Advancing in Schaaf-Yang syndrome pathophysiology: from bedside to subcellular analyses of truncated MAGEL2

Background Schaaf-Yang syndrome (SYS) is caused by truncating mutations in MAGEL2, mapping to the Prader-Willi region (15q11-q13), with an observed phenotype partially overlapping that of Prader-Willi syndrome. MAGEL2 plays a role in retrograde transport and protein recycling regulation. Our aim is to contribute to the characterisation of SYS pathophysiology at clinical, genetic and molecular levels. Methods We performed an extensive phenotypic and mutational revision of previously reported patients with SYS. We analysed the secretion levels of amyloid-β 1-40 peptide (Aβ1-40) and performed targeted metabolomic and transcriptomic profiles in fibroblasts of patients with SYS (n=7) compared with controls (n=11). We also transfected cell lines with vectors encoding wild- type (WT) or mutated MAGEL2 to assess stability and subcellular localisation of the truncated protein. Results Functional studies show significantly decreased levels of secreted Aβ1-40 and intracellular glutamine in SYS fibroblasts compared with WT. We also identified 132 differentially expressed genes, including non-coding RNAs (ncRNAs) such as HOTAIR, and many of them related to developmental processes and mitotic mechanisms. The truncated form of MAGEL2 displayed a stability similar to the WT but it was significantly switched to the nucleus, compared with a mainly cytoplasmic distribution of the WT MAGEL2. Based on the updated knowledge, we offer guidelines for the clinical management of patients with SYS. Conclusion A truncated MAGEL2 protein is stable and localises mainly in the nucleus, where it might exert a pathogenic neomorphic effect. Aβ1-40 secretion levels and HOTAIR mRNA levels might be promising biomarkers for SYS. Our findings may improve SYS understanding and clinical management

Fibroblastic SNAIL1 induces a metastasis-permisive tumor stroma by regulating fibronectin alternative splicing

[eng] SNAIL1 is a transcription factor with roles in repression of epithelial genes and enhancement of mesenchymal genes. As such, it plays a key role in Epithelial-to-mesenchymal transition and in fibroblast activation into myofibroblast. In this thesis, we characterize a novel role in splicing regulation, focusing on the induction of inclusion of the fibronectin extra domain A. This alternatively spliced domain, expressed in pathological situations such as wound healing and cancer, has various roles in inflammatory response, fibroblast activation and tumor cell migration. We show a correlation between SNAIL1 and EDA inclusion among a breast cancer PDX cohort and samples for 5 solid tumor types in advanced stages. Here, we describe a TGF/SNAIL1 dependent recruitment of the splicing factor SRSF1 to the EDA coding RNA, associated to increased inclusion. We focus on the effects of this increase in EDA inclusion in the context of fibronectin fibers assembled into the extracellular matrix. We use genetically modified fibroblasts, expressing specific isoforms of fibronectin, to derive matrices and demonstrate that, like fibroblastic SNAIL1 expression, the presence of fibronectin EDA is required to organize an aligned and stiffer extracellular matrix. The induced mechanical and topological ECM properties enhance tumor cell oriented individual migration, facilitate coordinated collective movement and induce a more efficient invasion. Depletion of the isoform completely prevents metastasis formation in an orthotopic cancer model. We use specific inhibitors to block EDA signaling reducing fibroblastic activation and leading to a restrictive matrix that limits tumor cell invasion. Therefore, our results demonstrate the role of the fibronectin EDA isoform in the conversion of a restrictive tumor stroma to a permissive one and its molecular control by the transcription factor SNAIL1.[cat] SNAIL1 és un factor de transcripció que regula la repressió de gens epitelials i l’activació de gens mesenquimals. En base a aquests rols, SNAIL1 és clau en la transició Epiteli-mesènquima i en l’activació de fibroblasts. En aquesta tesi, caracteritzem una nova funció com a regulador del “splicing”, centrant-nos especialment en la capacitat de promoure la inclusió del domini extra A (EDA) de la fibronectina. Aquest domini s’expressa en situacions patològiques com la cicatrització de ferides o el càncer i s’ha associat a la resposta inflamatòria, l’activació de fibroblasts i la migració de cèl·lules tumorals. Presentem l’existència d’una correlació entre els nivells de SNAIL1 i la inclusió d’EDA en una cohort de PDXs derivats de càncer de mama i en mostres de pacients de 5 tipus de tumors sòlids en estadis avançats. En aquest treball descrivim el reclutament del factor de “splicing” SRSF1 al RNA codificant per EDA, associat a un augment de la inclusió, de manera dependent de TGF i SNAIL1. El nostre estudi se centra en els efectes associats a aquest augment de la inclusió d’EDA en el context de fibres de fibronectina polimeritzades a la matriu extracel·lular. Usem fibroblasts modificats genèticament, induint l’expressió d’una única isoforma de fibronectina, per generar matrius extracel·lulars i demostrem que la presència de fibronectina EDA es necessària per a l’increment de l’alineament i la rigidesa de les matrius. Les propietats mecàniques i topològiques induïdes per la fibronectina EDA en la matriu extracel·lular augmenten la migració orientada de cèl·lules tumorals individuals, faciliten el moviment coordinat col·lectiu i indueixen una invasió més eficient. L’exclusió d’aquest domini en un model ortotòpic de càncer de mama porta a la completa eliminació de l’aparició de metàstasis. L’ús d’inhibidors específics per tal de bloquejar la senyalització d’EDA redueix l’activació de fibroblasts i indueix una matriu restrictiva que disminueix la invasió de les cèl·lules tumorals. Per tant, els nostres resultats demostren que el factor de transcripció SNAIL1 promou la inclusió de EDA en l’ARNm de fibronectina i el paper d’aquesta isoforma en la conversió de l’estroma tumoral de restrictiu a permissiu

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

This study was funded by grants awarded to AGH and JB by the Agencia Estatal de Investigación (AEI) del Ministerio de Ciencia e Innovación MCIN (PID2019-104698RB-I00 and PID2022-136968OB-I00).We thank Dr. Juan Valcárcel and Dr. Elena Martin (Centre de Regulació Genètica, PRBB, Barcelona, Spain) for their advice and help with the SANJUAN splicing analyses, Prof. Dr. Daniel Navajas (Unit of Biophysics and Bioengineering, UB, Spain) for his advice on micromechanical measurements and Drs. Antoni Celià, José Yelamos, and Joaquín Arribas (IMIM, Barcelona, Spain) for providing cell lines and PDXs. We also thank Jordi Vergés and Meritxell Torrent (IMIM, Barcelona, Spain) and Carmen Escudero (IMIM, Barcelona, Spain; currently at Vall d'Hebron Institut d'Oncologia, Barcelona, Spain) for their technical support.Background: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. Methods: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. Results: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. Conclusions: Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment

A de novo FOXP1 truncating mutation in a patient originally diagnosed as C syndrome

De novo FOXP1 mutations have been associated with intellectual disability (ID), motor delay, autistic features and a wide spectrum of speech difficulties. C syndrome (Opitz C trigonocephaly syndrome) is a rare and genetically heterogeneous condition, characterized by trigonocephaly, craniofacial anomalies and ID. Several different chromosome deletions and and point mutations in distinct genes have been associated with the disease in patients originally diagnosed as Opitz C. By whole exome sequencing we identified a de novo splicing mutation in FOXP1 in a patient, initially diagnosed as C syndrome, who suffers from syndromic intellectual disability with trigonocephaly. The mutation (c.1428 + 1 G > A) promotes the skipping of exon 16, a frameshift and a premature STOP codon (p.Ala450GLyfs*13), as assessed by a minigene strategy. The patient reported here shares speech difficulties, intellectual disability and autistic features with other FOXP1 syndrome patients, and thus the diagnosis for this patient should be changed. Finally, since trigonocephaly has not been previously reported in FOXP1 syndrome, it remains to be proved whether it may be associated with the FOXP1 mutation.The authors thank the patient and his family for wholehearted collaboration. They are also grateful to M. Cozar for technical assistance, and to CNAG for exome sequencing within the “300 exomes to elucidate rare diseases” program. Funding was from Associació Síndrome Opitz C, Terrassa, Spain; Spanish Ministerio de Economía y Competitividad (SAF2014-56562-R; SAF2016-75948-R, FECYT, crowdfunding PRECIPITA); ISCIII Ministerio de Economía y Competitividad (PT13/0001/0044), Catalan Government (2014SGR932) and from CIBERER (U720)

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

Abstract Background As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. Methods We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. Results In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix  that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. Conclusions Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment

A de novo FOXP1 truncating mutation in a patient originally diagnosed as C Syndrome

De novo FOXP1 mutations have been associated with intellectual disability (ID), motor delay, autistic features and a wide spectrum of speech difficulties. C syndrome (Opitz C trigonocephaly syndrome) is a rare and genetically heterogeneous condition, characterized by trigonocephaly, craniofacial anomalies and ID. Several different chromosome deletions and and point mutations in distinct genes have been associated with the disease in patients originally diagnosed as Opitz C. By whole exome sequencing we identified a de novo splicing mutation in FOXP1 in a patient, initially diagnosed as C syndrome, who suffers from syndromic intellectual disability with trigonocephaly. The mutation (c.1428 + 1 G > A) promotes the skipping of exon 16, a frameshift and a premature STOP codon (p.Ala450GLyfs*13), as assessed by a minigene strategy. The patient reported here shares speech difficulties, intellectual disability and autistic features with other FOXP1 syndrome patients, and thus the diagnosis for this patient should be changed. Finally, since trigonocephaly has not been previously reported in FOXP1 syndrome, it remains to be proved whether it may be associated with the FOXP1 mutation

Formation of an invasion-permissive matrix requires TGFβ/SNAIL1-regulated alternative splicing of fibronectin

Background: As in most solid cancers, the emergence of cells with oncogenic mutations in the mammary epithelium alters the tissue homeostasis. Some soluble factors, such as TGFβ, potently modify the behavior of healthy stromal cells. A subpopulation of cancer-associated fibroblasts expressing a TGFβ target, the SNAIL1 transcription factor, display myofibroblastic abilities that rearrange the stromal architecture. Breast tumors with the presence of SNAIL1 in the stromal compartment, and with aligned extracellular fiber, are associated with poor survival prognoses. Methods: We used deep RNA sequencing and biochemical techniques to study alternative splicing and human tumor databases to test for associations (correlation t-test) between SNAIL1 and fibronectin isoforms. Three-dimensional extracellular matrices generated from fibroblasts were used to study the mechanical properties and actions of the extracellular matrices on tumor cell and fibroblast behaviors. A metastatic mouse model of breast cancer was used to test the action of fibronectin isoforms on lung metastasis. Results: In silico studies showed that SNAIL1 correlates with the expression of the extra domain A (EDA)-containing (EDA+) fibronectin in advanced human breast cancer and other types of epithelial cancers. In TGFβ-activated fibroblasts, alternative splicing of fibronectin as well as of 500 other genes was modified by eliminating SNAIL1. Biochemical analyses demonstrated that SNAIL1 favors the inclusion of the EDA exon by modulating the activity of the SRSF1 splicing factor. Similar to Snai1 knockout fibroblasts, EDA- fibronectin fibroblasts produce an extracellular matrix that does not sustain TGFβ-induced fiber organization, rigidity, fibroblast activation, or tumor cell invasion. The presence of EDA+ fibronectin changes the action of metalloproteinases on fibronectin fibers. Critically, in an mouse orthotopic breast cancer model, the absence of the fibronectin EDA domain completely prevents lung metastasis. Conclusions: Our results support the requirement of EDA+ fibronectin in the generation of a metastasis permissive stromal architecture in breast cancers and its molecular control by SNAIL1. From a pharmacological point of view, specifically blocking EDA+ fibronectin deposition could be included in studies to reduce the formation of a pro-metastatic environment.This study was funded by grants awarded to AGH and JB by the Agencia Estatal de Investigación (AEI) del Ministerio de Ciencia e Innovación MCIN (Proyecto PID2019-104698RB-I00 financiado por MCIN/ AEI/10.13039/501100011033, y pProyecto PID2022-136968OB-I00 financiado por MCIN/AEI /10.13039/501100011033/ y por FEDER Una manera de hacer Europa)

A de novo nonsense mutation in MAGEL2 in a patient initially diagnosed as Opitz-C: similarities between Schaaf-Yang and Opitz-C syndromes

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes.Funding was from Associació Síndrome Opitz C, Terrassa, Spain; Spanish Ministerio de Economía y Competitividad (SAF2014-56562-R; FECYT, crowdfunding PRECIPITA); Catalan Government (2014SGR932) and from CIBERER (U720). We acknowledge support of the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017'

A de novo nonsense mutation in MAGEL2 in a patient initially diagnosed as Opitz-C: Similarities between Schaaf-Yang and Opitz-C syndromes

Opitz trigonocephaly C syndrome (OTCS) is a rare genetic disorder characterized by craniofacial anomalies, variable intellectual and psychomotor disability, and variable cardiac defects with a high mortality rate. Different patterns of inheritance and genetic heterogeneity are known in this syndrome. Whole exome and genome sequencing of a 19-year-old girl (P7), initially diagnosed with OTCS, revealed a de novo nonsense mutation, p.Q638*, in the MAGEL2 gene. MAGEL2 is an imprinted, maternally silenced, gene located at 15q11-13, within the Prader-Willi region. Patient P7 carried the mutation in the paternal chromosome. Recently, mutations in MAGEL2 have been described in Schaaf-Yang syndrome (SHFYNG) and in severe arthrogryposis. Patient P7 bears resemblances with SHFYNG cases but has other findings not described in this syndrome and common in OTCS. We sequenced MAGEL2 in nine additional OTCS patients and no mutations were found. This study provides the first clear molecular genetic basis for an OTCS case, indicates that there is overlap between OTCS and SHFYNG syndromes, and confirms that OTCS is genetically heterogeneous. Genes encoding MAGEL2 partners, either in the retrograde transport or in the ubiquitination-deubiquitination complexes, are promising candidates as OTCS disease-causing genes