Drosophila melanogaster as a model to study WNT pathway alteration in Cornelia de Lange Syndrome

Introduction: Cornelia de Lange syndrome (CdLS) is a rare genetic disorder affecting neurodevelopment, gastrointestinal and musculoskeletal systems. CdLS is caused by mutations within NIPBL, SMC1A, SMC3, RAD21, or HDAC8 genes. These genes codify for the cohesin complex, a multiprotein structure playing a role in chromatid adhesion, DNA repair and gene expression regulation. It has been demonstrated that a strong correlation exists between cohesin complex function and WNT signalling. Recently, it has been observed that chemical activation of the WNT pathway in nipblb-loss-of-function zebrafish embryos and in NIPBL-mutated patient fibroblasts rescued the adverse phenotype. Both embryos and fibroblasts present similar patterns of canonical WNT pathway alterations and CCND1 downregulation. Materials and Methods: Drosophila melanogaster is an inexpensive model to study CdLS and to screen in vivo for therapeutic compounds. Therefore, we have selected fly strains mutated in nipped-B and hdac3 genes (respectively NIPBL and HDAC8 in humans) for assessing the existing correlation between cohesin complex and WNT pathway and to screen for chemicals that revert the CdLS associated-phenotypes efficiently. Results: We have confirmed that mutated flies weight 5% less than wild type. Moreover, we have tested lithium chloride (LiCl) as WNT activator, demonstrating that 250mM is the highest concentration tolerated. Conclusions: Hence, we hypothesize that WNT pathway activation could improve mutant phenotype. We will be testing different doses of LiCl and other WNT activator to assess whether some of those chemical compounds could revert the syndrome-associated phenotype

Lithium as a positive modulator of defective WNT pathway in Cornelia de Lange Syndrome models

The cohesin complex is a multimeric system, highly conserved in the course of cellular evolution from the most primitive life forms to human cells. Cohesins are essential Structural Maintenance of Chromosomes (SMC), protein-containing complexes that interact with chromatin and modulate chromatin organization and gene expression. Genetic variants that cause structural and/or functional alterations induce an array of congenital pathologies named "cohesinopathies". It is believed that such malformations arise from deregulation of pivotal developmental molecular pathways. Canonical WNT pathway has been shown to be perturbed in association with central nervous system malformation in Cornelia de Lange Syndrome (CdLS), one of the most characterized cohesinopathy. In this study, we validated the relevance of canonical WNT pathway and assess the effect of LiCl-dependent activation of WNT pathway in three CdLS experimental models: Lymphoblastoid cell lines from patients, murine Neural Stem Cells (NSCs) and Drosophila melanogaster. Methods Lymphoblastoid cells (immortalized lines from CdLS patients) of patients carrying mutations of NIPBL or HDAC8 genes and healthy donors were used in these studies. These cells were treated with LiCl 1mM, 2,5mM and 5mM, and vehicle and proliferation rate were measured. Proliferation and differentiation capabilities were also assessed in CdLS NSCs upon LiCl treatment. Flies were grown upon food added with a different concentration of LiCl. Drosophila brains were analyzed for morphological evaluation. Results and conclusions Preliminary data on lymphoblastoid cells showed no effects on cell death rate in healthy donor following LiCl treatment. And, although with a patient-specific response, LiCl appeared to induce an increase in proliferation, especially in cell lines that were slow-growing compared to controls. NSCs showed reduced NSCs proliferation rate and differentiating capabilities. The presence of lithium could reduce the detrimental effects in a significant way. Drosophila mutants for nipped-B gene, the ortholog of human NIPBL, display malformations in mushroom bodies (MB), a structure involved in olfactory learning and memory. Treating subsequent generation of flies with 100mM of LiCl, MB morphology was restored in the offspring. All these data further confirm the hypothesis that in \u201ccohesinophaties\u201d is present an impairment of WNT pathway that could, in part, explain the typical neurodevelopmental alterations of this syndrome. Moreover, these studies could pave the way for future therapeutic strategies

Developmental abnormalities and cancer predisposition in neurofibromatosis type 1

Neurofibromatosis type 1 (NF1) is a developmental and cancer predisposing syndrome resulting from haploinsufficiency or alteration in neurofibromin, a multifunctional protein that acts in various signaling pathways affecting morphogenetic processes and cell proliferation. Neurofibromin deficiency deregulates Ras/Raf/MEK/ERK and Ras/PI3K/AKT/PKB/mTOR signaling networks and intersected pathways including the cAMP-dependent protein kinase A (PKA) and the Rho-cofillin which acts on actin cytoskeleton reorganization, cell motility and adhesion. As the neurofibromin-mediated pathways are associated with biological effects depending on the cell lineage, deregulation induced by NF1 mutation clearly has cell type-specific effects. This review summarizes our increasing knowledge of NF1 as a disease rooted in defective developmental mechanisms that can also influence the potential for malignant growth. The cardinal features of NF1 patients, at birth and during life involve the cardiovascular, connective/skeletal and central nervous systems, as they reflect the NF1 mutation sensitivity of cell lineages committed to specifying these systems during embryonic development. A switch to neoplastic transformation may also occur in both the prenatal and postnatal life in cancer initiating cells of defined lineages, with the cooperation of a genetically and epigenetically modified tumor microenvironment. We emphasize how much of our current knowledge of the pathomechanisms of NF1 clinical signs and cancer has come from engineered mouse models and in vitro primary cells and cell lines exposed to inhibitors of signaling molecules. Advances in our knowledge of the developmental defects primed by the loss neurofibromin should reveal further associations between given NF1 mutations and tissue-specific symptoms, thus improving the clinical management of the patients

X-linked Cornelia de Lange syndrome owing to SMC1L1 mutations

Cornelia de Lange syndrome is a multisystem developmental disorder characterized by facial dysmorphisms, upper limb abnormalities, growth delay and cognitive retardation. Mutations in the NIPBL gene, a component of the cohesin complex, account for approximately half of the affected individuals. We report here that mutations in SMC1L1 (also known as SMC1), which encodes a different subunit of the cohesin complex, are responsible for CdLS in three male members of an affected family and in one sporadic case

SHOX duplications found in some cases with type I Mayer-Rokitansky-Kuster-Hauser syndrome

Purpose: The Mayer-Rokitansky-Kuster-Hauser syndrome is defined as congenital aplasia of mullerian ducts derived structures in females with a normal female chromosomal and gonadal sex. Most cases with Mayer-Rokitansky-Kuster-Hauser syndrome are sporadic, although familial cases have been reported. The genetic basis of Mayer-Rokitansky-Kuster-Hauser syndrome is largely unknown and seems heterogeneous, and a small number of cases were found to have mutations in the WNT4 gene. The aim of this study was to identify possible recurrent submicroscopic imbalances in a cohort of familial and sporadic cases with Mayer-Rokitansky-Kuster-Hauser syndrome. Methods: Multiplex ligation-dependent probe amplification was used to screen the subtelomeric sequences of all chromosomes in 30 patients with Mayer-Rokitansky-Kuster-Hauser syndrome (sporadic, n = 27 and familial, n = 3). Segregation analysis and pyrosequencing were applied to validate the MLPA results in the informative family. Results: Partial duplication of the Xpter pseudoautosomal region 1 containing the short stature homeobox (SHOX) gene was detected in five patients with Mayer-Rokitansky-Kuster-Hauser syndrome (familial, n = 3 and sporadic, n = 2) and not in 53 healthy controls. The duplications were not overlapping, and SHOX was never entirely duplicated. Haplotyping in the informative family revealed that SHOX gene duplication was inherited from the unaffected father and was absent in two healthy sisters. Conclusions: Partial duplication of SHOX gene is found in some cases with both familial and sporadic Mayer-Rokitansky-Kuster-Hauser type I syndrome