RESUMO
Pathogenic variants in RAC3 cause a neurodevelopmental disorder with brain malformations and craniofacial dysmorphism, called NEDBAF. This gene encodes a small GTPase, which plays a critical role in neurogenesis and neuronal migration. We report a 31 weeks of gestation fetus with triventricular dilatation, and temporal and perisylvian polymicrogyria, without cerebellar, brainstem, or callosal anomalies. Trio whole exome sequencing identified a RAC3 (NM_005052.3, GRCh38) probably pathogenic de novo variant c.276 T>A p.(Asn92Lys). Eighteen patients harboring 13 different and essentially de novo missense RAC3 variants were previously reported. All the patients presented with corpus callosum malformations. Gyration disorders, ventriculomegaly (VM), and brainstem and cerebellar malformations have frequently been described. The only previous prenatal case associated with RAC3 variant presented with complex brain malformations, mainly consisting of midline and posterior fossa anomalies. We report the second prenatal case of NEDBAF presenting an undescribed pattern of cerebral anomalies, including VM and polymicrogyria, without callosal, cerebellar, or brainstem malformations. All neuroimaging data were reviewed to clarify the spectrum of cerebral malformations.
Assuntos
Hidrocefalia , Malformações do Sistema Nervoso , Polimicrogiria , Gravidez , Feminino , Humanos , Diagnóstico Pré-Natal , Agenesia do Corpo Caloso , Mutação de Sentido Incorreto , Proteínas rac de Ligação ao GTP/genéticaRESUMO
The formation of new myofibers in vertebrates occurs by myoblast fusion and requires fusogenic activity of the muscle-specific membrane protein myomaker. Here, using in silico (BLAST) genome analyses, we show that the myomaker gene from trout includes 14 minisatellites, indicating that it has an unusual structure compared with those of other animal species. We found that the trout myomaker gene encodes a 434-amino acid (aa) protein, in accordance with its apparent molecular mass (â¼40 kDa) observed by immunoblotting. The first half of the trout myomaker protein (1-220 aa) is similar to the 221-aa mouse myomaker protein, whereas the second half (222-234 aa) does not correspond to any known motifs and arises from two protein extensions. The first extension (â¼70 aa) apparently appeared with the radiation of the bony fish clade Euteleostei, whereas the second extension (up to 236 aa) is restricted to the superorder Protacanthopterygii (containing salmonids and pike) and corresponds to the insertion of minisatellites having a length of 30 nucleotides. According to gene expression analyses, trout myomaker expression is consistently associated with the formation of new myofibers during embryonic development, postlarval growth, and muscle regeneration. Using cell-mixing experiments, we observed that trout myomaker has retained the ability to drive the fusion of mouse fibroblasts with C2C12 myoblasts. Our work reveals that trout myomaker has fusogenic function despite containing two protein extensions.
Assuntos
Proteínas de Peixes , Regulação da Expressão Gênica/fisiologia , Proteínas de Membrana , Repetições Minissatélites , Proteínas Musculares , Oncorhynchus mykiss , Animais , Proteínas de Peixes/genética , Proteínas de Peixes/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Miofibrilas/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/metabolismoRESUMO
Bone morphogenetic protein 15 (BMP15) and growth and differentiation factor 9 (GDF9) are TGFbeta-like oocyte-derived growth factors involved in ovarian folliculogenesis as critical regulators of many granulosa cell processes and ovulation rate. Ovarian phenotypic effect caused by alterations in BMP15 and GDF9 genes appears to differ between species and may be relevant to their mono- or polyovulating status. Through phylogenetic analysis we recently showed that these two paralogous genes are strongly divergent and in rapid evolution as compared to other members of the TGFbeta superfamily. Here, we evaluate the amino acid substitution rates of a set of proteins implicated in the ovarian function, including BMP15 and GDF9, with special attention to the mono- or polyovulating status of the species. Among a panel of mono- and polyovulating mammals, we demonstrate a better conservation of some areas in BMP15 and GDF9 within mono-ovulating species. Homology modeling of BMP15 and GDF9 homodimer and heterodimer 3-D structures was suggestive that these areas may be involved in dimer formation and stability. A phylogenetic study of BMP15/GDF9-related proteins reveals that these two genes diverged from the same ancestral gene along with BMP3 and GDF10, two other paralogous genes. A substitution rate analysis based on this phylogenetic tree leads to the hypothesis of an acquisition of BMP15/GDF9-specific functions in ovarian folliculogenesis in mammals. We propose that high variations observed in specific areas of BMP15 and GDF9 in polyovulating species change the equilibrium between homodimers and heterodimers, modifying the biological activity and thus allowing polyovulation to occur.
Assuntos
Evolução Biológica , Proteína Morfogenética Óssea 15/metabolismo , Fator 9 de Diferenciação de Crescimento/metabolismo , Ovulação/fisiologia , Sequência de Aminoácidos , Animais , Proteína Morfogenética Óssea 15/genética , Feminino , Regulação da Expressão Gênica , Variação Genética , Fator 9 de Diferenciação de Crescimento/genética , Filogenia , Especificidade da EspécieRESUMO
Thyroid cancer is the most common endocrine malignancy and several genetic events have been described to promote the development of thyroid carcinogenesis. Besides the effects of specific mutations on thyroid cancer development, the molecular mechanisms controlling tumorigenesis, tumor behavior, and drug resistance are still largely unknown. Cancer organoids have been proposed as a powerful tool to study aspects related to tumor development and progression and appear promising to test individual responses to therapies. Here, using mESC-derived thyroid organoids, we developed a BrafV637E-inducible model able to recapitulate the features of papillary thyroid cancer in vitro. Overexpression of the murine BrafV637E mutation, equivalent to BrafV600E in humans, rapidly triggers to MAPK activation, cell dedifferentiation, and disruption of follicular organization. BrafV637E-expressing organoids show a transcriptomic signature for p53, focal adhesion, ECM-receptor interactions, EMT, and inflammatory signaling pathways. Finally, PTC-like thyroid organoids were used for drug screening assays. The combination of MAPK and PI3K inhibitors reversed BrafV637E oncogene-promoted cell dedifferentiation while restoring thyroid follicle organization and function in vitro. Our results demonstrate that pluripotent stem cells-derived thyroid cancer organoids can mimic tumor development and features while providing an efficient tool for testing novel targeted therapies.
Assuntos
Proteínas Proto-Oncogênicas B-raf , Neoplasias da Glândula Tireoide , Animais , Camundongos , Carcinogênese , Mutação , Organoides/patologia , Fosfatidilinositol 3-Quinases/genética , Câncer Papilífero da Tireoide/patologia , Neoplasias da Glândula Tireoide/tratamento farmacológico , Neoplasias da Glândula Tireoide/genética , Neoplasias da Glândula Tireoide/patologiaRESUMO
DNA methylation classifiers ("episignatures") help to determine the pathogenicity of variants of uncertain significance (VUS). However, their sensitivity is limited due to their training on unambiguous cases with strong-effect variants so that the classification of variants with reduced effect size or in mosaic state may fail. Moreover, episignature evaluation of mosaics as a function of their degree of mosaicism has not been developed so far. We improved episignatures with respect to three categories. Applying (i) minimum-redundancy-maximum-relevance feature selection we reduced their length by up to one order of magnitude without loss of accuracy. Performing (ii) repeated re-training of a support vector machine classifier by step-wise inclusion of cases in the training set that reached probability scores larger than 0.5, we increased the sensitivity of the episignature-classifiers by 30%. In the newly diagnosed patients we confirmed the association between DNA methylation aberration and age at onset of KMT2B-deficient dystonia. Moreover, we found evidence for allelic series, including KMT2B-variants with moderate effects and comparatively mild phenotypes such as late-onset focal dystonia. Retrained classifiers also can detect mosaics that previously remained below the 0.5-threshold, as we showed for KMT2D-associated Kabuki syndrome. Conversely, episignature-classifiers are able to revoke erroneous exome calls of mosaicism, as we demonstrated by (iii) comparing presumed mosaic cases with a distribution of artificial in silico-mosaics that represented all the possible variation in degree of mosaicism, variant read sampling and methylation analysis.
Assuntos
Anormalidades Múltiplas , Metilação de DNA , Humanos , Fenótipo , Anormalidades Múltiplas/genética , Alelos , MosaicismoRESUMO
BACKGROUND: Myostatin, a member of the TGFß superfamily, is well known as a potent and specific negative regulator of muscle growth. Targeting the myostatin signalling pathway may offer promising therapeutic strategies for the treatment of muscle-wasting disorders. In the last decade, various myostatin-binding proteins have been identified to be able to inhibit myostatin activity. One of these is GASP1 (Growth and Differentiation Factor-Associated Serum Protein-1), a protein containing a follistatin domain as well as multiple domains associated with protease inhibitors. Despite in vitro data, remarkably little is known about in vivo functions of Gasp1. To further address the role of GASP1 during mouse development and in adulthood, we generated a gain-of-function transgenic mouse model that overexpresses Gasp1 under transcriptional control of the human cytomegalovirus immediate-early promoter/enhancer. RESULTS: Overexpression of Gasp1 led to an increase in muscle mass observed not before day 15 of postnatal life. The surGasp1 transgenic mice did not display any other gross abnormality. Histological and morphometric analysis of surGasp1 rectus femoris muscles revealed an increase in myofiber size without a corresponding increase in myofiber number. Fiber-type distribution was unaltered. Interestingly, we do not detect a change in total fat mass and lean mass. These results differ from those for myostatin knockout mice, transgenic mice overexpressing the myostatin propeptide or follistatin which exhibit both muscle hypertrophy and hyperplasia, and show minimal fat deposition. CONCLUSIONS: Altogether, our data give new insight into the in vivo functions of Gasp1. As an extracellular regulatory factor in the myostatin signalling pathway, additional studies on GASP1 and its homolog GASP2 are required to elucidate the crosstalk between the different intrinsic inhibitors of the myostatin.
Assuntos
Proteínas de Transporte/genética , Fibras Musculares Esqueléticas/fisiologia , Hipertonia Muscular/genética , Miostatina/metabolismo , Músculo Quadríceps/fisiologia , Animais , Antígenos Virais/genética , Proteínas de Transporte/biossíntese , Citomegalovirus/genética , Folistatina/genética , Folistatina/metabolismo , Regulação da Expressão Gênica , Proteínas Imediatamente Precoces/genética , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Hipertonia Muscular/metabolismo , Miostatina/genética , Fenótipo , Regiões Promotoras Genéticas , RNA Mensageiro/biossíntese , Transcrição GênicaRESUMO
BACKGROUND/AIMS: Growth and differentiation factor-associated serum protein-1 (GASP-1) is a secreted protein known to be capable of binding and inhibiting the activity of several TGF-beta family members, including myostatin. The present study was designed to characterize murine GASP-1 post-translational modifications and to determine their influence on the biological activity of GASP-1. METHODS: We describe herein the site-directed mutagenesis of single N-glycosylation sites and combinations of them in 4 mutants of murine GASP-1. RESULTS: In vitro and in vivo analysis revealed that GASP-1 is a glycoprotein containing 2 N-glycans and several mucin-type O-glycans. Treatments by the recombinant murine GASP-1 protein enhance C2C12 proliferation and differentiation by inhibition of the myostatin pathway. The loss of N-glycans leads to a decrease in protein secretion rate but does not affect its ability to activate myogenesis. CONCLUSION: Analysis of structure-function relationships of murine GASP-1 provides insights into the involvement of the carbohydrate moiety of mGASP-1 on its biological activity.
Assuntos
Proteínas de Transporte/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Células COS , Proteínas de Transporte/genética , Diferenciação Celular , Linhagem Celular , Proliferação de Células , Chlorocebus aethiops , Glicosilação , Peptídeos e Proteínas de Sinalização Intracelular , Espectrometria de Massas , Camundongos , Mutagênese Sítio-Dirigida , Mioblastos/citologia , Mioblastos/metabolismo , Peptídeos/análise , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , TransfecçãoRESUMO
PTH resistance is characterized by elevated parathyroid hormone (PTH) levels, hypocalcemia, hyperphosphatemia and it is classically associated with GNAS locus genetic or epigenetic defects. Inactivating PTH/PTHrP signaling disorders (iPPSD) define overlapping phenotypes based on their molecular etiology. iPPSD1 is associated with PTH1R variants and variable phenotypes including ossification anomalies and primary failure of tooth eruption but no endocrine disorder. Here we report on a 10-month-old child born from consanguineous parents, who presented with mild neurodevelopmental delay, seizures, enlarged fontanelles, round face, and bilateral clinodactyly. Hand x-rays showed diffuse delayed bone age, osteopenia, short metacarpal bones and cone-shaped distal phalanges. A diagnosis of PTH resistance was made on the basis of severe hypocalcemia, hyperphosphatemia, elevated PTH and normal vitamin D levels on blood sample. The patient was treated with calcium carbonate and alfacalcidol leading to rapid bio-clinical improvement. Follow-up revealed multiple agenesis of primary teeth and delayed teeth eruption, as well as Arnold-Chiari type 1 malformation requiring a ventriculoperitoneal shunt placement. GNAS gene analysis showed no pathogenic variation, but a likely pathogenic homozygous substitution c.723C>G p.(Asp241Glu) in PTH1R gene was found by trio-based whole exome sequencing. We studied the deleterious impact of the variant on the protein conformation with bioinformatics tools. In conclusion, our study reports for the first time PTH resistance in a child with a biallelic PTH1R mutation, extending thereby the clinical spectrum of iPPSD1 phenotypes.
Assuntos
Hiperfosfatemia , Hipocalcemia , Pseudo-Hipoparatireoidismo , Humanos , Hipocalcemia/complicações , Hormônio Paratireóideo/metabolismo , Proteína Relacionada ao Hormônio Paratireóideo , Pseudo-Hipoparatireoidismo/diagnóstico , Pseudo-Hipoparatireoidismo/genéticaRESUMO
The thyroid gland captures iodide in order to synthesize hormones that act on almost all tissues and are essential for normal growth and metabolism. Low plasma levels of thyroid hormones lead to hypothyroidism, which is one of the most common disorder in humans and is not always satisfactorily treated by lifelong hormone replacement. Therefore, in addition to the lack of in vitro tractable models to study human thyroid development, differentiation and maturation, functional human thyroid organoids could pave the way to explore new therapeutic approaches. Here we report the generation of transplantable thyroid organoids derived from human embryonic stem cells capable of restoring plasma thyroid hormone in athyreotic mice as a proof of concept for future therapeutic development.
Assuntos
Hipotireoidismo , Organoides , Humanos , Animais , Camundongos , Hipotireoidismo/terapia , Células-Tronco Embrionárias , Hormônios TireóideosRESUMO
The understanding of muscle tissue formation and regeneration is essential for the development of therapeutic approaches to treat muscle diseases or loss of muscle mass and strength during ageing or cancer. One of the critical steps in muscle formation is the fusion of muscle cells to form or regenerate muscle fibres. To identify new genes controlling myoblast fusion, we performed a siRNA screen in c2c12 myoblasts. The genes identified during this screen were then studied in vivo by knockdown in zebrafish using morpholino. We found that N-alpha-acetyltransferase 15 (Naa15) knockdown enhanced c2c12 myoblast fusion, suggesting that Naa15 negatively regulates myogenic cell fusion. We identified two Naa15 orthologous genes in the zebrafish genome: Naa15a and Naa15b. These two orthologs were expressed in the myogenic domain of the somite. Knockdown of zebrafish Naa15a and Naa15b genes induced a "U"-shaped segmentation of the myotome and alteration of myotome boundaries, resulting in the formation of abnormally long myofibres spanning adjacent somites. Taken together, these results show that Naa15 regulates myotome formation and myogenesis in fish.
Assuntos
Desenvolvimento Muscular/fisiologia , Mioblastos/metabolismo , Acetiltransferase N-Terminal A/metabolismo , Acetiltransferase N-Terminal E/metabolismo , Proteínas de Peixe-Zebra/metabolismo , Peixe-Zebra/embriologia , Animais , Fusão Celular , Técnicas de Inativação de Genes , Camundongos , Mioblastos/citologia , Acetiltransferase N-Terminal A/genética , Acetiltransferase N-Terminal E/genética , Proteínas de Peixe-Zebra/genéticaRESUMO
The WFIKKN (WAP, Follistatin/kazal, Immunoglobulin, Kunitz and Netrin domain-containing) protein family is composed of two multidomain proteins: WFIKKN1 and WFIKKN2. They were formed by domain shuffling and are likely present in deuterostoms. The WFIKKN (also called GASP) proteins are well known for their function in muscle and skeletal tissues, namely, inhibition of certain members of the transforming growth factor beta (TGFB) superfamily such as myostatin (MSTN) and growth and differentiation factor 11 (GDF11). However, the role of the WFIKKN proteins in other tissues is still poorly understood in spite of evidence suggesting possible action in the inner ear, brain and reproduction. Further, several recent studies based on next generation technologies revealed differential expression of WFIKKN1 and WFIKKN2 in various tissues suggesting that their function is not limited to MSTN and GDF11 inhibition in musculoskeletal tissue. In this review, we summarize current knowledge about the WFIKKN proteins and propose that they are "companion" proteins for various growth factors by providing localized and sustained presentation of TGFB proteins to their respective receptors, thus regulating the balance between the activation of Smad and non-Smad pathways by TGFB.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas de Transporte/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Domínios Proteicos , Proteínas/genética , Fator de Crescimento Transformador beta/antagonistas & inibidoresRESUMO
Antibody affinity maturation relies on activation-induced cytidine deaminase (AID)-dependent somatic hypermutation (SHM) of immunoglobulin (Ig) loci. Class switch recombination (CSR) can in parallel occur between AID-targeted, transcribed, spliced and repetitive switch (S) regions. AID thus initiates not only mutations but also double-strand breaks (DSBs). What governs the choice between those two outcomes remains uncertain. Here we explore whether insertion of transcribed intronic S regions in a locus (Igκ) strongly recruiting AID is sufficient for efficient CSR. Although strongly targeted by AID and carrying internal deletions, the knocked-in S regions only undergo rare CSR-like events. This model confirms S regions as exquisite SHM targets, extending AID activity far from transcription initiation sites, and shows that such spliced and repetitive AID targets are not sufficient by themselves for CSR. Beyond transcription and AID recruitment, additional IgH elements are thus needed for CSR, restricting this hazardous gene remodelling to IgH loci.
Assuntos
Afinidade de Anticorpos/fisiologia , Linfócitos B/fisiologia , Citidina Desaminase/metabolismo , Animais , Diversidade de Anticorpos , Citidina Desaminase/genética , Citometria de Fluxo , Regulação Enzimológica da Expressão Gênica , Técnicas de Introdução de Genes , Switching de Imunoglobulina , Camundongos , Baço/citologiaRESUMO
Bone Morphogenetic Protein 15 (BMP15) is a TGFß-like oocyte-derived growth factor involved in ovarian folliculogenesis as a critical regulator of many granulosa cell processes. Alterations of the BMP15 gene have been found associated with different ovarian phenotypic effects depending on the species, from sterility to increased prolificacy in sheep, slight subfertility in mouse or associated with primary ovarian insufficiency (POI) in women. To investigate the evolving role of BMP15, a phylogenetic analysis of this particular TGFß family member was performed. A maximum likelihood phylogenetic tree of several TGFß/BMP family members expressed by the ovary showed that BMP15 has a very strong divergence and a rapid evolution compared to others. Moreover, among 24 mammalian species, we detected signals of positive selection in the hominidae clade corresponding to F146, L189 and Y235 residues in human BMP15. The biological importance of these residues was tested functionally after site directed-mutagenesis in a COV434 cells luciferase assay. By replacing the positively selected amino acid either by alanine or the most represented residue in other studied species, only L189A, Y235A and Y235C mutants showed a significant increase of BMP15 signaling when compared to wild type. Additionally, the Y235C mutant was more potent than wild type in inhibiting progesterone secretion of ovine granulosa cells in primary culture. Interestingly, the Y235C mutation was previously identified in association with POI in women. In conclusion, this study evidences that the BMP15 gene has evolved faster than other members of the TGFß family and was submitted to a positive selection pressure in the hominidae clade. Some residues under positive selection are of great importance for the normal function of the protein and thus for female fertility. Y235 represents a critical residue in the determination of BMP15 biological activity, thus indirectly confirming its role in the onset of POI in women.
Assuntos
Proteína Morfogenética Óssea 15/genética , Insuficiência Ovariana Primária/genética , Animais , Células Cultivadas , Feminino , Células da Granulosa/efeitos dos fármacos , Humanos , Camundongos , Mutação , Progesterona , RatosRESUMO
Growth and differentiation factor Associated Serum Protein (GASP) 1 and 2 are proteins known to be involved in the control of myostatin activity at least in vitro. Most deuterostome GASPs share a modular organization including WAP, follistatin/kazal, IGc2, two kunitz, and NTR domains. Based on an exon shuffling model, we performed independent phylogenetic analyses on these modules and assessed that papilin is probably a sister sequence to GASP with a divergence date estimated from the last common ancestor to bilateria. The final organization was acquired by the addition of the FS domain in early deuterostomes. Our study revealed that Gasp genes diverged during the first round of genome duplication in early vertebrates. By evaluating the substitution rate at different sites on the proteins, we showed a better conservation of the follistatin/kazal domain of GASP1 than GASP2 in mammals, suggesting a stronger interaction with myostatin. We also observed a progressive increase in the conservation of follistatin and kunitz domains from the ancestor of Ciona to early vertebrates. In situ hybridization performed on mouse embryos showed a weak Gasp1 expression in the formed somites at 10.5 dpc and in limb buds from embryonic E10.0 to E12.5. Similar results were obtained for zebrafish embryos. We propose a synthetic view showing possible interactions between GASP1 and myostatin and highlighting the role of the second kunitz domain in preventing myostatin proteolysis.