A TCF4-dependent gene regulatory network confers resistance to immunotherapy in melanoma.

To better understand intrinsic resistance to immune checkpoint blockade (ICB), we established a comprehensive view of the cellular architecture of the treatment-naive melanoma ecosystem and studied its evolution under ICB. Using single-cell, spatial multi-omics, we showed that the tumor microenvironment promotes the emergence of a complex melanoma transcriptomic landscape. Melanoma cells harboring a mesenchymal-like (MES) state, a population known to confer resistance to targeted therapy, were significantly enriched in early on-treatment biopsies from non-responders to ICB. TCF4 serves as the hub of this landscape by being a master regulator of the MES signature and a suppressor of the melanocytic and antigen presentation transcriptional programs. Targeting TCF4 genetically or pharmacologically, using a bromodomain inhibitor, increased immunogenicity and sensitivity of MES cells to ICB and targeted therapy. We thereby uncovered a TCF4-dependent regulatory network that orchestrates multiple transcriptional programs and contributes to resistance to both targeted therapy and ICB in melanoma.

Deciphering novel TCF4-driven mechanisms underlying a common triplet repeat expansion-mediated disease.

Fuchs endothelial corneal dystrophy (FECD) is an age-related cause of vision loss, and the most common repeat expansion-mediated disease in humans characterised to date. Up to 80% of European FECD cases have been attributed to expansion of a non-coding CTG repeat element (termed CTG18.1) located within the ubiquitously expressed transcription factor encoding gene, TCF4. The non-coding nature of the repeat and the transcriptomic complexity of TCF4 have made it extremely challenging to experimentally decipher the molecular mechanisms underlying this disease. Here we comprehensively describe CTG18.1 expansion-driven molecular components of disease within primary patient-derived corneal endothelial cells (CECs), generated from a large cohort of individuals with CTG18.1-expanded (Exp+) and CTG 18.1-independent (Exp-) FECD. We employ long-read, short-read, and spatial transcriptomic techniques to interrogate expansion-specific transcriptomic biomarkers. Interrogation of long-read sequencing and alternative splicing analysis of short-read transcriptomic data together reveals the global extent of altered splicing occurring within Exp+ FECD, and unique transcripts associated with CTG18.1-expansions. Similarly, differential gene expression analysis highlights the total transcriptomic consequences of Exp+ FECD within CECs. Furthermore, differential exon usage, pathway enrichment and spatial transcriptomics reveal TCF4 isoform ratio skewing solely in Exp+ FECD with potential downstream functional consequences. Lastly, exome data from 134 Exp- FECD cases identified rare (minor allele frequency <0.005) and potentially deleterious (CADD>15) TCF4 variants in 7/134 FECD Exp- cases, suggesting that TCF4 variants independent of CTG18.1 may increase FECD risk. In summary, our study supports the hypothesis that at least two distinct pathogenic mechanisms, RNA toxicity and TCF4 isoform-specific dysregulation, both underpin the pathophysiology of FECD. We anticipate these data will inform and guide the development of translational interventions for this common triplet-repeat mediated disease.

AMD1 promotes breast cancer aggressiveness via a spermidine-eIF5A hypusination-TCF4 axis.

BACKGROUND: Basal-like breast cancer (BLBC) is the most aggressive subtype of breast cancer due to its aggressive characteristics and lack of effective therapeutics. However, the mechanism underlying its aggressiveness remains largely unclear. S-adenosylmethionine decarboxylase proenzyme (AMD1) overexpression occurs specifically in BLBC. Here, we explored the potential molecular mechanisms and functions of AMD1 promoting the aggressiveness of BLBC. METHODS: The potential effects of AMD1 on breast cancer cells were tested by western blotting, colony formation, cell proliferation assay, migration and invasion assay. The spermidine level was determined by high performance liquid chromatography. The methylation status of CpG sites within the AMD1 promoter was evaluated by bisulfite sequencing PCR. We elucidated the relationship between AMD1 and Sox10 by ChIP assays and quantitative real-time PCR. The effect of AMD1 expression on breast cancer cells was evaluated by in vitro and in vivo tumorigenesis model. RESULTS: In this study, we showed that AMD1 expression was remarkably elevated in BLBC. AMD1 copy number amplification, hypomethylation of AMD1 promoter and transcription activity of Sox10 contributed to the overexpression of AMD1 in BLBC. AMD1 overexpression enhanced spermidine production, which enhanced eIF5A hypusination, activating translation of TCF4 with multiple conserved Pro-Pro motifs. Our studies showed that AMD1-mediated metabolic system of polyamine in BLBC cells promoted tumor cell proliferation and tumor growth. Clinically, elevated expression of AMD1 was correlated with high grade, metastasis and poor survival, indicating poor prognosis of breast cancer patients. CONCLUSION: Our work reveals the critical association of AMD1-mediated spermidine-eIF5A hypusination-TCF4 axis with BLBC aggressiveness, indicating potential prognostic indicators and therapeutic targets for BLBC.

scRNA sequencing uncovers a TCF4-dependent transcription factor network regulating commissure development in mouse.

Transcription factor 4 (TCF4) is a crucial regulator of neurodevelopment and has been linked to the pathogenesis of autism, intellectual disability and schizophrenia. As a class I bHLH transcription factor (TF), it is assumed that TCF4 exerts its neurodevelopmental functions through dimerization with proneural class II bHLH TFs. Here, we aim to identify TF partners of TCF4 in the control of interhemispheric connectivity formation. Using a new bioinformatic strategy integrating TF expression levels and regulon activities from single cell RNA-sequencing data, we find evidence that TCF4 interacts with non-bHLH TFs and modulates their transcriptional activity in Satb2+ intercortical projection neurons. Notably, this network comprises regulators linked to the pathogenesis of neurodevelopmental disorders, e.g. FOXG1, SOX11 and BRG1. In support of the functional interaction of TCF4 with non-bHLH TFs, we find that TCF4 and SOX11 biochemically interact and cooperatively control commissure formation in vivo, and regulate the transcription of genes implicated in this process. In addition to identifying new candidate interactors of TCF4 in neurodevelopment, this study illustrates how scRNA-Seq data can be leveraged to predict TF networks in neurodevelopmental processes.

FUT8-AS1/miR-944/Fused in Sarcoma/Transcription Factor 4 Feedback Loop Participates in the Development of Oral Squamous Cell Carcinoma through Activation of Wnt/ß-Catenin Signaling Pathway.

As a common type of head and neck squamous cell carcinoma, oral squamous cell carcinoma (OSCC) is a lethal and deforming disease. Long noncoding RNAs have emerged as critical modulators in different malignancies. However, the role of fucosyltransferase 8 antisense RNA 1 (FUT8-AS1) in OSCC still remains elusive. In this study, quantitative RT-PCR and Western blot were used for the measurement of RNAs and proteins. Mechanism assays explored the putative correlation among genes. In vitro assays evaluated the changes in OSCC cell malignant phenotype, whereas in vivo assays highlighted the influence of FUT8-AS1 on tumor growth. FUT8-AS1, aberrantly up-regulated in OSCC tissues and cells, could exacerbate OSCC cell malignant behaviors. The cancerogenic property of FUT8-AS1 in OSCC was further confirmed via animal experiments. Furthermore, FUT8-AS1 enhanced the expression of transcription factor 4 (TCF4) via sponging miR-944 and recruiting fused in sarcoma (FUS), thus affecting OSCC cell biological behaviors via modulation on Wnt/ß-catenin signaling activity. In addition, TCF4 was validated as the transcriptional activator of FUT8-AS1. To conclude, TCF4-mediated FUT8-AS1 could exacerbate OSCC cell malignant behaviors and facilitate tumor growth via modulation on miR-944/FUS/TCF4.

Pathogenic variants in SOX11 mimicking Pitt-Hopkins syndrome phenotype.

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder characterised by severe intellectual disability (ID), distinctive facial features and autonomic nervous system dysfunction, caused by TCF4 haploinsufficiency. We clinically diagnosed with PTHS a 14 6/12 -year-old female, who had a normal status of TCF4. The pathogenic c.667del (p.Asp223MetfsTer45) variant in SOX11 was identified through whole exome sequencing (WES). SOX11 variants were initially reported to cause Coffin-Siris syndrome (CSS), characterised by growth restriction, moderate ID, coarse face, hypertrichosis and hypoplastic nails. However, recent studies have provided evidence that they give rise to a distinct neurodevelopmental disorder. To date, SOX11 variants are associated with a variable phenotype, which has been described to resemble CSS in some cases, but never PTHS. By reviewing both clinically and genetically 32 out of 82 subjects reported in the literature with SOX11 variants, for whom detailed information are provided, we found that 7/32 (22%) had a clinical presentation overlapping PTHS. Furthermore, we made a confirmation that overall SOX11 abnormalities feature a distinctive disorder characterised by severe ID, high incidence of microcephaly and low frequency of congenital malformations. Purpose of the present report is to enhance the role of clinical genetics in assessing the individual diagnosis after WES results.

Psychiatric risk gene Transcription Factor 4 (TCF4) regulates the density and connectivity of distinct inhibitory interneuron subtypes.

Transcription factor 4 (TCF4) is a basic helix-loop-helix transcription factor that is implicated in a variety of psychiatric disorders including autism spectrum disorder (ASD), major depression, and schizophrenia. Autosomal dominant mutations in TCF4 are causal for a specific ASD called Pitt-Hopkins Syndrome (PTHS). However, our understanding of etiological and pathophysiological mechanisms downstream of TCF4 mutations is incomplete. Single cell sequencing indicates TCF4 is highly expressed in GABAergic interneurons (INs). Here, we performed cell-type specific expression analysis (CSEA) and cellular deconvolution (CD) on bulk RNA sequencing data from 5 different PTHS mouse models. Using CSEA we observed differentially expressed genes (DEGs) were enriched in parvalbumin expressing (PV+) INs and CD predicted a reduction in the PV+ INs population. Therefore, we investigated the role of TCF4 in regulating the development and function of INs in the Tcf4+/tr mouse model of PTHS. In Tcf4+/tr mice, immunohistochemical (IHC) analysis of subtype-specific IN markers and reporter mice identified reductions in PV+, vasoactive intestinal peptide (VIP+), and cortistatin (CST+) expressing INs in the cortex and cholinergic (ChAT+) INs in the striatum, with the somatostatin (SST+) IN population being spared. The reduction of these specific IN populations led to cell-type specific alterations in the balance of excitatory and inhibitory inputs onto PV+ and VIP+ INs and excitatory pyramidal neurons within the cortex. These data indicate TCF4 is a critical regulator of the development of specific subsets of INs and highlight the inhibitory network as an important source of pathophysiology in PTHS.

Evaluation of Nav1.8 as a therapeutic target for Pitt Hopkins Syndrome.

Pitt Hopkins Syndrome (PTHS) is a rare syndromic form of autism spectrum disorder (ASD) caused by autosomal dominant mutations in the Transcription Factor 4 (TCF4) gene. TCF4 is a basic helix-loop-helix transcription factor that is critical for neurodevelopment and brain function through its binding to cis-regulatory elements of target genes. One potential therapeutic strategy for PTHS is to identify dysregulated target genes and normalize their dysfunction. Here, we propose that SCN10A is an important target gene of TCF4 that is an applicable therapeutic approach for PTHS. Scn10a encodes the voltage-gated sodium channel Nav1.8 and is consistently shown to be upregulated in PTHS mouse models. In this perspective, we review prior literature and present novel data that suggests inhibiting Nav1.8 in PTHS mouse models is effective at normalizing neuron function, brain circuit activity and behavioral abnormalities and posit this therapeutic approach as a treatment for PTHS.

A patient with Pitt-Hopkins syndrome with concomitant common variable immunodeficiency.

In patients with 18q deletion syndrome (18q-), immunodeficiency, autoimmunity, and allergies have been described in a subset. Pitt-Hopkins syndrome represents a specific subset of patients with 18q- who have a proximal deletion involving the TCF4 gene or a TCF4 variant. Immunodeficiency has been reported in the overall 18q- population; however, immunodeficiency with Pitt-Hopkins syndrome has not been highlighted. This case report details the immunologic evaluations and the associated infections seen in a young adult with Pitt-Hopkins syndrome to underscore the challenges of managing adults with a complex phenotype who develop frequent infections. This patient with Pitt-Hopkins syndrome ultimately fulfilled the diagnostic criteria for common variable immunodeficiency. Immunoglobulin replacement has led to a somewhat improved infection pattern, although she continues to have aspiration events leading to pneumonia. This case highlights the clinical evolution of Pitt-Hopkins syndrome and serves as a reminder that immunodeficiency can occur in this syndrome.

DNA methylation changes and increased mRNA expression of coagulation proteins, factor V and thrombomodulin in Fuchs endothelial corneal dystrophy.

Late-onset Fuchs endothelial corneal dystrophy (FECD) is a disease affecting the corneal endothelium (CE), associated with a cytosine-thymine-guanine repeat expansion at the CTG18.1 locus in the transcription factor 4 (TCF4) gene. It is unknown whether CTG18.1 expansions affect global methylation including TCF4 gene in CE or whether global CE methylation changes at advanced age. Using genome-wide DNA methylation array, we investigated methylation in CE from FECD patients with CTG18.1 expansions and studied the methylation in healthy CE at different ages. The most revealing DNA methylation findings were analyzed by gene expression and protein analysis. 3488 CpGs had significantly altered methylation pattern in FECD though no substantial changes were found in TCF4. The most hypermethylated site was in a predicted promoter of aquaporin 1 (AQP1) gene, and the most hypomethylated site was in a predicted promoter of coagulation factor V (F5 for gene, FV for protein). In FECD, AQP1 mRNA expression was variable, while F5 gene expression showed a ~ 23-fold increase. FV protein was present in both healthy and affected CE. Further gene expression analysis of coagulation factors interacting with FV revealed a ~ 34-fold increase of thrombomodulin (THBD). THBD protein was detected only in CE from FECD patients. Additionally, we observed an age-dependent hypomethylation in elderly healthy CE.Thus, tissue-specific genome-wide and gene-specific methylation changes associated with altered gene expression were discovered in FECD. TCF4 pathological methylation in FECD because of CTG18.1 expansion was ruled out.

Nanopore sequencing method for CTG18.1 expansion in TCF4 in late-onset Fuchs endothelial corneal dystrophy and a comparison of the structural features of cornea with first-degree relatives.

BACKGROUND: To evaluate the relationship between the number of trinucleotide repeats (TNR) in late-onset Fuchs corneal endothelial dystrophy (FCED) and to compare the endothelial properties of FCED, first-degree relatives, and controls. METHODS: Blood samples were collected from FCEDs to determine TNR number. The FCED patients, first-degree relatives, and controls were examined with specular microscopy for central corneal thickness (CCT), endothelial cell density (ECD), pleomorphism and polymegatism, and with corneal topography for specific indicators such as (i) displacement of thinnest point of cornea, (ii) loss of isopachs, (iii) focal posterior surface depression towards anterior chamber. RESULTS: This study included 92 patients with FCED, 92 first-degree relatives, and 96 controls. CCT was thickest in FCEDs (558.0 µm) (p < 0.05) while there was no difference between relatives (533.0 µm) and controls (530.4 µm) (p = 0.845). ECD was decreased in both FCED (2069.2 mm2) and relatives (2171.4 mm2) than controls (2822.9 mm2) (p < 0.05 in both). The presence of pleomorphism and polymegatism was significant in patients with FCED (93.4% and 93.4%, respectively), relatives (86.9% and 86.04%, respectively), and controls (8.33% and 1.04%, respectively) (p < 0.05). Specific topographic indicators differed among the groups (p < 0.05). The mean repeat number of the FCED patients was 17.48 ± 4.54 (12-25) times. The TNR number of FCED cases correlated with the relative CCT (p < 0.05, R = 0.615) and cell density (p = 0.009, R = -0.499). CONCLUSIONS: A strong association between the corneal endothelium in relatives and TNR number of FCEDs was defined. Relatives tended to have fewer corneal endothelial cells, even though they did not have clinical findings.

Altered ratio of dendritic cell subsets in skin-draining lymph nodes promotes Th2-driven contact hypersensitivity.

Plasmacytoid dendritic cells (pDCs) specialize in the production of type I IFN (IFN-I). pDCs can be depleted in vivo by injecting diphtheria toxin (DT) in a mouse in which pDCs express a diphtheria toxin receptor (DTR) transgene driven by the human CLEC4C promoter. This promoter is enriched for binding sites for TCF4, a transcription factor that promotes pDC differentiation and expression of pDC markers, including CLEC4C. Here, we found that injection of DT in CLEC4C-DTR+ mice markedly augmented Th2-dependent skin inflammation in a model of contact hypersensitivity (CHS) induced by the hapten fluorescein isothiocyanate. Unexpectedly, this biased Th2 response was independent of reduced IFN-I accompanying pDC depletion. In fact, DT treatment altered the representation of conventional dendritic cells (cDCs) in the skin-draining lymph nodes during the sensitization phase of CHS; there were fewer Th1-priming CD326+ CD103+ cDC1 and more Th2-priming CD11b+ cDC2. Single-cell RNA-sequencing of CLEC4C-DTR+ cDCs revealed that CD326+ DCs, like pDCs, expressed DTR and were depleted together with pDCs by DT treatment. Since CD326+ DCs did not express Tcf4, DTR expression might be driven by yet-undefined transcription factors activating the CLEC4C promoter. These results demonstrate that altered DC representation in the skin-draining lymph nodes during sensitization to allergens can cause Th2-driven CHS.

Complex Craniosynostosis in Pitt-Hopkins Syndrome: Case Report in Twins.

INTRODUCTION: Pitt-Hopkins syndrome (PTHS) is a rare genetic syndrome associated with neurodevelopmental disorders and craniofacial dysmorphisms caused by variations in the TCF4 transition factor. The aim of this article was to report the case of two twin infants diagnosed with PTHS, confirmed by the identification of a heterozygous pathogenic variant in the TCF4 gene through DNA extracted from a buccal swab. CASE PRESENTATION: Both infants presented with craniofacial asymmetry with a metopic crest and cranial deformity. During the diagnostic investigation, computed tomography with three-dimensional reconstruction of the skull showed premature fusion of the left coronal and metopic sutures in both twins. They underwent craniofacial reconstruction at the 9th month of age using a combination of techniques. The postoperative outcomes were satisfactory in both cases. CONCLUSION: To the best of our knowledge, this is the first case report to describe the occurrence of complex craniosynostosis (CCS) in children with PTHS. Further studies are needed to determine whether the co-occurrence of PTHS and CCS described here indicates an association or is explained by chance.

CTG18.1 expansion in transcription factor 4 (TCF4) in corneal graft failure: preliminary study.

Fuchs endothelial corneal dystrophy (FECD) is caused by a corneal endothelial cell loss, leading to corneal edema and visual impairment. The most significant genetic risk factor for FECD is an expansion of the CTG18.1 locus in transcription factor 4 (TCF4). The current treatment for severe FECD is corneal transplantation, with Descemet stripping automated keratoplasty (DSAEK) as a common surgical method. Although successful in most cases, the risk for transplant failure due to diverse causes must be considered. In this study, we investigated if presence of TCF4 CTG18.1 expansion with more than 31 (n ≥ 31) repeats in donated corneal grafts could be a reason for corneal transplant failure after DSAEK. For this, nine consecutively failed DSAEK corneal grafts were genotyped for CTG18.1 repeat length. One-sided Mann-Whitney U test was performed to evaluate if failed DSAEK corneal grafts had longer CTG18.1 repeats than healthy controls from the same population. All failed corneal grafts had CTG18.1 n ≤ 27 with a median of 18 (IQR 8.0) repeats for the longest allele. There was no statistical difference in CTG18.1 repeat lengths between failed corneal grafts and the geographically matched healthy control group. In conclusion, none of the nine failed corneal grafts in our material had CTG18.1 repeat lengths ≥ 31, a cut-off known to have a biological relevance in FECD. Thus, our results suggest that the assessment of donors and inspection of the corneal tissue before the decision for procurement is sufficient, in terms of recognizing FECD in the donor.

Expanding the phenotype in Pitt-Hopkins syndrome; description of new oral finding and dental management considerations.

BACKGROUND: Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder with physical, cognitive, and behavioral characteristics that is caused by heterozygous mutations in the TCF4 gene. Patients with PTHS might present a unique challenge for oral healthcare professionals because of the associated comorbidities. CASE REPORT: Here we describe a new case of PTHS in a 13-year-old girl with particular emphasis on oro-dental findings and oral healthcare management. Observed oro-dental findings in our case included shallow palate, absence of lingual frenum, gingival enlargement, thick lips and relative microdontia. The patient was unable to tolerate dental care under local anesthesia. Therefore, comprehensive dental treatment was performed under general anesthesia after a careful pre-anesthetic cardio-respiratory, neurological, and hematological evaluation. The patient was closely monitored intra-operatively for breathing rhythm, O2 saturation, and signs of respiratory distress. The patient was observed for 24 h post-op for respiratory distress and was discharged then uneventfully. CONCLUSION: Dental treatment under general anesthesia in these patients might be complicated by the abnormal breathing rhythm, and close monitoring and follow up for signs of respiratory distress after general anesthesia is necessary. Recognition of oral and dental findings might help to expand the phenotype and better characterize rare syndromes.

MPZL1 suppresses the cancer stem-like properties of lung cancer through ß-catenin/TCF4 signaling.

This study was designed to explore the influence of myelin protein zero-like protein 1 (MPZL1) on the stem-like properties of cancer cells and the underlying mechanism in lung adenocarcinoma. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to evaluate mRNA expression level. CCK8, wound healing, and transwell assays were applied to assess cell proliferation, migration, and invasion. Tumorsphere-formation assay was utilized to assess cancer stem cell-like properties. LF3 was used to block the ß-catenin/Transcription factor 4 (TCF-4) signaling. Xenograft nude mouse model was conducted; tumor weight and volume were recorded. Western blot assay was utilized to detect the expression levels of CD44, CD133, ß-catenin, TCF-4, and MPZL1. Following MPZL1 knockdown, the mRNA expression levels of MPZL1, ß-catenin, and TCF-4 were inhibited, while the mRNA expression levels of the above genes were increased after the MPZL1 overexpression. MPZL1 knockdown suppressed cell proliferation, migration, and invasion, reduced the tumorsphere-formation capacity, and restrained the expression levels of CD44 and CD133. However, MPZL1 overexpression promoted the cell proliferation, migration, and invasion, enhanced the tumorsphere-formation capacity, and increased the expression levels of CD44 and CD133. Interestingly, LF3 treatment partially revised the effect of MPZL1 overexpression. These findings were further corroborated by in vivo experiments. We concluded that MPZL1 could suppress the lung adenocarcinoma cells' proliferation, migration, invasion, and lung cancer stem cells characteristics. The underlying mechanism is involved in the activation of ß-catenin/TCF-4 signaling.

TCF7L2 regulates postmitotic differentiation programmes and excitability patterns in the thalamus.

Neuronal phenotypes are controlled by terminal selector transcription factors in invertebrates, but only a few examples of such regulators have been provided in vertebrates. We hypothesised that TCF7L2 regulates different stages of postmitotic differentiation in the thalamus, and functions as a thalamic terminal selector. To investigate this hypothesis, we used complete and conditional knockouts of Tcf7l2 in mice. The connectivity and clustering of neurons were disrupted in the thalamo-habenular region in Tcf7l2-/- embryos. The expression of subregional thalamic and habenular transcription factors was lost and region-specific cell migration and axon guidance genes were downregulated. In mice with a postnatal Tcf7l2 knockout, the induction of genes that confer thalamic terminal electrophysiological features was impaired. Many of these genes proved to be direct targets of TCF7L2. The role of TCF7L2 in terminal selection was functionally confirmed by impaired firing modes in thalamic neurons in the mutant mice. These data corroborate the existence of master regulators in the vertebrate brain that control stage-specific genetic programmes and regional subroutines, maintain regional transcriptional network during embryonic development, and induce terminal selection postnatally.

A typical variant in TCF4 exon 18 is not associated with Pitt-Hopkins syndrome but with a familial case of mild and nonspecific neurodevelopmental disorder.

TCF4 gene encodes a class I helix-loop-helix transcription factor critical for the developing brain. Common polymorphisms in TCF4 and disruptive variants in the proximal region of the gene have been linked to relatively mild neuropsychiatric or neurodevelopmental disorders. In contrast, variants impacting distal exons are associated with Pitt-Hopkins syndrome (PTHS), a severe autosomal dominant condition characterized by profound intellectual disability, developmental delay, limited or absent speech, distinctive facies, and disordered breathing. Although phenotypic variability has been observed in PTHS, intellectual impairment and significant speech and motor delays are invariably present. In contrast to the typical de novo variants causing TCF4-related disorder and PTHS, we report a familial form of TCF4-related disorder where the missense variant arose de novo in the father and was inherited by two of his children. Although this family's variant's position in exon 18 predicted a typical PTHS phenotype, none of the affected individuals met the clinical diagnostic criteria for PTHS suggested by Zollino et al. in the first international consensus statement (as in the study by Zollino et al. in 2019). Rather, the three affected family members exhibited remarkably variable and milder phenotypes than would have been predicted from the position of their TCF4 variant. Thus, the clinical spectrum of PTHS-associated TCF4 variants may be broader than previously reported.

Fatal gastrointestinal complications in Pitt-Hopkins syndrome.

Pitt-Hopkins syndrome (PTHS) is a rare neurodevelopmental disorder caused by mutations of the transcription factor 4 (Tcf4) gene. Individuals with PTHS often suffer from severe abdominal bloating and constipation. In this short communication, we discuss two individuals with PTHS who died unexpectedly due to gastrointestinal complications. We aim to increase awareness among healthcare professionals who care for individuals with PTHS, to ensure adequate screening and management of gastrointestinal symptoms in this population. Moreover, we discuss how fatal gastrointestinal complications may be related to PTHS and provide an overview of the literature.

Salt-like transcription factor 4 promotes laryngeal cancer progression through transcriptional activation of ubiquitin-specific protease 21 to stabilize Yin Yang 1.

Laryngeal cancer (LC) is a rare and challenging clinical problem. Our aim was to investigate the mechanism of salt-like transcription factor 4 (SALL4) in LC. LC tissue and paracancerous tissue were collected. Relative mRNA or protein levels were measured by quantitative real-time polymerase chain reaction or Western blot. MTT, wound healing, and transwell assay were performed to evaluate cell proliferation, migration and invasion. The binding relationship between SALL4 and USP21 promoter was verified by dual-luciferase assay and ChIP. Co-IP and glutathione-S-transferase (GST)-pull down were performed to measure the protein interaction between USP21 and YY1. Additionally, YY1 ubiquitination level was analyzed. It was found that SALL4 mRNA and SALL4 protein levels were elevated in LC clinical tissues and various LC cells. Knockdown of SALL4 inhibited epithelial-mesenchymal transition (EMT) of LC cells. USP21 was transcriptionally activated by SALL4. Co-IP and GST-pull down confirmed USP21 interacted with YY1. USP21 protected YY1 from degradation through deubiquitination. Furthermore, overexpression of USP21 reversed the effect of knockdown of SALL4 on YY1 and EMT in LC cells. In general, SALL4 facilitated EMT of LC cells through modulating USP21/YY1 axis.