRNAs by Majorbio Bio-pharm Technology Co., Ltd. The terms and pathways with Padj < 0.05 were regarded as significant enrichments.GO  according to the manufacturer’s instructions. GAPDH for mRNAs and lncRNAs and U6 for miRNAs were used as endogenous controls. All primers used in RT-qPCR are designed using Primer Premier 5 software and exhibited in −ΔΔCT method, and one-way ANOVA in SPSS 20.0 was used to analyze the data. Differences were considered significant at p < 0.05.To verify the accuracy and reliability of differentially expressed RNAs (DE RNAs) screened by RNA-seq, six DE mRNAs, six DE lncRNAs, and six DE miRNAs were randomly selected for RT-qPCR. We used the PrimeScript™ RT Reagent Kit with gDNA Eraser   to convert RNAs to cDNAs. The random hexamers were used for DE mRNAs and DE lncRNAs, and stem-loop RT primers were used for DE miRNAs. RT-qPCR was performed using the TB GreenAll tools, databases, and related information used in this study are shown in After the quality control for raw reads, a total of 1,051,297,502 clean reads for long RNA-seq and 97,422,413 clean reads for small RNA-seq were obtained from all samples. The percentages of Q20 and Q30 were above 97.8% and 93.91%, respectively. The average GC content of all samples was approximately 45.71%, and the average base error rate was below 0.0227% . The cleAt the three development stages, there are 29,615 mRNAs, 12,399 lncRNAs , and 1,216 miRNAs (containing 944 known and 272 novel miRNAs). The length of lncRNAs was mainly longer than 3,000 bp , and the2(fold change) in E10 vs. E14 are summarized in Three comparisons  between E10, E14, and E18 were performed during the skeletal muscle growth and development of Tibetan chickens. In E10 vs. E14, 1,600 DE mRNAs , 210 DE Padj ≤ 0.05) of the top 20 are displayed in GO and KEGG functional enrichment analyses were carried out with DE mRNAs and target mRNAs of DE lncRNAs and DE miRNAs between E10 and E14. Significantly enriched GO terms and KEGG pathways  in E10 vs. E18 are summarized in A total of 4,610 DE mRNAs , 573 DE Padj ≤ 0.05) in the top 20 mainly contained striated muscle contraction, embryonic forelimb morphogenesis, intermediate filament organization, sarcomere organization, and ATP biosynthetic process. The significantly enriched KEGG pathways were the citrate cycle (TCA cycle), DNA replication, valine, leucine, and isoleucine degradation, glycolysis/gluconeogenesis, and some pathways associated with diseases  in E14 vs. E18 are summarized in In comparison between E14 and E18, we screened 2,166 DE mRNAs , 234 DE lncRNAs (126 upregulation and 108 downregulation), and 33 DE miRNAs (22 upregulation and 11 downregulation) . The topSimilarly, GO and KEGG functional analyses were performed. The top 20 significantly enriched GO terms and KEGG pathways of DE mRNAs are shown in There were 1,561 cis- and trans-target mRNAs identified for 234 DE lncRNAs. Significantly enriched top 20 GO terms for the target mRNAs of DE lncRNAs primarily included sarcomere organization, ATP biosynthetic process, reactive oxygen species metabolic process, and other terms related to cell respiration. Significantly enriched top 20 KEGG pathways were primarily associated with the regulation of diseases, amino acid metabolism, TCA cycle, glycolysis/gluconeogenesis, and steroid biosynthesis . We predTo further explore the regulation mechanisms of coding and non-coding RNAs in chicken leg muscle growth and development, the common DE RNAs that were differentially expressed in all three comparisons  were screened by Venn analysis. Therefore, 531 common DE mRNAs, 16 common DE lncRNAs, and eight common DE miRNAs were obtained . SubsequThe GO enrichment analysis for common DE mRNAs suggested that significantly enriched top 20 GO terms mainly included sarcomere organization, actomyosin structure organization, striated muscle cell development, actin cytoskeleton organization, muscle system process, muscle fiber development, Z disc, myofibril, and muscle contraction. The KEGG enrichment analysis revealed only eight significant KEGG pathways, which mainly included focal adhesion, ECM–receptor interaction, PI3K-Akt signaling pathway, and other pathways related to cardiac muscle .The target mRNAs of common DE lncRNAs and DE miRNAs were predicted from the three comparisons. Significantly enriched top 20 GO terms for target mRNAs of common DE lncRNAs were primarily involved in sarcomere organization, muscle cell development, actomyosin structure organization, muscle fiber development, muscle contraction, regulation of skeletal muscle contraction, actin filament-based process, and sarcolemma. Furthermore, the significantly enriched KEGG pathways were similar to the pathways of common DE mRNAs . The tarDE mRNAs were selected from the significantly enriched GO terms associated with muscle growth and development. DE lncRNAs and DE miRNAs related to muscle growth and development were obtained through targeted relationships. Based on the three comparisons , we constructed three ceRNA networks related to muscle growth and development. In the comparison group E10 vs. E14, 205 DE mRNAs, 41 DE miRNAs, and 100 DE lncRNAs together constitute 370 lncRNA–miRNA–mRNA interaction pairs . A totalThe ceRNA regulatory network related to muscle growth and development was constructed from 50 common DE mRNAs, 13 DE miRNAs , and six common DE lncRNAs  that yielded 67 pairs of candidate ceRNAs (lncRNA–miRNA–mRNA) .MYL3, CSRP3, LDB3, MAT1A, KLHL31, and MYH1B) (The relative expression levels of six DE mRNAs (d MYH1B) , six DE d MYH1B) , and sixd MYH1B)  were meaOver the past 50 years, chickens have become the most consumed meat in the world . SkeletaFor the GO enrichment analysis of three comparisons, there were many GO terms related to skeletal muscle growth and development and limb formation in the comparison group E10 vs. E14, such as forelimb morphogenesis, sarcomere organization, muscle structure development, striated muscle cell development, and embryonic limb morphogenesis. In addition, many target mRNAs of DE lncRNAs were enriched in GO terms such as myofibril assembly, muscle fiber development, striated muscle contraction, M band, and muscle cell development. Many target mRNAs of DE miRNAs were enriched in the GO terms related to morphogenesis; the reason could be that during embryonic muscle development, the myogenic progenitor cells derived from the myotome continue to migrate and proliferate to the limbs, thereby promoting limb morphogenesis . These rIGF2BP3 (insulin-like growth factor-2 mRNA-binding protein 3) via the IGF2-PI3K/Akt signaling pathway (PPP1R13B (protein phosphatase 1 regulatory subunit 13B) via the PI3K-Akt signaling pathway  and CACNA1S . The TPM2 gene produced the Tpm2.2 protein isoform that played a regulatory role in the contractile thin filaments . It had been reported that gga-miR-22-3p inhibited proliferation and promoted differentiation of skeletal muscle cells in porcine  and AR (androgen receptor) and suppress the expression of RHOBTB1 (Rho-related BTB domain containing 1). Some studies suggested that CASQ was involved in muscle growth and development  regulatory network, we found six common DE lncRNAs, including downregulated lnc-45.2 (NONGGAT000245.2), upregulated first and then downregulated lnc-778 (ENSGALT00000097778), and upregulated lnc-74.3 (MSTRG.5174.3), lnc-75.25 (MSTRG.7875.25), lnc-73.4 (MSTRG.1173.4), and lnc-019 (ENSGALT00000096019). lnc-45.2 could combine with the gga-let-7 family . ilaments . CACNA1S poultry . Similar porcine , sheep , among which the DUSP10 gene was a member of the dual-specificity MAPK phosphatase (DUSP) enzyme family. Various DUSP enzymes were vital in the regulation of mitogen-activated protein kinase (MAPK) signaling pathways; the activation states of MAPKs were primarily regulated by a family of DUSPs , MYOCD (myocardin), CLIP4 (CAP-Gly domain containing linker protein family member 4), ASPN (asporin), and CACNA1S by targeting gga-miR-145-5p. NEXN was mainly related to actin binding, MYOCD was involved in the regulation of muscle contraction and muscle system processes, CLIP4 was related to cytoskeletal development, and ASPN was involved in calcium ion binding and tissue development. lnc-019 could bind to gga-miR-302d, upregulating CAV2 (caveolin 2), MYBPC1 (myosin-binding protein C1), and ASPN and downregulating HSPH1 (heat shock protein family H (Hsp110) member 1), MYH15 (myosin heavy chain 15), and LAMA1 . These mRNAs were all associated with muscle growth and development. For example, the myosin-binding protein C translated by MYBPC1 was present in the A band of the sarcomere, regulating the cycling of actomyosin cross-bridges during muscle fiber contraction by interacting with the thick and thin filaments of the sarcomere  and TNNI2 . MATN2 played an important role in the formation of primary and secondary myofibers (TNNI2 as a candidate gene regulating myoblast proliferation and differentiation in chickens. Later, TNNI2 gradually increased with muscle regeneration. Finally, lnc-75.25 acted as a “sponge” for the novel miRNA 1_4798, which could target 14 genes. According to the GO enrichment analysis, these genes were mainly related to the regulation of actomyosin, striated muscle, myofibril development, and regulation of calcium ion transport and binding, and most genes were upregulated. We speculated that this novel miRNA may be involved in skeletal muscle growth and development by regulating gene expression.For upregulated lncRNAs, lnc74.3 acted as a sponge for gga-miR-1769-3p to increase the expression of of DUSPs . Many stof DUSPs . Therefoarcomere . It is sntiation . Therefoyofibers . NihashiIn summary, we constructed a ceRNA network that functions in chicken skeletal muscle. In the regulatory process, many lncRNAs acted as ceRNAs competing with mRNAs to bind miRNAs, indirectly regulating the expression levels of mRNAs related to skeletal muscle growth and development and ultimately affecting skeletal muscle growth and development, such as lnc-778–gga-miR-22-3p–CASQ, lnc-778–gga-miR-22-3p–AR, lnc-778–gga-miR-22-3p–RHOBTB1, lnc74.3–gga-miR-1769-3p–DUSP10, and lnc-019–gga-miR-302d–MYBPC. However, the biological function of the lncRNA–miRNA–mRNA interactions mentioned in this study necessitated further validation.In this study, we used RNA-seq to systematically analyze the expression of mRNAs, lncRNAs, and miRNAs in the embryonic leg muscles of Tibetan chickens at three developmental stages. The functions of DE mRNAs, DE lncRNAs, and DE miRNAs in three comparisons were annotated by GO and KEGG enrichment analyses. The expression levels of various RNAs during skeletal muscle growth and development in chicken embryos were comprehensively revealed, which provided a genomic resource for a better understanding of the molecular mechanisms underlying skeletal muscle growth and development in the embryonic stage of chickens. In addition, we constructed a ceRNA network related to skeletal muscle growth and development and revealed several candidate lncRNA–miRNA–mRNA interaction pairs that may regulate skeletal muscle growth in chickens. However, the specific role of these candidate RNAs needs to be further validated. These results provide a theoretical basis for further studies on the mechanisms underlying skeletal muscle growth and development in chickens.


The JAK/STAT signaling pathway plays a key role in cytokine signaling and is involved in development, immunity, and tumorigenesis for nearly any cell. At first glance, the JAK/STAT signaling pathway appears to be straightforward. However, on closer examination, the factors influencing the JAK/STAT signaling activity, such as cytokine diversity, receptor profile, overlapping JAK and STAT specificity among non-redundant functions of the JAK/STAT complexes, positive regulators , and negative regulators , demonstrate the complexity of the pathway’s architecture, which can be quickly disturbed by mutations. The JAK/STAT signaling pathway has been, and still is, subject of basic research and offers an enormous potential for the development of new methods of personalized medicine and thus the translation of basic molecular research into clinical practice beyond the use of JAK inhibitors. Gain-of-function and loss-of-function mutations in the three immunologically particularly relevant signal transducers STAT1, STAT3, and STAT6 as well as JAK1 and JAK3 present themselves through individual phenotypic clinical pictures. The established, traditional paradigm of loss-of-function mutations leading to immunodeficiency and gain-of-function mutation leading to autoimmunity breaks down and a more differentiated picture of disease patterns evolve. This review is intended to provide an overview of these specific syndromes from a clinical perspective and to summarize current findings on pathomechanism, symptoms, immunological features, and therapeutic options of STAT1, STAT3, STAT6, JAK1, and JAK3 loss-of-function and gain-of-function diseases. The JAK/STAT signaling pathway is a prime example of the transmission of signals from extracellular ligands to the nucleus. It can explain how messenger substances such as cytokines and growth factors can mediate their functions into the cell in a diverse but specific manner. To date, more than 50 cytokines, growth factors, and hormones are known to trigger cell responses, using the signaling module of Janus tyrosine kinase (JAK) and signal transducer and activators of transcription (STAT) molecules .2 domains of STATs. STATs are normally present as inactive monomers or unphosphorylated dimers in the cytoplasm before being recruited into the JAK/receptor complex . As. As21]. + T cells into TH17 helper cells [H17 cells contribute significantly to the recruitment of effector cells to sites of inflammation caused by bacterial and fungal pathogens. By activating neutrophil granulocytes, this cell group enables effective control of bacteria and fungi in healthy individuals [STAT3, impairing the function of the gene product in different biochemical ways. The resulting molecules cause loss-of-function of the JAK/STAT3 pathway almost always by exerting a dominant-negative (DN) effect on functional STAT3 monomers sequestering them into defect STATWT:STATmut dimers.Among all seven STAT molecules, STAT3 is a key transcription factor for the immune system. For example, STAT3 is crucial for the differentiation of naïve CD4er cells . TH17 ceividuals . HeterozSTAT3 mediates cytokine signaling responses of, for example, IL-6, IL-9, IL-10, IL-11, IL-17, IL-19, IL-20, IL-21, IL-22, IL-23, leukemia inhibitory factor (LIF), oncostatin M (OSM), interferon-α (IFN-α), IFN-β, or IFN-γ in development and the immune system. In many ways, the transcription factor thereby maintains an immunological balance between inflammation and immunodeficiency.STAT3 DN mutations are inherited or de novo missense mutations, but in-frame deletions, in-frame insertions, essential splice-site mutations, or even frameshift mutations have been documented. Most of the autosomal dominant DN mutations lay in the DNA binding domain or SH2 domain and impair STAT3 dimerization or the homodimers’ ability to bind to DNA in order to activate gene transcription [STAT3 lead to embryonic lethality in STAT3−/− knock-out mice, but this have not been reported in humans, whereas heterozygous STAT3+/- mice appear phenotypically normal, at least when kept under specific-pathogen-free conditions. As STAT3 not only has key functions in the immune system but is also essential for development and cell survival, human biallelic LOF mutations are not compatible with life [STAT3 variant S381* led to a reduction of STAT3 activity by 50% due to nonsense-mediated decay [STAT3 variants revealed that > 95.3% of all variants exert a DN effect through aberrant proteins  rather than a haploinsufficient effect [STAT3 DN mutations is postulated, although intrafamilial phenotypic variation and at least three cases of unaffected patients are reported [STAT3. Instead, defects in other molecules transducing or modulating STAT3 signaling may also cause a phenotype that is reminiscent of HIES. During the last years, additional monogenetic defects have been described, such as an autosomal recessive LOF mutation in different subunits of the IL-6 receptor , impairing STAT3 function [341 (ZNF341) gene show significantly reduced STAT3 mRNA levels due to an impaired ability to bind to the STAT3 promoter. ZNF341 is a key transcription factor regulating STAT3 expression. The subsequently impaired positive regulation of transcription of STAT3 mimics a STAT3 deficiency. ZNF341 LOF patients present with similar clinical manifestations to those described in patients with STAT3 DN mutations. Therefore, biallelic ZNF341 LOF mutations are considered as an autosomal recessive cause of hyper-IgE syndrome (HIES) [DOCK8, PMG3, or SPINK5, may also cause an HIES-like phenotype, however, with additional phenotypic complications [Most of the cription -27. As Scription . Homozygith life , 30. Unled decay . Howevert effect . Full pereported -35. Possreported , 36. Notfunction -39. Patie (HIES) , 41. Autications . STAT6 GStaphylococcus aureus, Streptococcus pneumoniae, Haemophilus influenzae, non-tuberculous mycobacteria, gram-negative bacteria such as Pseudomonas aeruginosa) are not effective, chronic infections with bronchiectasis or pneumatoceles may develop, which increase morbidity and mortality and should therefore be prevented. The use of antibiotic and antifungal drugs is therefore indicated for prophylaxis but also for therapy. Besides the immunological abnormalities, extra-hematopoietic symptoms also exist. HIES patients present with a characteristic facial morphology including prominent forehead and chin, broad nose, and wide-set eyes. The majority of patients have a high-arched palate, retained primary teeth, and hyperextensible joints, as well as scoliosis, arthritis, and osteopenia or osteoporosis, resulting in bone fractures after minor trauma. The vascular system is also affected: Compared to healthy individuals, there is a higher incidence of aneurysms, vascular tortuosity, dilatation, and thickened walls of the coronary or cerebral vessels, which increases the risk of myocardial infarction, subarachnoid hemorrhage, or other bleedings. Patients should therefore undergo routine MRI screening on a regular basis [The attenuated transduction of extracellular cytokine signals by STAT3 leads to a stereotypical phenotype called Job’s syndrome or HIES. The syndrome’s clinical manifestations have been first described in 1966  and sincar basis . The risar basis -51; hencH17 cells [STAT3 DN mutations. STAT3-mediated transcription of genes encoding IL-17 and the transcription factor RAR-related orphan receptor gamma-t (RORγt) is essential since they define TH17 differentiation [Pneumocystis jirovecii [Histoplasmosis, Cryptococcus) or meningitis  also occur significantly more often compared to the normal population [Patients show eosinophilia > 700/μl), low numbers of memory T cells and memory B cells, and almost no T17 cells . As alrentiation -57. Theintiation , 58. Oppirovecii  and disspulation . The redpulation . It is wpulation . Closer pulation , 64. Herpulation , 65-70. 0/μl, lowS. aureus) or topical antiseptics can also be used. Antifungal prophylaxis is indicated in recurrent or chronic candidiasis; likewise, anti-Aspergillus prophylaxis is indicated in predilected patients  due to the increased risk of aspergillomas. As soon as lung contamination is present, it should be treated antifungally. Education in airway clearance techniques for patients with bronchiectasis is also important to prevent infections in the first place. If patients suffer from recurrent respiratory tract infections despite prophylaxis, immunoglobulins can be administered intravenously [Since a curative therapy for HIES is not yet available, the most important goal at present is the intensive symptomatic treatment of infections, and even more so the prophylaxis of infections. Therefore, early diagnosis with analysis of the clinical phenotype by the application of the HIES score , 71 and venously -74. Prelvenously . ConsideH17 lymphocytes have been demonstrated. Although the results could only be shown in a small group of 14 patients, it can be postulated that allogeneic hematopoietic stem cell transplantation (HSCT) in principle can correct the immunodeficiency and patients thereby benefit clinically [Because current therapies do not provide a causal solution but are limited to continuous antimicrobial prophylaxis, research is being conducted into other treatment options. Although allogenic peripheral stem cell transplantation did not seem to be effective when performed initially -77, majoinically , 79. Howinically , 80, 81.inically . Althouginically , 84. It STAT3 DN mutations can lead to autosomal dominant HIES with a characteristic immunodeficiency triad of elevated IgE, cyst-forming pneumonia, and recurrent cold skin abscesses caused by deficient STAT3-mediated TH17 differentiation. In addition to non-immunological symptoms, recent reports of autoimmunity expand the clinical picture. Autosomal recessive mutations in signaling elements influencing the JAK/STAT3 pathway, such as ZNF341, manifest with similar characteristics and can be poorly distinguished in clinical diagnostics with the HIES score. The focus is on prophylaxis to prevent life-threatening infections or chronic diseases and is complemented by causal therapy methods such as HSCT and future techniques.Overall, STAT3 GOF germline mutations lead to an early childhood onset multiorgan autoimmune disorder  known as infantile-onset multisystem autoimmune disease-1 (ADMIO1). It is characterized by a wide spectrum of clinical and immunological features which were first described in five unrelated patients in 2014 by Flanagan et al. [STAT3 gene have been described [Heterozygous n et al. . Since tescribed .STAT3 GOF mutations present with autoimmune cytopenia (immune thrombocytopenic purpura (ITP), 24/42 described patients), autoimmune hemolytic anemia , and neutropenia (9/28). Other features are lymphoproliferation disorders such as lymphadenopathy (23/42), hepatosplenomegaly (23/42), Hodgkin’s lymphoma (1/42), or T cell large granular lymphocyte leukemia  but also autoimmune-associated disorders like enteropathy (24/42), interstitial lung disease (15/42), hypothyroidism (13/42), and type I diabetes mellitus (10/42). In contrast to STAT3 DN syndrome, immunodeficiency is not always the salient feature of patients, although a large number of patients required immune system related therapy (see below). The majority of the patients showed an increased susceptibility to (myco-)bacterial, viral, and fungal infections, which mainly affected the lower and upper respiratory tract. The described symptoms can be complemented by postnatal growth failure, delayed onset of puberty, and cutaneous , inflammatory , musculoskeletal , renal , or ocular  manifestations. The diversity of patients’ symptoms supports the suspicion of a STAT3 GOF syndrome [A systematic review by Fabre et al. in 2019 provided great insight into characteristic symptoms of patients with STAT3 GOF mutations, which include immunological as well as systemic features . More thsyndrome .The different phenotypes make precise and early diagnosis difficult. It is important to consider the patients in the context of all their symptoms, so that an experienced immunologist can make the correct diagnosis, especially in the presence of other symptoms that result in a STAT3 GOF syndrome. However, it should be noted that occasionally asymptomatic and only mildly affected patients have been described; hence, the incomplete penetrance should also be taken into consideration (see below) , 88-92.STAT3 gene locus in suspected congenital autoimmune disease, associated with other symptoms of STAT3 GOF syndrome, is currently the most valid diagnostic test as there are currently no established biomarker or pathognomonic immune cell alterations associated with STAT3 GOF. Hence, only cloning of the variant and subsequent in vitro functional testing can provide information about the pathogenicity of variants that have not been previously identified [2 domain, the transactivation domain, and the coiled-coil domain [STAT3 LOF missense mutations also occur at the same sites in the STAT3 gene, so that the effect on STAT3 function seems to depend largely on the amino acid inserted and its charge (see below).Genetic testing of the entified . Activatl domain . Most ofl domain . InteresSTAT3 GOF mutation carriers were identified in family studies [STAT3 mutations alone may not be sufficient for disease onset, but that other, elusive factors must be present. Because of the variable phenotype of patients with STAT3 GOF mutations and the different possible pathomolecular mechanisms of STAT3 hyperactivation, the question of genotype–phenotype correlation arises; however, no clear correlation could be demonstrated so far. However, it seems that mutations within the DNA binding domain present with a more severe phenotype. Further case reports and systematic reviews may contribute significantly to the elucidation of this question, thus advancing patient-centered medicine. Why STAT3 GOF and STAT3 DN present with different penetrance patterns remains unknown. It is plausible that the LOF phenotype may be less broad than the GOF phenotype what might be one major distinguishing feature of STAT LOF and STAT GOF mutations. The influence of hyperactivated STAT3 molecules at different pathway levels is manifold . Thus, one can imagine many more ways in which activating mutations can dysregulate a signaling pathway.In addition, 15 unaffected  studies . The obs2 domain were shown to lead to increased sensitivity to cytokines and enhanced dimerization [STAT1 GOF mutations, see below [STAT3 GOF variants with regard to their molecular behavior [STAT3 mutations into functional groups now appears biologically and clinically meaningful. However, the authors point to other hitherto unknown factors such as additional mutations, epigenetic changes, crosstalk with other pathways, or environmental factors , which probably also influence penetrance.Generally, amino acid substitution in the DNA binding domain was shown to increase DNA binding affinity and to lead to enhanced transactivation due to changes in the electrical charge of the STAT3 molecule , 93; mutrization ; mutatiorization , 94. Onlee below , 95, 96)ee below , 97. In behavior . They weSTAT3 GOF mutations are characterized by several hallmarks: More than half of the patients present with hypogammaglobulinemia and more than a quarter with NK cell lymphopenia, T cell lymphopenia, or B cell lymphopenia. In particular, regulatory T cells (Tregs) and switched memory B cells are decreased, although these numbers may vary widely between patients [H17 cells, a cell population known to induce autoimmunity when overactivated [STAT3 mutations are therefore predisposed to autoimmunity via overactivation. More generally, autoimmunity is thought to arise from a dysregulation of the equilibrium between TH17 and Treg cells [reg numbers observed in STAT3 GOF are consistent with the role of STAT3 in mediating forkhead box protein 3 (FOXP3)-inhibiting cytokines and concomitant inhibition of differentiation of naïve T cells into autoimmunity-controlling Treg cells [reg cells, as well as the inhibition of induced Treg (iTreg) cell polarization from I CD4+ T cells, are relevant [reg cells [STAT3 GOF and STAT5B LOF mutations, like cytopenia, short stature, and enteropathy [reg cells as a result of decreased CD25 and FOXP3 expression and reduced IL-2 signaling [reg cells may explain autoimmunity processes via the lack of negative regulation of TH17 cells [H17 counts have been affected by immunosuppressive therapy [H17 development in untreated STAT3 GOF patients would be beneficial. Since STAT3 GOF and immunodysregulation polyendocrinopathy enteropathy X-linked (IPEX) syndrome share symptoms such as autoimmunity, together with reduced Treg cell frequency, it is arguable that STAT3 GOF belongs to the spectrum of “Tregopathies” [reg defects are major drivers of STAT3 GOF syndrome, but collect evidence for selective deficiency in generation of iTreg cells and an expansion of effector CD8+ T cell population [reg frequency and suppressive activity are found. This study provides further evidence for the role of the Treg compartment in the development of autoimmunity by revealing cytokine hypersensitivity through deficient negative regulation of STATs. It should be noted that compared to STAT3 GOF, there was incomplete penetrance but also differences in manifestation (SLE in SOCS1 haploinsufficiency vs. enteropathy and diabetes in STAT3 GOF).From an immunological perspective, patients with patients . STAT3, ctivated . STAT3 meg cells -103. Theeg cells . STAT3-deg cells . The effrelevant . Howeverrelevant , 108. Ineg cells . It is aeropathy . The attignaling -113. Imp17 cells , 113. Fa therapy . Therefopathies” . A very pulation . In thispulation . In thisSTAT3 GOF mutations was initially limited to immunosuppressive and immune-modulatory therapy. In most cases, patients receive steroids, mycophenolate mofetil, tacrolimus, rituximab, sirolimus, and anti-TNF-α antibodies. Respiratory bacterial infections can be prevented or treated with antibiotics, and treatment can be supplemented with respiratory physiotherapy. Intravenous or subcutaneous immunoglobulin substitution may help patients with hypogammaglobulinemia [STAT1 GOF mutations, HSCT so far did not reveal a good outcome: only in two out of six treated patients, HSCT could meet the objective of a causal curative treatment with complete resolution of autoimmune manifestations [STAT3 mRNA and downregulate STAT3 expression [Treatment of patients with ulinemia . Howeverulinemia . In combstations , 117. Instations , 118. Thstations , 120. Instations -123. Addpression , 119. HeSTAT3 GOF mutations) is often associated with a worse prognosis and accelerated disease progression [STAT3 GOF mutations can be detected in 30–40% of T cell large granuloma lymphocytic (LGL) leukemia and chronic lymphoproliferative disorder of natural killer cells (CLPD-NK) [+ large granular lymphocytes in T-LGL) [2 domain, resulting in increased dimerization, activation of the STAT3 protein, and transcriptional activation [STAT3-deficient mouse models. They suggest that it may act as a negative regulator in certain tumor entities  -132. Botn T-LGL) , 134. Thn T-LGL) . The muttivation . Howevere cancer , glioblae cancer , intestie cancer , lung cae cancer , and thye cancer ).STAT1 may cause autosomal dominant or autosomal recessive diseases. STAT1 is heavily involved in mediating cytokine signaling of type I (IFN-α/β), type II (IFN-γ), and type III (IFN-λ) interferon as well as IL-6, IL-9, IL-10, IL-11, IL-12, IL-19, IL-21, IL-27, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), ciliary neurotrophic factor (CNTF), LIF, and OSM [H17 cells producing IL-17A, IL-17F, and IL-22. STAT1 has been described to have activating GOF but also LOF mutations.Mutations in  and OSM , 143. IFSTAT1 LOF mutations can also affect the patient’s immune responses in a characteristic manner. The defects that occur can be classified into three categories: autosomal recessive complete STAT1 deficiency, autosomal recessive partial STAT1 deficiency, and autosomal dominant STAT1 deficiency [As STAT1 is central for the integrity of the immune system, ficiency -147. TheSTAT1 gene causing autosomal recessive complete STAT1 deficiency [STAT1 mRNA analysis. These can reveal potential STAT1 expression reductions caused, for example, by mutations in splicing regulators leading to aberrant pre-mRNA splicing and consecutive protein degradation. Protein analysis and STAT1 functional testing can follow to assess the final impact of an intronic variant on protein level.Patients with autosomal recessive complete STAT1 deficiency completely lack STAT1 expression and therefore the ability of pSTAT1 induction after IFN type I, II, and III as well as interleukin stimulation . Only 24 patients with an autosomal recessive complete STAT1 deficiency (16 different mutations) have been described and 17 died, most likely because of its profound consequences for the immune system (see below). Interestingly, in 2020, Sakata et al. described the first intronic mutation in the ficiency . Since iSTAT1 null mutations suffer from extensive infections of intracellular bacteria  and viruses  as well as in some cases secondary hemophagocytic syndrome. Most of these complications occur after a few months and may be lethal in the first two years of life. A STAT1 LOF mutation must be considered when evaluating patients with the clinical picture of severe mycobacterial and viral infections.Patients with these Mycobacterium kansasii, HSV-1, CMV) [Pathomolecularly, the lack of a STAT1-mediated response to IFN-γ, IFN-α/β, IFN-λ, and IL-27 is the cause of an inadequate activation of effector cells, which are supposed to fight off the actually weakly virulent pathogens  .Out of 24 published patients, ten patients received HSCT of whom only seven patients are alive , 150-152In addition, patients with a milder form of STAT1 LOF have been reported . Both th2 domain [STAT1 alleles do not lead to a predisposition to viral infections, which can be explained by an unaffected response to IFN-α/β in heterozygous cells. The IFN-γ response, on the other hand, appears to be limited by a heterozygous mutation [Since 2001, 31 patients with autosomal dominant STAT1 deficiency have been documented , 154. Th2 domain , 157, or2 domain , which i2 domain , 160. Thmutation , 159-161mutation .As can be seen, too little STAT1 immunity  is associated with a susceptibility to mycobacterial and viral infections. A balanced equilibrium of STAT1 signaling is central to avoid severe and possibly life-threatening infections, because STAT1 GOF typically leads to autoimmunity and CMC. Since its first description in 2011 by Van de Veerdonk et al., the STAT1 GOF phenotype, characterized by immunodeficiency and autoimmunity, has been well described .Candida albicans, although invasive infections with Candida albicans, Cryptococcus spp., Pneumocystis jirovecii, Aspergillus spp., and Penicillium marneffei can also be observed in approximately 20% of cases. Overall, around half of all CMC cases appear to be attributable to STAT1 GOF mutations [STAT1 GOF mutation. In addition to fungal pathogens, patients with STAT1 GOF mutations are susceptible to recurrent lower respiratory tract bacterial infections (LRTI). Infections with S. aureus, Streptococcus spp., Pseudomonas aeruginosa, or H. influenzae can - in some circumstances - lead to long-lasting LRTI that can result in severe pneumonia, bronchitis, interstitial pneumonia, or more persistently, bronchiectasis. Mycobacterial diseases may occur after infections of tuberculous  or non-tuberculous mycobacteria  as well as BCG vaccine, including tuberculosis, skin disease, and adenitis [Herpesviridae  have also been commonly described [STAT1 GOF mutations are associated with an increased incidence of certain tumor entities, some of which may be explained by persistent mucocutaneous fungal infections that develop into squamous cell carcinoma . STAT1 GOF patients also present with melanoma, lymphoma, leukemia, prostate cancer, or papillary thyroid cancer [The most prominent symptom of patients is the presence of CMC, which can be observed in about 90% of all patients before the age of 10 years (early-onset CMC), yet the lifetime risk of CMC is nearly 100% , 163. Thutations -166, higd cancer . Chronicd cancer .STAT1 GOF mutations have a deficiency of TH17 cells, which is due to a poor development of IL-17A-, IL-17F-, and IL-22-producing cells [H17 cells, which are necessary for preventing CMC, cannot be observed in all patients (82%), whereas 98% of patients develop CMC [H17 counts. There are individual case reports showing impaired production of IL-6, IL-17A, and IL-22 and increased production of IL-4 in comparison to healthy individuals [H17 cell-mediated immunity via IL-23 silencing [reg cells or FOXP3 mutations could be found in STAT1 GOF, another mechanism must underlie this phenotype (see below) [+ and CD8+ T cells are observed , CD19+ or CD20+ B cell numbers are low in 23.9%, and most patients have reduced numbers of CD19+CD27+ memory B cells. CD16+CD56+ NK cells are low in 32% and show a reduced NK cell toxicity. In addition, humoral immunodeficiency with low serum IgG, IgA, or IgM levels was described in 6.5%, 18.3%, and 6.3% of patients, respectively [The symptoms of the patients can be partly explained by the changes in immune cell subpopulations. Eighty-seven percent of patients with ng cells . Increasng cells , 168-172elop CMC , 173-184ividuals , 186. Inilencing . Decreasilencing , cannot ilencing -191. Becectively . Elevateectively .2 domain [The underlying genetic causes for STAT1 GOF are activating mutations in mostly the coiled-coil domain but also in the DNA binding domain, linker domain , or SH2 2 domain . More th2 domain .+ monocytes with concomitant high levels of total STAT1 expression [STAT1 mRNA levels [STAT1R247W), decreased mobility , or immobility in the nucleus (STAT1T419R) as the cause of STAT1 hyperactivation [STAT1 GOF mutations as fewer IFN-γ activation site (GAS) motifs are detectable in promoters of STAT1 GOF-specific genes. The ratio of GAS present to GAS absent in promoters of STAT1-regulated genes decreased significantly for the STAT1T419R variant in chromatin immunoprecipitation (ChIP)-Seq experiments [STAT1 variants resulted in different mutation-specific fingerprints and offers a possible explanation for the wide range of phenotypic variation. Reasonably well described is the observation that GOF mutations in the coiled-coil domain and DNA binding domain impair the stabilization of the STAT1 homodimer in an antiparallel conformation. Physiologically, conversion from parallel conformation to antiparallel conformation is required for dephosphorylation at pY701 by phosphatases. By remaining in the parallel conformation and lacking access to pY701, STAT1 GOF homodimers remain phosphorylated and become resistant to dephosphorylation [The resulting mechanisms for hyperactivation of STAT1 are the subject of current research and are not yet fully understood in their complexity. Various, sometimes opposing, hypotheses exist for the increased molecular activity of the STAT1 protein. Easily understood are results that imply an impaired cycle of phosphorylation or dephosphorylation, where either the phosphorylation of STAT1 occurs faster or the nuclear dephosphorylation is slowed down . In expepression , 194-196A levels , 198. OtA levels , 199, intivation . There aeriments . In RNA-rylation , 202.H17 cells since those cytokines are STAT1-dependent repressors for TH17 differentiation in mice and humans [STAT1 GOF mutations, enhanced IL-27 signaling upregulates the expression of programmed death-ligand 1 (PD-L1) [H17 differentiation, this process may contribute to defect induction of mucosal immunity against fungal pathogens [STAT1 and STAT3 mutations, respectively, on the process of heterodimerization, and how different mutations affect STAT1:STAT3 heterodimer formation. As STAT3 is the key transcription factor for TH17 development (see above), this concept provides an explanation for the high prevalence of CMC in both patients with STAT3 LOF mutations and patients with STAT1 GOF mutations. The negative effect of STAT1 GOF on STAT3-driven TH17 cell induction is the most likely reason behind the overlapping symptoms of both syndromes  , 203. Asathogens , 205. Baathogens -209, it erferons . These oerferons , 210.reg cell number and function including FOXP3 wild type. LOF mutations in CD25, STAT5B, and ITCH were identified as other causes, which can be differentiated from each other by specific symptoms. Since FOXP3-dependent Treg development does not appear to be defective, the central functions of the IPEX-like associated proteins for Treg function suggest an immune dysregulation of the same [reg cells [STAT1 GOF, STAT3 LOF, IL-10 LOF, and IL21R LOF mutations can alter circulating TFH (cTFH) cell differentiation by producing fewer total cells or different cTFH cell subsets [reg counts and functional tests, but the latter were limited to only two patients [reg frequency compared to controls could be detected [reg-mediated suppression in particular were not investigated [STAT1 mutants destabilizing the Treg cells by reprogramming them into TH1-like cells, altering transcriptomes, and producing functional IL-10 deficiencies [reg cell development and altered STAT1-mediated gene transcription were detected in a STAT1 GOF patient with common variable immunodeficiency (CVID) symptoms - but without classical IPEX-like symptoms [reg cells, in particular by collecting evidence for increased Treg expansion in the absence of STAT1 [STAT1 GOF is one cause of an IPEX-like phenotype, hence IPEX-typical symptoms of primarily normal Tthe same . For exaeg cells , 212. Theg cells , 214. Theg cells -217. Thieg cells , 219. It subsets . The res subsets , 221. Thpatients . Even indetected . Moreovestigated . Accordiciencies , 223. Insymptoms . Pathomeof STAT1 -228.STAT3 GOF mutations, STAT1-induced SOCS1-mediated suppression of STAT5 may occur with STAT1 GOF mutations. Since STAT5 is known to be involved in terminal NK cell differentiation, NK cell toxicity, and perforin expression, this may explain increased viral susceptibility in patients with STAT1 GOF mutations [Similar to utations , 230. Liutations .In light of the characteristic symptoms, treating physicians should consider the differential diagnosis STAT1 GOF in patients with CMC alone, CMC with lower respiratory tract infections, or CMC with autoimmune thyroid disease. Altered lymphocyte subpopulations can be determined by lymphopanel analyses. In this context, various genetic and functional tests are useful for strengthening the diagnosis of STAT1 GOF, e.g., subcellular distribution or levels of STAT1, pSTAT1, interferon-stimulated gene factor 3, and γ-activated factor (GAF) in unstimulated and in IFN-α-, IFN-γ-, or IL-27-stimulated conditions. Also, gene transcription induced by interferons or in vitro luciferase STAT1 reporter assays may be helpful for diagnosing STAT1 GOF. Eventually, DNA sequencing can provide information about the exact genotypic variation of the patient. Integrating such findings could serve to identify possible genotype–phenotype correlations. According to Zhang et al. the median onset of symptoms in STAT1 GOF patients is 1 year, but the median diagnosis does not occur until 6.2 years, implying a noteworthy delay of diagnosis .STAT1 GOF mutations has advanced considerably over the past 10 years. While the initial focus has been on symptomatic treatment of patients with abatement of infection-related symptoms, treatment options nowadays aim at a more causal approach. Topical or systemic antifungal therapy (preferably triazoles such as fluconazole), prophylactic or ad hoc antibiotic therapy, and immunoglobulin replacement therapy especially for the prophylaxis or treatment of pneumonia is commonly used. Eczematous dermatitis may be treated with topical steroids or topical application of calcineurin inhibitors.The treatment of patients with Cytomegalovirus (VZV and CMV) exacerbation due to higher risk during JAK inhibitor treatment and the already increased risk of infections in patients with STAT1 GOF mutation, a prophylactic treatment with aciclovir should be considered [With the advent of JAK inhibitors (jakinibs) such as ruxolitinib and baricitinib (both inhibiting predominantly JAK1 and JAK2), targeted therapeutic options are available for STAT1 GOF patients and have improved symptoms to even resolution of CMC in 12/20 patients , 70, 229nsidered , 232. Thnsidered , 192.STAT1 GOF mutations waiting for HSCT to reduce post-transplant complications [STAT1 GOF mutations. Mechanistically, this could help to downregulate uncontrolled inflammation and therefore minimize the risk of secondary graft failure. The purpose of the concomitant and bridging therapy is to restore the STAT1-mediated immune response to a normal state until HSCT can correct the underlying genetic defect. The studied patient showed stabilization by therapy with ruxolitinib  but persistent TH17 deficiency. Subsequent HSCT normalized STAT1 phosphorylation/dephosphorylation, dysregulated gene expression, the IFN signature, and T, B, and NK cell function. Further studies are necessary to better understand the challenging problems in transplanting patients with GOF mutations in which - at least theoretically - full conditioning and a 100% donor chimerism need to be achieved for a sustained therapeutic success.In recent studies, ruxolitinib was considered a useful drug for bridging therapy in patients with ications . Approxiications . RuxolitH2 differentiation, B cell proliferation, IgE class switch, as well as generation and activation of eosinophil granulocytes and mast cells. Consequently, STAT6 is involved in the pathophysiology of atopic diseases such as allergy, asthma, or atopic dermatitis. While somatic STAT6 polymorphisms are mainly associated with various tumor entities such as Hodgkin lymphoma, only recently patients with germline STAT6 GOF disorders have been reported [STAT6 is involved in the signaling pathways of several cytokines such as IL-3, IL-15, IFN-α, and PDGF, but plays a key role in the signal transduction of IL-4 and IL-13. IL-4 and IL-13 are significantly relevant to Treported , 234. A STAT6 GOF mutations were identified [2 domain, and near the phosphorylation site Y641 in the transactivation domain. Sharma et al. showed that all variants are located near the protein-DNA interaction region or in the STAT6-specific protein-DNA recognition site [In 2022 and 2023, 21 patients from 13 unrelated kindreds with germline heterozygous entified -237. Theion site . They weSTAT6 GOF mutations [STAT6 GOF is a multisystem disorder characterized by its key symptoms early-onset multiple severe food allergic disease and severe treatment-resistant atopic dermatitis -237. Theutations , 240. Inutations STAT6 GOF variants studied showed increased pSTAT6 levels before and after stimulation compared to healthy controls, and there are reports of physiological but also slowed dephosphorylation kinetics [STAT6 variant [STAT6 variants showed increased transcriptional activity in luciferase reporter assays in both the absence and presence of IL-4 [IL4R, CISH, and XBP1 (involved in plasma cell and eosinophil differentiation), as well as an increase of TH2-inducing factors. For variants E382Q and D419G, partly unique transcriptional profiles were observed that are not known in wild type suggesting more GOF mechanism than only enhanced DNA binding. Patients typically show normal T, B, and NK cell counts, with high levels of TH2 lymphocytes next to signs of chronic inflammation [H2 cytokines IL-4, IL-5, and IL-13, which are major contributors to the pathogenesis of atopic diseases, were significantly elevated in TH2 patient cells. TH2 skewing as well as suppressed TH1 and TH17 responses were evident in STAT6 GOF patients [+ T cells as well as non-class switched and class switched memory B cells. Consistent with the physiological function of STAT6, high serum IgE levels and hypereosinophilia were key features of STAT6 GOF patients.The kinetics , 237. Su variant . Through of IL-4 , 237. Trammation . The TH2patients , 237. FuD419N knock-in mice spontaneous development of dermatitis with skin thickening, eosinophilic skin infiltration, elevated serum IgE levels, and mRNA expression of type 2 cytokines could also be observed [In vitro, normalization of STAT6 activation by IL-4/STAT6 axis inhibitors ruxolitinib, tofacitinib, and the anti-IL-4Rα antibody dupilumab could be demonstrated . In accoobserved .STAT3, ZNF341, DOCK8, and IL6ST can cause HIES, raising the suggestion of Sharma et al. to consider STAT6 GOF as another cause of HIES. The overlapping phenotype further strengthens the role of genetic testing as the most reliable diagnostic method for identifying STAT6 variants followed by functional testing, if necessary.Sharma et al. propose a set of clinical findings for which the presence of STAT6 GOF should be further investigated . These iH2 gene signature, IL-4Rα expression, and eosinophilia was observed.Prior to the knowledge of the dysregulated STAT6 pathway, most patients were offered therapy with systemic corticosteroids, tacrolimus, methotrexate, and mepolizumab, which had little or no satisfactory effect on symptoms. Targeted therapies with ruxolitinib, tofacitinib, and dupilumab resulted in symptom control with improvement in atopic dermatitis, growth velocity, and eosinophilic esophagitis in numerous patients. Thereupon, total weaning from steroids was possible in some patients. Immunologically, reversion of STAT6 hyperactivation after IL-4 stimulation and faster STAT6 dephosphorylation were seen , 237. NoJAK1 LOF mutation was described, instead a perinatal lethal phenotype was shown in JAK1 knock-out mice [For a long time, no out mice . In in vFirst in 2016, the only patient so far with two homozygous missense germline mutations was described, of which the P733L mutation has been undescribed and the P832S variant has already been characterized in four heterozygous individuals .The patient, who died at the age of 23, is characterized by various immunodeficiency symptoms , cardiomyopathy, pleural and mediastinal fibrosis, pulmonary macrophage infiltration, and in particular by atypical mycobacterial infections and high-grade metastatic bladder carcinoma. Susceptibility to these atypical mycobacterial infections is most likely due to T cell lymphopenia, impaired IFN-γ production by existing T cells, and a reduced JAK1-STAT1 signaling response .Functional tests show reduced pSTAT levels after stimulation . T cell stimulation assays also reveal extensive immune dysregulation, especially of IFN-γ production. Further transfection experiments investigate the individual effect of the two mutations located in the pseudokinase domain, whereby P733L emerges as the probable disease-causing variant.Eletto et al. conclude that the JAK1 pseudokinase domain, in the interaction with JAK2, takes on an essential role in the activation of the latter, allowing IFN-γ-stimulated STAT1 phosphorylation. A recent follow-up to this research addresses the question of why the patient did not suffer from viral infections (apart from flat warts) although JAK1 is significantly involved in the IFN-α-mediated immune response against viruses. It was shown that in myeloid cells JAK1 has non-redundant functions in the IFN-γ response to control intracellular bacterial infection, and - despite reduced IFN-α responses - does not lead to increased viral susceptibility in all cell types . It remaJAKs  [JAK1 have been described in oncological pathologies such as acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), and solid-organ cancers [JAKs have been reported, ranging from immunodeficiency to autoimmunity and lymphoproliferation. Not least because these mutations interfere so massively with the immune system, such immunodeficiencies occur very rarely and can only be identified through careful observation, detailed clinical investigation, and advanced functional analysis.As early as the 1990s, mutations in the Janus kinases located upstream in the JAK/STAT signaling pathway have been described, which manifest themselves in altered signaling responses to IFN-α/β and IFN-γ . Somaticnd TYK2) . In part cancers -250. In JAK1 in a mother and her two sons, whereas previously, a somatic mutation at the identical site with a malignant phenotype was reported [JAK1 (A634D) could be identified in the index patient and confirmed in her children. The mutation is localized in the inhibitory pseudokinase domain of JAK1, a domain which is considered to be of functional importance due to its high conservation across multiple species. Functional assays on immortalized B cells and primary CD3+ T cells revealed increased pSTAT1 and pSTAT3 levels both in the unstimulated setting and upon IFN and IL-6 stimulation, respectively. In vitro treatments with ruxolitinib showed normalized levels of pSTAT1 and pSTAT3, so in conjunction with the knowledge of previously described somatic mutation with a malignant phenotype [JAK1A634D mouse model with a similar phenotype [In 2017, Del Bel et al. identified the first human germline mutation of reported . Phenotyhenotype , 252-254henotype , 256. RuS703I genotype provided further insights into the role of the pseudokinase domain in which the mutation was localized [+ monocytes approximately 15% S703I+, I T cells 55% S703I+, CD56hi NK cells 69% S703I+), with additional monoallelic expression bias in the transcription of JAK1 observed. The knowledge of JAK1S703I mosaicism with cell type-specific allele distribution as well as the mechanism of monoallelic bias helps to understand the diffuse phenotype with different manifestation severity of autoinflammatory and atopic symptoms in different organs [The 2020 report of a JAK1 GOF patient with combined autoinflammatory and atopic phenotype in the case of mosaic JAK1ocalized . On the t organs -260. TheJAK1 mutations. While Shomali et al. were the first to identify an acquired somatic mutation also in the pseudokinase domain (R629_S632delinsSA) with activation of the JAK1-STAT3/STAT5 pathway and clonal eosinophilic neoplasm [JAK1 mutation of unclear somatic or germline origin, with both mutations showing (hyper-)eosinophilia [JAK1 mutation H596D, which was found in a patient with a multiorgan immunodysregulatory phenotype featuring autoinflammation keratinization disease, hepatitis, autism, and atopic dermatitis. In addition, the patient presented with mild erythema, hepatosplenomegaly, failure to thrive, motor and learning impairments, glycogen storage disease type 4, and hyperlipidemia as well as lymphocytic infiltration of liver and upper dermis, epidermal hyperplasia, and eosinophilia. At the age of 3 years, acute liver failure necessitated a liver transplant; she died of unknown causes at the age of 22. A Jak1 knock-in mouse model  showed a hyperkeratotic and scaling phenotype  and histopathologically lymphocytic infiltrates of the liver as well as a skin phenotype, which is overall consistent with the patient’s phenotype. By immunohistochemistry, elevated levels of pSTAT3, pSTAT6, and pJAK1 in the skin as well as pSTAT1, pSTAT3, pSTAT5, pSTAT6, and pJAK1 in the liver could be detected. Using the results of RNA sequencing of knock-in mouse samples, the authors conclude that the JAK1 variant H596D exerts a GOF effect on the JAK/STAT signaling response [JAK1 mutation and activation of inflammatory STAT-mediated signaling pathways such as the IL-6, TNF-α, or IFN-γ axis to effects such as inflammatory liver infiltration, atopic dermatitis, and eosinophilia. Since atopic dermatitis can also occur in STAT3 GOF patients  by dendritic cells. STAT3 can inhibit STAT6-mediated chemokine production in these cells. In dendritic cells, IL-4 and IL-13 can activate STAT3 and STAT6 via the IL-4Rα/IL-13Rα1 receptor complex and its associated JAK1. In epidermal keratinocytes, this signaling axis leads to an increased production of T cell proliferation- and TH2 differentiation-promoting thymic stromal lymphopoietin, as well as IL-25 and IL-33. It also downregulates filaggrin and loricrin expression, two proteins that are indispensable to the skin barrier. The deficiency of filaggrin and loricrin, leading to the destruction of the skin barrier, is characteristic of AD. It can be overcome by the anti-IL-4Rα antibody dupilumab, which disrupts IL-13/IL-4 signaling, highlighting the central role of IL-13/IL-4 signaling dysregulation in AD pathogenesis [H2 chemokines, and a restoration of barrier-related protein expression. Therefore, in addition to corticosteroids, the IL-4Rα antibody dupilumab and JAK inhibitors have been tested for treatment. The topical JAK inhibitor delgocitinib is approved in some countries for the treatment of atopic dermatitis. By interfering with the IL-4Rα/γc, IL-4Rα/IL-13Rα1, and IL-31RA/OSMR signaling responses, skin lesions, associated itch, and the skin barrier itself can be reliably controlled. In the future, instead of steroids, combined treatment of IL-13/IL-4-JAK-STAT6/STAT3 axis inhibitors together with IL-13/IL-4 interfering antibodies may be used. As STAT3 has been shown to take on similar functions as STAT6 in lymphocytes and keratinocytes, one might learn from the STAT6 GOF genotype. Overexpression of STAT6 in mice is also associated with AD-like skin symptoms, which is consistent with atopic symptoms in STAT6 GOF patients. Based on this knowledge, AD can also be understood as cutaneous inflammation due to elevated IL-13/IL-4 signaling, which might be related to dysregulated JAK1 and STAT3 responses.Then, in 2021 and 2022, Shomali et al. and Takeichi et al. described two more activating neoplasm , Takeichnophilia . Shomaliresponse . Their hogenesis . In keraJAK1 mutations leading to a malignant phenotype such as T cell neoplasms, ALL, myeloproliferative neoplasm [JAK1 are described to be found in association with mutations in other genes, such as NOTCH1 or PTPRC [JAK3 GOF mutations can be found concordantly with mutations in TP53, NOTCH1, or CDKN2A [JAK1 and JAK3 mutations with other genetic alterations has also been observed in solid cancers, such as breast cancer. In melanoma, mutations in BRAF have been found to accompany JAK3 mutations [TP53 and NOTCH1, as well as MYC amplification, have been identified alongside JAK1 mutations [JAK mutations in addition to other known oncogenes is likely promoting tumorigenesis. In addition, mutations of the pseudokinase domain of JAK2, a structural homologue of JAK1, are found in patients with chronic eosinophilic leukemia and a consequent polycythemia [c receptor, JAK1/TYK2 via IFN-α/β receptor and gp130 receptors).It is interesting to note that the four variants described were all found in the pseudokinase domain of JAK1. In addition, somatic neoplasm -266, AMLneoplasm -250 haveor PTPRC , 267. Sir CDKN2A . Such coutations , while iutations . This cocythemia . It can cythemia , 273. Hocythemia . In partcythemia . This reIL2RG gene encoding the common gamma-chain (γc) as the cause of X-linked severe combined immunodeficiency (X-SCID), an immunodeficiency characterized by the absence of T cells and NK cells in the presence of non-functional B cells [JAK3 mutations were identified as the cause of autosomal recessive T−B+NK–SCID [JAK3 mutations [IL7R (IL-7Rα) (< 10%) or IL2RG (γc) (25–46%) [A few years after the discovery of mutations in the  B cells , JAK3 mu+NK–SCID , 278. Beutations -281. In (25–46%) .c and IL-7Rα to mediate the signaling response of interleukins IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21 acting through this pathway [c/JAK3 signaling [Pathomolecularly, impaired T and NK cell development and B cell function may be due to reduced or abolished protein expression, protein stability, or receptor binding of the JAK3 protein, which physiologically interacts with γ pathway . Contrarignaling .As a result of the defect, patients who appear healthy for a short time after birth suffer from a massive restriction of the immune defense; therefore, this clinical picture must be considered life-threatening. Humoral and cellular immune responses are extremely reduced due to the absence of T cells, resulting in a life-threatening infection susceptibility. In particular, recurrent or persistent infections of the respiratory tract and gastrointestinal tract with viruses and fungi , chronic infections with opportunistic pathogens, chronic diarrhea, and failure to thrive determine the fatal outcome of the disease in early childhood if not treated adequately .To protect patients from ubiquitous pathogens, infants used to be isolated in sterile plastic tents, so they were known as “bubble babies”. Fortunately, over the last decades, HSCT has been optimized as the curative therapy of choice to such an extent that a long-term survival of up to 95% can be achieved with an optimally matched donor-recipient pairing. Compared to other forms of SCID with B cell loss, the presence of B cells has a positive effect on the outcome of HSCT . Since HJAK3 GOF mutations have been reported. The novel germline heterozygous JAK3 variant Q507P in a mother and her son was phenotypically associated with vasculitis and CVID, recurrent multilineage autoimmune cytopenia, psoriasis, hypogammaglobulinemia, and NK cell lymphoproliferation as well as elevated numbers of aberrant NK cells, large granular lymphocytes with benign morphology, reduced memory, and isotype-switched B cells [Activating germline mutations of JAK3 were previously undescribed, only somatic GOF mutations were known to exist in T cell proliferative leukemia , 288, th B cells . The Q50 B cells  and was JAK3R840C variant was first found in association with a new heterozygous LOF CTLA4Y139C variant with the clinical picture of PID. As the mutation localizes within the JH1 domain in close proximity to the ATP-binding site, Sic et al. suggest a possible altered N-lobe conformation and interaction with the pseudokinase domain resulting in overactivation of the protein [JAK3A572V - stands out and manifests in an immunodysregulatory phenotype. The latter is characterized by early bronchiolitis, recurrent upper respiratory tract infections, lymphadenopathy, splenomegaly, pancytopenia, severe hypogammaglobulinemia, insufficient vaccination response, hypothyroidism, and hypercellular bone marrow. The authors show that a large proportion of the present T cells are CXCR5+PD-1+TIGIT+ TFH cells, which, however, can only insufficiently produce the cytokines necessary for plasma cell and memory B cell maturation. The combination of the individually harmless JAK3 and CTLA4 variants results in a TFH cell defect with symptoms such as lymphadenopathy, autoimmunity, and hypogammaglobulinemia.In a second case report, the rare heterozygous  protein . When coPneumocystis, Cryptococcus, or Coccidioides), and mycobacterial infections . Thus, rational use of JAK inhibitors must be considered when treating STAT1 GOF or STAT3 GOF patients, in which the risk for viral and fungal infections or cancer is already elevated, respectively, in those patients.The integration of clinical studies and basic science research creates an understanding of the pathomolecular processes and thus allows patient-centered and individualized therapies. In terms of autoimmune diseases, JAK inhibitors have already successfully been introduced into clinic  . These i2 domain, one research focus lies on the development of non-peptide or peptidomimetic agents to disturb SH2 domain interactions with other STAT molecules as well as inhibition of tyrosine and serine phosphorylation. Further obstacles are poor bioavailability of peptide agents and side effects in the form of, for example, cytopenia and dose-limiting vomiting with the STAT3 inhibitor OPB-51602 and OPB-31121. In addition to N-terminal inhibitors, DNA binding domain inhibitors and decoy oligodeoxynucleotides are also being investigated; the latter are designed to mimic the STAT DNA binding sequence in order to sequester STATs and downregulate STAT-dependent gene transcription. Similar approaches are being pursued with small interfering RNAs and antisense oligonucleotides to induce STAT degradation. Since this region is highly conserved between STATs, unselectivity such as off-target STAT1 blockade by a STAT3 inhibitor remains a challenge in development [In addition to the small molecules inhibiting JAK directly, there are also interleukin inhibitors and interleukin-receptor inhibitors (mostly antibodies), STAT inhibitors, and SOCS mimetics, which are currently at different stages of development . Oral STAT3 inhibitors have already been developed for solid or hematopoietic tumors but have not yet entered clinical use due to severe adverse effects and poor selectivity. As many mutations are located in the SHelopment -298.In the last decade, in vitro research has been performed to develop SOCS mimetics and SOCS antagonists, which have shown encouraging results in mouse models of autoimmune-associated diseases like diabetes and multiple sclerosis. The underlying design principle involves the development of a peptide that contains the kinase inhibitory region (KIR), which is naturally present in SOCS1 and SOCS3. Physiologically, KIRs take over the main role of SOCS1 and SOCS3 in inhibiting JAK2, so that the activation of downstream targets such as STAT1 is reduced. In this way, the use of small peptide SOCS mimetics containing KIR sequences allows the immune balance to be shifted toward inhibition of cytokine responses with the consequence of reduced autoimmunity. Conversely, SOCS antagonists, which act mainly via proteolytic degradation, can promote the expression of proinflammatory cytokines via enhanced STAT signaling responses, thereby enhancing viral immunity through, e.g., SOCS1 inhibition. So far, there are no known clinical trials for SOCS mimetics or antagonists. Similar problems in development as with JAK inhibitors and STAT inhibitors are anticipated: The KIR sequences of SOCS1 and SOCS3 are partially homologous, so cross-regulation is feasible. An essential part of the development of such therapeutics will be the balance of homeostasis between excessive and insufficient immunity in an already dysregulated organism via phenotype-adapted dose-specific SOCS regulation -301.STAT3 DN mutation) apart from stem cell transplantation, which carries considerable side effects and risks. The immunosuppressive effects of conditioning therapy used in HSCT increase the infection susceptibility of patients, which can cause significant morbidity. Prophylactic measures such as antibiotics and immunoglobulin therapy are necessary to prevent these life-threatening infections. While with GvHD, the graft immune system attacks host tissue, failure to engraft is another complication, leaving the patient immunocompromised and in need of a second HSCT. The side effects of conditioning therapy with chemotherapeutic agents to deplete bone medullary stem cells, among others, can be diverse and serious, possibly with long-term consequences for vulnerable organs such as the brain, lungs, kidney, and liver. The comparatively rare use of HSCT in germline JAK/STAT diseases with unknown long-term efficacy must be taken into account. Obviously, non-hematological disease manifestations  or irreversible remodeling processes (such as bronchiectasis/pneumatoceles in STAT3 DN or vascular changes in STAT1 GOF) cannot be successfully treated by HSCT. Consequently, in order to consider HSCT for diseases with high disease-related morbidity and mortality (such as STAT1 LOF), an early disease diagnosis including prognosis assessment is indispensable. Across the different JAK/STAT diseases, different outcomes are reported with about 30% survival in STAT3 GOF, 40% in STAT1 GOF, 70% in STAT1 LOF to up to 95% in JAK3 LOF. In general, the risk of disease progression and disease complications associated with HSCT and mortality must be weighed individually for each patient, and a decision must be made for the most beneficial therapy [Up to know, there are no therapies available for an attenuated JAK/STAT signal , NET formation, and anti-NET autoantibodies. NETs are extracellular formations of mainly neutrophil DNA that bind pathogens and are therefore considered one of the major defense mechanisms of neutrophils. Nowadays, NETs are considered to have a significant role in the etiology of lupus autoantigens and in driving proinflammatory mechanisms, thus providing a pathomechanistic link between autoimmunity and STAT3 DN in this context [First, in 2021, the lupus-like autoimmune phenotype was described in a cohort of ten STAT3 DN patients . A new s context . Moreove context . It remaregs in STAT3 GOF. The pathogenesis of increased susceptibility to infections caused by bacteria, viruses, mycobacteria and fungi is largely not understood. Although decreased counts of dendritic, Treg, TH17, and NK cells are measured in patients, molecular mechanisms behind the increased infection susceptibility in the presence of, e.g., normal NK cell function , are not understood. The mechanisms of deficiency of switched memory B cells resulting in hypogammaglobulinemia are also not yet understood [Second, the main characteristics of STAT3 GOF are autoimmune cytopenia, lymphadenopathy, hepatosplenomegaly, autoimmune enteropathy, interstitial lung disease, autoimmune hypothyroidism, type I diabetes mellitus, and other inflammatory manifestations such as AD. Nevertheless, it is now known that cellular and humoral immunodeficiencies occur in 37% and 51% of patients, respectively . Advancederstood . Interesderstood . This isCandida albicans infection [H17 cells play an important role in the immunity against C. albicans. Therefore, in the case of STAT1 hyperactivation, there is enhanced IL-27-mediated upregulation of PD-L1, which inhibits TH17 differentiation. Altered STAT3 promoter binding through STAT1 GOF-dependent epigenetic alteration of acetylation status also plays a role in IL-17 dysregulation by decreasing STAT3-dependent transcription that facilitates TH17 differentiation (RORγt/IL-17/IL-22/IL-10/c-Fos/SOCS3/c-Myc) [regs necessary for the control of the immune responses play a relevant role in the IPEX-like syndrome, but functional tests and Treg counting have not yet provided any significant evidence for this hypothesis. The clinical picture of STAT1 GOF exemplifies how the seemingly paradoxical coexistence of immunodeficiency and autoimmunity is reality for many patients. Until now, the mechanisms are still poorly understood. We are left with the question on the different penetrance of the different symptom complexes: why do almost all patients develop CMC and bacterial infections, but less than half develop autoimmune/autoinflammatory symptoms?Third, almost every STAT1 GOF patient will develop CMC in his or her lifetime, predominantly as a result of nfection . Not untnfection , 165. Th3/c-Myc) . NotablyThis new understanding implies three important aspects that will influence the management of JAK/STAT diseases in the future: There is a need to acknowledge that certain mutations in proteins offer great possibility for dysregulation not only of the canonical pathway, but also of many pathways that are as yet poorly understood or not recognized at all Fig. . Throughregs). Only through a deeper understanding of the pathomechanism at the molecular level it will be possible to develop effective causal therapies (beyond non-specific immunosuppression or immunoglobulin administration). JAK inhibitors have ushered in a new era of immunotherapies, with the ability to be truly targeted in the case of JAK/STAT deficiencies. Integral personalized therapy is only possible if both immunodeficiency and autoimmunity are addressed in treatment when they co-occur. In such cases, dose titration makes sense, as does the use of highly specific JAK inhibitors as opposed to broad-acting ones to treat only the dysregulated protein or pathway. In the future, there will probably also be - so far paradoxical - therapeutic approaches, such as JAK inhibition in SLE-like phenotype in STAT3 DN [Further, this creates a need for meticulous case reports when novel mutations are identified and reviews to identify clustered symptoms that may be rare. Moreover, this requires derived functional studies that investigate the molecular pathomechanisms behind the clinical phenotype  and to apply them in the diagnosis of new patients. A far-sighted and open-minded research attitude is of great benefit if unexpected results (such as SLE-like autoimmunity in STAT3 DN mutations) can be assigned to these diseases and new explanatory approaches for a holistic understanding of the syndromes arise. For several years now, JAK inhibitors have also been able to be used selectively in cases of overactivation of the signaling pathways, and more causal approaches such as HSCT and, in the future, gene therapies are developing. For all therapies, the risk of adverse events must be considered, such as increased incidence of viral infections in STAT1 GOF during JAK inhibitor use or fatal outcomes in HSCT of STAT3 GOF patients. This review provided a distinguished overview of recent findings of syndromes associated with STAT1, STAT3, STAT6, JAK1, or JAK3 germline mutations emphasizing the relation of clinical signs, symptoms, laboratory findings, and the pathomolecular etiology. The understanding of JAK/STAT dysregulation as a model on a one-dimensional spectrum between the extremes of immunodeficiency and autoimmunity is now becoming outdated. Instead, progress in pathomechanistic understanding and clinical and immunological manifestations is leading to multidimensional representations that reconcile the coexistence of symptoms from both ends of the immunity spectrum. We therefore advocate a two-dimensional understanding of JAK/STAT LOF and GOF diseases, with the intention of identifying novel molecular pathomechanisms, to discover and provide explanations for previously opaque phenotypes and to develop new therapeutic approaches for affected patients.Since the JAK/STAT signaling pathway plays a central role in mediating fundamental biological processes such as in development and in the immune system, genetic defects in these players may lead to dramatic clinical consequences. Over the past years, new results in JAK/STAT research improved the pathomolecular understanding of the syndromes. Case reports of new patients with, e.g., JAK1 GOF, reveal novel disease patterns that were previously undetected due to their rarity or their life-threatening nature. The improved pathophysiological understanding leads to focused diagnostic procedures and targeted treatment approaches as seen in STAT6 GOF. The reporting of more patients with rare diseases is essential to systematically identify sporadic complications (such as mycobacterial and viral infections in autosomal recessive complete


Lynch syndrome (LS)-associated glioblastoma (GBM) is rare in clinical practice, and simultaneous occurrence with cutaneous porokeratosis is even rarer. In this study, we analyzed the clinicopathological and genetic characteristics of LS-associated GBMs and concurrent porokeratosis, as well as evaluated the tumor immune microenvironment (TIME) of LS-associated GBMs.Immunohistochemical staining was used to confirm the histopathological diagnosis, assess MMR and PD-1/PD-L1 status, and identify immune cell subsets. FISH was used to detect amplification of EGFR and PDGFRA, and deletion of 1p/19q and CDKN2A. Targeted NGS assay analyzed somatic variants, MSI, and TMB status, while whole-exome sequencing and Sanger sequencing were carried out to analyze the germline mutations.ARID1A, TP53, ATM, and NF1 genes. All GBMs had TMB-H but not MSI-H. CD8+ T cells and CD163+ macrophages were abundant in each GBM tissue. The primary and recurrent GBMs of II:1 showed mesenchymal characteristics with high PD-L1 expression. The family members harbored a novel heterozygous germline mutation in MSH2 and FDPS genes, confirming the diagnosis of LS and disseminated superficial actinic porokeratosis.In the LS family, three members  were affected by GBM. GBMs with loss of MSH2 and MSH6 expression displayed giant and multinucleated bizarre cells, along with mutations in LS-associated GBM exhibits heterogeneity in clinicopathologic and molecular genetic features, as well as a suppressive TIME. The presence of MMR deficiency and TMB-H may serve as predictive factors for the response to immune checkpoint inhibitor therapy in GBMs. The identification of LS-associated GBM can provide significant benefits to both patients and their family members, including accurate diagnosis, genetic counseling, and appropriate screening or surveillance protocols. Our study serves as a reminder to clinicians and pathologists to consider the possibility of concurrent genetic syndromes in individuals or families. MLH1, MSH2, MSH6, or PMS2) or deletions in EPCAM (Lynch syndrome (LS) is an autosomal dominant condition caused by a germline heterozygous pathogenic variant in one of four DNA mismatch repair (MMR) genes , The First Affiliated Hospital of Shandong First Medical University (II:4), and Shandong Cancer Hospital and Institute (I:1). We also collected peripheral blood samples from family members for whole-genome sequencing and Sanger sequencing.Immunohistochemical (IHC) staining was used to confirm the histopathological diagnosis, assess MMR and PD-1/PD-L1 status, and identify immune cell subsets in the tumor microenvironment. The antibodies utilized in this study are listed in 500 ng of DNA extracted from FFPE tissues was modified by sodium bisulfite, which converts unmethylated cytosine to uracil, following manufacturer’s instructions of MGMT plus Kit . PCR was performed on the modified DNA and the products were sequenced. The average methylation of the 10 CpG sites was calculated using the PyroMark Q96 ID system (Qiagen). An analytical cut-off of 5% was used to distinguish methylated from unmethylated samples, as suggested by the MGMT plus Kit.The 163-bp fragment was amplified using KAPA2G Robust HotStart ReadyMix (Sigma) with the primers 5’-GTCCTGCCCCTTCACCTT-3’ and 5’-CAGCGCTGCCTGAAACTC-3’. The purified PCR products were sequenced by GATC Biotech , and the chromatograms were manually interpreted using 4Peaks Software .EGFR and PDGFRA amplification and 1p/19q and CDKN2A deletion were identified using FISH. The FISH assay was performed using locus-specific probes for EGFR (7p12), 1p36, 19q13, PDGFRA (4q12), CDKN2A (9p21), and corresponding reference centromeric probes recommended by the manufacturer , following previously described method .A next-generation sequencing (NGS) assay that targets 550 somatic genes, as well as microsatellite instability (MSI) and tumor mutation burden (TMB) status, was performed at Di’An Diagnostics  using an Illumina Hiseq4000 platform . Whole-exome sequencing (WES) was performed on DNA samples obtained from peripheral leucocytes of family members. The validation of germline mutations was analyzed using Sanger sequencing. Genomic DNA isolation, sequencing and data analysis procedures were described in previous studies .DNA was extracted from tumors and subjected to genome-wide DNA methylation analysis using Illumina Human MethylationEPIC BeadChip (850k) assay . Data processing and quality checks were performed using the GenomeStudio Methylation Module. To evaluate subgroups, hierarchical clustering, t-distributed stochastic neighbor embedding (tSNE), and principal component analysis were conducted using the R package Rtsne, and pheatmap was used with the WPGMA linkage method and Euclidean distance .2/day). The patient then underwent six cycles of adjuvant TMZ . After completing chemotherapy, MRI examination revealed no signs of recurrence at the surgical site. However, at 5 months after the completion of chemotherapy (1 year after the first surgery), the patient underwent MRI due to worsening headaches and cognitive impairment, which showed the presence of abnormal enhancement shadows in the original surgical area, indicating tumor recurrence. On June 18, 2019, he underwent the second intracranial tumor resection (II:1r). Two months after the second surgery, imaging examination showed recurrence once again. The patient declined PD-1 targeted immunotherapy and radiotherapy and received only TMZ treatment (200 mg/m2/day),. However, due to aggravated headaches and vomiting, the patient discontinued TMZ after two cycles treatment and passed away on November 26, 2019. Additionally, he underwent resection for colonic adenocarcinoma at the age of 35 years and penile squamous cell carcinoma (SCC) at the with loss of MSH2 and MSH6 expression  revealed a well-defined nodular mass, measuring 5.4×5.0×5.0 cm in the right temporal lobe, with hyperintense T1 weighted image (WI) and a typical ring-enhancing pattern (2/day). Subsequently, the patient underwent six cycles of adjuvant TMZ chemotherapy . Due to residual tumor suspected on imaging, an additional 7 sessions of radiation therapy (DT 60 Gy) were administered on January 5, 2022. The patient’s TMZ chemotherapy was discontinued in June 2022 after 6 cycles chemotherapy. On August 31, 2022, imaging examination revealed progression of the tumor in the left temporal lobe. Chemotherapy combined with targeted therapy was initiated, consisting of bevacizumab  and erlotinib . On April 23, 2023, MRI examination showed edema in the lesion area, and a single dose of bevacizumab (300 mg) was administered for treatment. The patient is currently under regular follow-up, with no evidence of tumor recurrence. She developed colonic adenomatous polyps at the age of 46 years with loss of MSH2 and MSH6 expression , the family declined further treatment. The patient experienced tumor recurrence four months after the surgery and passed away two months later. He had rectal carcinoma at the age of 67 years.Histological analysis showed that the primary tumor of II:1 was composed of giant and multinucleated bizarre cells, intermixed with smaller and mononuclear cells  promoter mutation, CDKN2A deletion, EGFR and PDGFRA amplification, as well as chromosome 7 gain and 10 loss, were not identified in any of the three GBMs  in I:1 and II:1r, and microsatellite-stable (MSS) in II:4. Furthermore, the TMB in I:1 (78.87 Mut/Mb), II:1r (754.23 Mut/Mb) and II:4 (42.25 Mut/Mb) was identified as TMB-H, based on the cut-off of 10 Mut/Mb.To analyze somatic genetic variations in GBMs, we conducted NGS panel sequencing. As shown in Based on DNA methylation status, GBMs in II:1r and II:4 were classified as GBM-mesenchymal (MES) and high grade glioma (HGG)-receptor tyrosine kinase type I (RTKI), respectively (MSH2 gene (chr2:47656751-47657242) using WES. According to the variant interpretation guidelines of the American College of Medical Genetics and Genomics (ACMG), this variant is confirmed to be a pathogenic mutation. Furthermore, with familial clustering of cancers (We identified a novel heterozygous germline 492 bp deletion in the 2). The numbers of CD8+ T cells were higher than CD4+ T cells in each GBM tissue, with the highest densities observed in the tumors of II:1 (440.37 ± 192.39/mm2) and II:1r (504.44 ± 263.05/mm2). Additionally, macrophages were highly enriched throughout the tumors in all four GBMs, as shown by CD163 immunostaining. Representative photomicrographs of lymphocytes and macrophages infiltration are shown in To assess the immune microenvironment of GBMs, we performed immunostaining of tissue sections for lymphocytes and macrophages. We observed few scanty CD4+ T cells in all four GBMs, with the highest density observed in the tumor of II:4 , and PolyPhen-2 (http://genetics.bwh.harvard.edu/pph2/) predicted it as a possible pathogenic variant with a score of 0.933, resulting in a nonfunctional protein product. We have presented the family pedigree of DSAP in We observed multiple family members with skin lesions characterized by many superficial, annular and brownish macules on their faces, necks, limbs and trunks occurring after the age of 30. The skin lesion on II:2 was notable due to excessive sunlight exposure as a farmer , with a lifetime risk estimated to be between of 1% to 6% , 9. Howe was GBM . Identif was GBM . [2] His was GBM . However was GBM , 13. The was GBM . TherefoARID1A, TP53, ATM, SETD2, and NF1 in four GBMs, consistent with the genetic analysis of Kim, et al.  biosynthesis in the mevalonate pathway. This pathway is known to be critical for cholesterol synthesis to maintain GBM cell growth, including the preservation of GBM stemness and the prevention of apoptosis . The patients/participants provided their written informed consent to participate in this study.Z-GY and FH wrote the original manuscript. Z-GY, FH and G-HZ design the study. Y-JX, Z-HY, Y-CN, X-KL, Z-MZ and M-QZ coordinated patient recruitment and sample collection. Y-HH performed the methylation analysis. Y-HH, X-HG and CM contributed to the genetic testing and data interpretation. ZW and G-HZ took and interpreted histological images as well as made the final pathological diagnosis. ZW, G-CL and G-HZ critically revised the manuscript. All authors contributed to the article and approved the submitted version.