Pathogenic variants in the SPTLC1 gene cause hyperkeratosis lenticularis perstans.

BACKGROUND: Hyperkeratosis lenticularis perstans (HLP), also known as Flegel disease, is a rare skin disease presenting with asymptomatic small hyperkeratotic papules. The lesions often appear on the dorsal feet and lower legs, and typically develop after the fourth decade of life. A genetic basis for HLP is suspected; however, so far no gene defect linked to the development of HLP has been identified. OBJECTIVES: We aimed to identify the genetic cause of HLP. METHODS: For mutational analysis we studied a cohort of five patients with HLP using next-generation sequencing (NGS). We used DNA -extracted from fresh skin biopsies alongside ethylenediamine tetraacetic acid (EDTA) blood samples from two patients, and formalin-fixed -paraffin-embedded skin biopsy material from three patients. In addition, immunofluorescence staining of HLP lesions from four patients was investigated. RESULTS: In all samples from the five patients with HLP we identified by NGS rare variants in the SPTLC1 gene. In four patients we detected small deletions/frameshift variants and in one patient a splicing variant, predicted to disturb the splicing process. In blood samples the detected variants were heterozygous with an allele frequency of 49% and 50%, respectively. In skin biopsies the allele frequency was within the range of 46-62%. Immunofluorescence staining revealed reduced SPTLC1 protein levels in skin of patients. CONCLUSIONS: Our findings suggest that pathogenic variants in the SPTLC1 gene are the underlying genetic cause of HLP. Of note, the identified variants were either frameshift- or splicing variants probably leading to nonsense-mediated mRNA decay and thus reduced SPTLC1 protein levels. We conclude that diminished SPTLC1, the key enzyme in sphingolipid biosynthesis, leads to the development of HLP, which highlights the sphingolipid pathway as a new therapeutic target.

Marfan Syndrome Caused by Disruption of the FBN1 Gene due to A Reciprocal Chromosome Translocation.

Marfan syndrome (MFS) is a hereditary connective tissue disease caused by heterozygous mutations in the fibrillin-1 gene (FBN1) located on chromosome 15q21.1. A complex chromosomal rearrangement leading to MFS has only been reported in one case so far. We report on a mother and daughter with marfanoid habitus and no pathogenic variant in the FBN1 gene after next generation sequencing (NGS) analysis, both showing a cytogenetically reciprocal balanced translocation between chromosomes 2 and 15. By means of fluorescence in situ hybridization of Bacterial artificial chromosome (BAC) clones from the breakpoint area on chromosome 15 the breakpoint was narrowed down to a region of approximately 110 kb in FBN1. With the help of optical genome mapping (OGM), the translocation breakpoints were further refined on chromosomes 2 and 15. Sequencing of the regions affected by the translocation identified the breakpoint of chromosome 2 as well as the breakpoint of chromosome 15 in the FBN1 gene leading to its disruption. To our knowledge, this is the first report of patients with typical clinical features of MFS showing a cytogenetically reciprocal translocation involving the FBN1 gene. Our case highlights the importance of structural genome variants as an underlying cause of monogenic diseases and the useful clinical application of OGM in the elucidation of structural variants.

Porokeratosis Plantaris, Palmaris et Disseminata Caused by Con- genital Pathogenic Variants in the MVD Gene and Loss of Hetero-zygosity in Affected Skin.

Porokeratoses are a heterogeneous group of keratinization disorders. For linear porokeratosis and disseminated superficial actinic porokeratosis, a heterozygous pathogenic germline variant in a mevalonate pathway gene and a postzygotic second hit mutation present in affected skin have been shown to be the patho-genetic mechanism for the development of the lesions. However, the molecular mechanism leading to development of porokeratosis plantaris, palmaris et disseminata is not known. This study analysed a cohort of 4 patients with linear porokeratosis and 3 patients with porokeratosis plantaris, palmaris et disseminata, and performed mutation analyses of DNA extracted from blood samples and skin biopsies. All of the study patients carried the heterozygous germline variant c.70+5G>A in the MVD gene. Loss of heterozygosity due to a second hit mutation was found in affected skin of 3 patients with linear porokeratosis and 2 patients with porokeratosis plantaris, palmaris et disseminata. These results suggest that porokeratosis plantaris, palmaris et disseminata shares the same pathogenetic mechanism as other porokeratosis subtypes and belongs to the phenotypic spectrum of MVD-associated porokeratosis.

A distinct CD38+CD45RA+ population of CD4+, CD8+, and double-negative T cells is controlled by FAS.

The identification and characterization of rare immune cell populations in humans can be facilitated by their growth advantage in the context of specific genetic diseases. Here, we use autoimmune lymphoproliferative syndrome to identify a population of FAS-controlled TCRαß+ T cells. They include CD4+, CD8+, and double-negative T cells and can be defined by a CD38+CD45RA+T-BET- expression pattern. These unconventional T cells are present in healthy individuals, are generated before birth, are enriched in lymphoid tissue, and do not expand during acute viral infection. They are characterized by a unique molecular signature that is unambiguously different from other known T cell differentiation subsets and independent of CD4 or CD8 expression. Functionally, FAS-controlled T cells represent highly proliferative, noncytotoxic T cells with an IL-10 cytokine bias. Mechanistically, regulation of this physiological population is mediated by FAS and CTLA4 signaling, and its survival is enhanced by mTOR and STAT3 signals. Genetic alterations in these pathways result in expansion of FAS-controlled T cells, which can cause significant lymphoproliferative disease.

A Family with Palmar and Plantar Hyperkeratosis: A Quiz.

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Distinct molecular response patterns of activating STAT3 mutations associate with penetrance of lymphoproliferation and autoimmunity.

Germline STAT3 gain-of-function (GOF) mutations have been linked to poly-autoimmunity and lymphoproliferation with variable expressivity and incomplete penetrance. Here we studied the impact of 17 different STAT3 GOF mutations on the canonical STAT3 signaling pathway and correlated the molecular results with clinical manifestations. The mutations clustered in three groups. Group 1 mutants showed altered STAT3 phosphorylation kinetics and strong basal transcriptional activity. They were associated with the highest penetrance of lymphoproliferation and autoimmunity. Group 2 mutants showed a strongly inducible transcriptional reporter activity and were clinically less penetrant. Group 3 mutants were mostly located in the DNA binding domain and showed the strongest DNA binding affinity despite a poor transcriptional reporter response. Thus, the GOF effect of STAT3 mutations is determined by a heterogeneous response pattern at the molecular level. The correlation of response pattern and clinical penetrance indicates a significant contribution of mutation-determined effects on disease manifestations.

SNAIL1-mediated downregulation of FOXA proteins facilitates the inactivation of transcriptional enhancer elements at key epithelial genes in colorectal cancer cells.

Phenotypic conversion of tumor cells through epithelial-mesenchymal transition (EMT) requires massive gene expression changes. How these are brought about is not clear. Here we examined the impact of the EMT master regulator SNAIL1 on the FOXA family of transcription factors which are distinguished by their particular competence to induce chromatin reorganization for the activation of transcriptional enhancer elements. We show that the expression of SNAIL1 and FOXA genes is anticorrelated in transcriptomes of colorectal tumors and cell lines. In cellular EMT models, ectopically expressed Snail1 directly represses FOXA1 and triggers downregulation of all FOXA family members, suggesting that loss of FOXA expression promotes EMT. Indeed, cells with CRISPR/Cas9-induced FOXA-deficiency acquire mesenchymal characteristics. Furthermore, ChIP-seq data analysis of FOXA chromosomal distribution in relation to chromatin structural features which characterize distinct states of transcriptional activity, revealed preferential localization of FOXA factors to transcriptional enhancers at signature genes that distinguish epithelial from mesenchymal colon tumors. To validate the significance of this association, we investigated the impact of FOXA factors on structure and function of enhancers at the CDH1, CDX2 and EPHB3 genes. FOXA-deficiency and expression of dominant negative FOXA2 led to chromatin condensation at these enhancer elements. Site-directed mutagenesis of FOXA binding sites in reporter gene constructs and by genome-editing in situ impaired enhancer activity and completely abolished the active chromatin state of the EPHB3 enhancer. Conversely, expression of FOXA factors in cells with inactive CDX2 and EPHB3 enhancers led to chromatin opening and de novo deposition of the H3K4me1 and H3K27ac marks. These findings establish the pioneer function of FOXA factors at enhancer regions of epithelial genes and demonstrate their essential role in maintaining enhancer structure and function. Thus, by repressing FOXA family members, SNAIL1 targets transcription factors at strategically important positions in gene-regulatory hierarchies, which may facilitate transcriptional reprogramming during EMT.

ZEB1 is neither sufficient nor required for epithelial-mesenchymal transition in LS174T colorectal cancer cells.

Epithelial-mesenchymal transition (EMT) is implicated in metastases formation and acquired therapy resistance in several tumor entities. The two transcription factors SNAIL1 and ZEB1 are thought to be master regulators of EMT and to form a core regulatory network required for EMT-associated transcriptional reprogramming. Yet, inducible EMT models show the sequential upregulation first of SNAIL1 and only subsequently of ZEB1. Therefore, SNAIL1 and ZEB1 might be differentially needed for the onset and propagation of EMT. Here we used LS174T colorectal adenocarcinoma cells which do not express endogenous EMT-inducing transcription factors, to investigate whether ZEB1 is an obligatory downstream mediator of Snail1-induced EMT, and to test whether ZEB1 could elicit an EMT in a background of naïve epithelial cells by itself. However, CRISPR/Cas9-mediated knockout of ZEB1 did not affect the ability of ectopically expressed Snail1 to trigger a complete EMT in ZEB1-deficient LS174T cells. In contrast to Snail1, ectopic ZEB1 had only minor effects on cell morphology and invasive growth in three-dimensional spheroid cultures. In agreement with this, expression of ZEB1 did not lead to repression of epithelial marker genes, and mesenchymal markers were not upregulated by ZEB1. Likewise, ectopic ZEB1 expression did not confer increased chemoresistance. We conclude that ZEB1 is neither required nor sufficient for EMT in LS174T colorectal cancer cells.

Enhancer decommissioning by Snail1-induced competitive displacement of TCF7L2 and down-regulation of transcriptional activators results in EPHB2 silencing.

Transcriptional silencing is a major cause for the inactivation of tumor suppressor genes, however, the underlying mechanisms are only poorly understood. The EPHB2 gene encodes a receptor tyrosine kinase that controls epithelial cell migration and allocation in intestinal crypts. Through its ability to restrict cell spreading, EPHB2 functions as a tumor suppressor in colorectal cancer whose expression is frequently lost as tumors progress to the carcinoma stage. Previously we reported that EPHB2 expression depends on a transcriptional enhancer whose activity is diminished in EPHB2 non-expressing cells. Here we investigated the mechanisms that lead to EPHB2 enhancer inactivation. We show that expression of EPHB2 and SNAIL1 - an inducer of epithelial-mesenchymal transition (EMT) - is anti-correlated in colorectal cancer cell lines and tumors. In a cellular model of Snail1-induced EMT, we observe that features of active chromatin at the EPHB2 enhancer are diminished upon expression of murine Snail1. We identify the transcription factors FOXA1, MYB, CDX2 and TCF7L2 as EPHB2 enhancer factors and demonstrate that Snail1 indirectly inactivates the EPHB2 enhancer by downregulation of FOXA1 and MYB. In addition, Snail1 induces the expression of Lymphoid enhancer factor 1 (LEF1) which competitively displaces TCF7L2 from the EPHB2 enhancer. In contrast to TCF7L2, however, LEF1 appears to repress the EPHB2 enhancer. Our findings underscore the importance of transcriptional enhancers for gene regulation under physiological and pathological conditions and show that SNAIL1 employs a combinatorial mechanism to inactivate the EPHB2 enhancer based on activator deprivation and competitive displacement of transcription factors.

SNAIL1 combines competitive displacement of ASCL2 and epigenetic mechanisms to rapidly silence the EPHB3 tumor suppressor in colorectal cancer.

EPHB3 is a critical cellular guidance factor in the intestinal epithelium and an important tumor suppressor in colorectal cancer (CRC) whose expression is frequently lost at the adenoma-carcinoma transition when tumor cells become invasive. The molecular mechanisms underlying EPHB3 silencing are incompletely understood. Here we show that EPHB3 expression is anti-correlated with inducers of epithelial-mesenchymal transition (EMT) in primary tumors and CRC cells. In vitro, SNAIL1 and SNAIL2, but not ZEB1, repress EPHB3 reporter constructs and compete with the stem cell factor ASCL2 for binding to an E-box motif. At the endogenous EPHB3 locus, SNAIL1 triggers the displacement of ASCL2, p300 and the Wnt pathway effector TCF7L2 and engages corepressor complexes containing HDACs and the histone demethylase LSD1 to collapse active chromatin structure, resulting in rapid downregulation of EPHB3. Beyond its impact on EPHB3, SNAIL1 deregulates markers of intestinal identity and stemness and in vitro forces CRC cells to undergo EMT with altered morphology, increased motility and invasiveness. In xenotransplants, SNAIL1 expression abrogated tumor cell palisading and led to focal loss of tumor encapsulation and the appearance of areas with tumor cells displaying a migratory phenotype. These changes were accompanied by loss of EPHB3 and CDH1 expression. Intriguingly, SNAIL1-induced phenotypic changes of CRC cells are significantly impaired by sustained EPHB3 expression both in vitro and in vivo. Altogether, our results identify EPHB3 as a novel target of SNAIL1 and suggest that disabling EPHB3 signaling is an important aspect to eliminate a roadblock at the onset of EMT processes.

Silencing of the EPHB3 tumor-suppressor gene in human colorectal cancer through decommissioning of a transcriptional enhancer.

The protein tyrosine kinase Ephrin type-B receptor 3 (EPHB3) counteracts tumor-cell dissemination by regulating intercellular adhesion and repulsion and acts as tumor/invasion suppressor in colorectal cancer. This protective mechanism frequently collapses at the adenoma-carcinoma transition due to EPHB3 transcriptional silencing. Here, we identify a transcriptional enhancer at the EPHB3 gene that integrates input from the intestinal stem-cell regulator achaete-scute family basic helix-loop-helix transcription factor 2 (ASCL2), Wnt/ß-catenin, MAP kinase, and Notch signaling. EPHB3 enhancer activity is highly variable in colorectal carcinoma cells and precisely reflects EPHB3 expression states, suggesting that enhancer dysfunction underlies EPHB3 silencing. Interestingly, low Notch activity parallels reduced EPHB3 expression in colorectal carcinoma cell lines and poorly differentiated tumor-tissue specimens. Restoring Notch activity reestablished enhancer function and EPHB3 expression. Although essential for intestinal stem-cell maintenance and adenoma formation, Notch activity seems dispensable in colorectal carcinomas. Notch activation even promoted growth arrest and apoptosis of colorectal carcinoma cells, attenuated their self-renewal capacity in vitro, and blocked tumor growth in vivo. Higher levels of Notch activity also correlated with longer disease-free survival of colorectal cancer patients. In summary, our results uncover enhancer decommissioning as a mechanism for transcriptional silencing of the EPHB3 tumor suppressor and argue for an antitumorigenic function of Notch signaling in advanced colorectal cancer.

Acetylation of human TCF4 (TCF7L2) proteins attenuates inhibition by the HBP1 repressor and induces a conformational change in the TCF4::DNA complex.

The members of the TCF/LEF family of DNA-binding proteins are components of diverse gene regulatory networks. As nuclear effectors of Wnt/ß-catenin signaling they act as assembly platforms for multimeric transcription complexes that either repress or activate gene expression. Previously, it was shown that several aspects of TCF/LEF protein function are regulated by post-translational modification. The association of TCF/LEF family members with acetyltransferases and deacetylases prompted us to investigate whether vertebrate TCF/LEF proteins are subject to acetylation. Through co-expression with p300 and CBP and subsequent analyses using mass spectrometry and immunodetection with anti-acetyl-lysine antibodies we show that TCF4 can be acetylated at lysine K150 by CBP. K150 acetylation is restricted to TCF4E splice variants and requires the simultaneous presence of ß-catenin and the unique TCF4E C-terminus. To examine the functional consequences of K150 acetylation we substituted K150 with amino acids representing the non-acetylated and acetylated states. Reporter gene assays based on Wnt/ß-catenin-responsive promoter regions did not indicate a general role of K150 acetylation in transactivation by TCF4E. However, in the presence of CBP, non-acetylatable TCF4E with a K150R substitution was more susceptible to inhibition by the HBP-1 repressor protein compared to wild-type TCF4E. Acetylation of K150 using a bacterial expression system or amino acid substitutions at K150 alter the electrophoretic properties of TCF4E::DNA complexes. This result suggests that K150 acetylation leads to a conformational change that may also represent the mechanism whereby acetylated TCF4E acquires resistance against HBP1. In summary, TCF4 not only recruits acetyltransferases but is also a substrate for these enzymes. The fact that acetylation affects only a subset of TCF4 splice variants and is mediated preferentially by CBP suggests that the conditional acetylation of TCF4E is a novel regulatory mechanism that diversifies the transcriptional output of Wnt/ß-catenin signaling in response to changing intracellular signaling milieus.