RESUMEN
INTRODUCTION: KCTD15 encodes an oligomeric BTB domain protein reported to inhibit neural crest formation through repression of Wnt/beta-catenin signalling, as well as transactivation by TFAP2. Heterozygous missense variants in the closely related paralogue KCTD1 cause scalp-ear-nipple syndrome. METHODS: Exome sequencing was performed on a two-generation family affected by a distinctive phenotype comprising a lipomatous frontonasal malformation, anosmia, cutis aplasia of the scalp and/or sparse hair, and congenital heart disease. Identification of a de novo missense substitution within KCTD15 led to targeted sequencing of DNA from a similarly affected sporadic patient, revealing a different missense mutation. Structural and biophysical analyses were performed to assess the effects of both amino acid substitutions on the KCTD15 protein. RESULTS: A heterozygous c.310G>C variant encoding p.(Asp104His) within the BTB domain of KCTD15 was identified in an affected father and daughter and segregated with the phenotype. In the sporadically affected patient, a de novo heterozygous c.263G>A variant encoding p.(Gly88Asp) was present in KCTD15. Both substitutions were found to perturb the pentameric assembly of the BTB domain. A crystal structure of the BTB domain variant p.(Gly88Asp) revealed a closed hexameric assembly, whereas biophysical analyses showed that the p.(Asp104His) substitution resulted in a monomeric BTB domain likely to be partially unfolded at physiological temperatures. CONCLUSION: BTB domain substitutions in KCTD1 and KCTD15 cause clinically overlapping phenotypes involving craniofacial abnormalities and cutis aplasia. The structural analyses demonstrate that missense substitutions act through a dominant negative mechanism by disrupting the higher order structure of the KCTD15 protein complex.
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Dominio BTB-POZ , Anomalías Craneofaciales , Cara , Humanos , Anomalías Múltiples , Proteínas Co-Represoras/genética , Anomalías Craneofaciales/genética , Displasia Ectodérmica , Cara/anomalías , Mutación Missense/genética , SíndromeRESUMEN
PURPOSE: Studies have previously implicated PRRX1 in craniofacial development, including demonstration of murine Prrx1 expression in the preosteogenic cells of the cranial sutures. We investigated the role of heterozygous missense and loss-of-function (LoF) variants in PRRX1 associated with craniosynostosis. METHODS: Trio-based genome, exome, or targeted sequencing were used to screen PRRX1 in patients with craniosynostosis; immunofluorescence analyses were used to assess nuclear localization of wild-type and mutant proteins. RESULTS: Genome sequencing identified 2 of 9 sporadically affected individuals with syndromic/multisuture craniosynostosis, who were heterozygous for rare/undescribed variants in PRRX1. Exome or targeted sequencing of PRRX1 revealed a further 9 of 1449 patients with craniosynostosis harboring deletions or rare heterozygous variants within the homeodomain. By collaboration, 7 additional individuals (4 families) were identified with putatively pathogenic PRRX1 variants. Immunofluorescence analyses showed that missense variants within the PRRX1 homeodomain cause abnormal nuclear localization. Of patients with variants considered likely pathogenic, bicoronal or other multisuture synostosis was present in 11 of 17 cases (65%). Pathogenic variants were inherited from unaffected relatives in many instances, yielding a 12.5% penetrance estimate for craniosynostosis. CONCLUSION: This work supports a key role for PRRX1 in cranial suture development and shows that haploinsufficiency of PRRX1 is a relatively frequent cause of craniosynostosis.
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Craneosinostosis , Proteínas de Homeodominio , Animales , Humanos , Ratones , Secuencia de Bases , Suturas Craneales/patología , Craneosinostosis/genética , Genes Homeobox , Proteínas de Homeodominio/genética , PenetranciaRESUMEN
INTRODUCTION: Replication of the nuclear genome is an essential step for cell division. Pathogenic variants in genes coding for highly conserved components of the DNA replication machinery cause Meier-Gorlin syndrome (MGORS). OBJECTIVE: Identification of novel genes associated with MGORS. METHODS: Exome sequencing was performed to investigate the genotype of an individual presenting with prenatal and postnatal growth restriction, a craniofacial gestalt of MGORS and coronal craniosynostosis. The analysis of the candidate variants employed bioinformatic tools, in silico structural protein analysis and modelling in budding yeast. RESULTS: A novel homozygous missense variant NM_016095.2:c.341G>T, p.(Arg114Leu), in GINS2 was identified. Both non-consanguineous healthy parents carried this variant. Bioinformatic analysis supports its classification as pathogenic. Functional analyses using yeast showed that this variant increases sensitivity to nicotinamide, a compound that interferes with DNA replication processes. The phylogenetically highly conserved residue p.Arg114 localises at the docking site of CDC45 and MCM5 at GINS2. Moreover, the missense change possibly disrupts the effective interaction between the GINS complex and CDC45, which is necessary for the CMG helicase complex (Cdc45/MCM2-7/GINS) to accurately operate. Interestingly, our patient's phenotype is strikingly similar to the phenotype of patients with CDC45-related MGORS, particularly those with craniosynostosis, mild short stature and patellar hypoplasia. CONCLUSION: GINS2 is a new disease-associated gene, expanding the genetic aetiology of MGORS.
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Proteínas Cromosómicas no Histona , Microtia Congénita , Craneosinostosis , Micrognatismo , Proteínas de Ciclo Celular/genética , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Microtia Congénita/genética , Craneosinostosis/genética , Trastornos del Crecimiento/genética , Humanos , Micrognatismo/genética , Rótula/anomalías , Saccharomyces cerevisiae/genéticaRESUMEN
Next-generation sequencing is a powerful tool for the discovery of genes related to neurodevelopmental disorders (NDDs). Here, we report the identification of a distinct syndrome due to de novo or inherited heterozygous mutations in Tousled-like kinase 2 (TLK2) in 38 unrelated individuals and two affected mothers, using whole-exome and whole-genome sequencing technologies, matchmaker databases, and international collaborations. Affected individuals had a consistent phenotype, characterized by mild-borderline neurodevelopmental delay (86%), behavioral disorders (68%), severe gastro-intestinal problems (63%), and facial dysmorphism including blepharophimosis (82%), telecanthus (74%), prominent nasal bridge (68%), broad nasal tip (66%), thin vermilion of the upper lip (62%), and upslanting palpebral fissures (55%). Analysis of cell lines from three affected individuals showed that mutations act through a loss-of-function mechanism in at least two case subjects. Genotype-phenotype analysis and comparison of computationally modeled faces showed that phenotypes of these and other individuals with loss-of-function variants significantly overlapped with phenotypes of individuals with other variant types (missense and C-terminal truncating). This suggests that haploinsufficiency of TLK2 is the most likely underlying disease mechanism, leading to a consistent neurodevelopmental phenotype. This work illustrates the power of international data sharing, by the identification of 40 individuals from 26 different centers in 7 different countries, allowing the identification, clinical delineation, and genotype-phenotype evaluation of a distinct NDD caused by mutations in TLK2.
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Estudios de Asociación Genética , Patrón de Herencia/genética , Mutación con Pérdida de Función/genética , Trastornos del Neurodesarrollo/genética , Proteínas Quinasas/genética , Adolescente , Adulto , Secuencia de Bases , Línea Celular , Niño , Preescolar , Facies , Femenino , Humanos , Lactante , Masculino , ARN Mensajero/genética , ARN Mensajero/metabolismo , Translocación Genética , Adulto JovenRESUMEN
SUMMARY: amplimap is a command-line tool to automate the processing and analysis of data from targeted next-generation sequencing experiments with PCR-based amplicons or capture-based enrichment systems. From raw sequencing reads, amplimap generates output such as read alignments, annotated variant calls, target coverage statistics and variant allele counts and frequencies for each target base pair. In addition to its focus on user-friendliness and reproducibility, amplimap supports advanced features such as consensus base calling for read families based on unique molecular identifiers and filtering false positive variant calls caused by amplification of off-target loci. AVAILABILITY AND IMPLEMENTATION: amplimap is available as a free Python package under the open-source Apache 2.0 License. Documentation, source code and installation instructions are available at https://github.com/koelling/amplimap.
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Secuenciación de Nucleótidos de Alto Rendimiento , Programas Informáticos , Alelos , Reacción en Cadena de la Polimerasa , Reproducibilidad de los ResultadosRESUMEN
Curry-Jones syndrome (CJS) is a multisystem disorder characterized by patchy skin lesions, polysyndactyly, diverse cerebral malformations, unicoronal craniosynostosis, iris colobomas, microphthalmia, and intestinal malrotation with myofibromas or hamartomas. Cerebellar medulloblastoma has been described in a single affected individual; in another, biopsy of skin lesions showed features of trichoblastoma. The combination of asymmetric clinical features, patchy skin manifestations, and neoplastic association previously led to the suggestion that this could be a mosaic condition, possibly involving hedgehog (Hh) signaling. Here, we show that CJS is caused by recurrent somatic mosaicism for a nonsynonymous variant in SMO (c.1234C>T [p.Leu412Phe]), encoding smoothened (SMO), a G-protein-coupled receptor that transduces Hh signaling. We identified eight mutation-positive individuals (two of whom had not been reported previously) with highly similar phenotypes and demonstrated varying amounts of the mutant allele in different tissues. We present detailed findings from brain MRI in three mutation-positive individuals. Somatic SMO mutations that result in constitutive activation have been described in several tumors, including medulloblastoma, ameloblastoma, and basal cell carcinoma. Strikingly, the most common of these mutations is the identical nonsynonymous variant encoding p.Leu412Phe. Furthermore, this substitution has been shown to activate SMO in the absence of Hh signaling, providing an explanation for tumor development in CJS. This raises therapeutic possibilities for using recently generated Hh-pathway inhibitors. In summary, our work uncovers the major genetic cause of CJS and illustrates strategies for gene discovery in the context of low-level tissue-specific somatic mosaicism.
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Anomalías Craneofaciales/etiología , Intestinos/anomalías , Mutación/genética , Anomalías Cutáneas/etiología , Receptor Smoothened/genética , Sindactilia/etiología , Preescolar , Anomalías Craneofaciales/patología , Femenino , Humanos , Lactante , Recién Nacido , Intestinos/patología , Masculino , Transducción de Señal , Anomalías Cutáneas/patología , Sindactilia/patologíaRESUMEN
DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.
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Proteínas de Ciclo Celular/genética , Microtia Congénita/genética , Craneosinostosis/genética , Trastornos del Crecimiento/genética , Micrognatismo/genética , Mutación , Rótula/anomalías , Adolescente , Adulto , Alelos , Empalme Alternativo/genética , Secuencia de Aminoácidos , Amnios/citología , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/deficiencia , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Células Cultivadas , Niño , Preescolar , Análisis Mutacional de ADN , Replicación del ADN , Exoma/genética , Exones/genética , Femenino , Estudios de Asociación Genética , Humanos , Masculino , Modelos Moleculares , Conformación Proteica , Síndrome , Adulto JovenRESUMEN
Human ZIC1 (zinc finger protein of cerebellum 1), one of five homologs of the Drosophila pair-rule gene odd-paired, encodes a transcription factor previously implicated in vertebrate brain development. Heterozygous deletions of ZIC1 and its nearby paralog ZIC4 on chromosome 3q25.1 are associated with Dandy-Walker malformation of the cerebellum, and loss of the orthologous Zic1 gene in the mouse causes cerebellar hypoplasia and vertebral defects. We describe individuals from five families with heterozygous mutations located in the final (third) exon of ZIC1 (encoding four nonsense and one missense change) who have a distinct phenotype in which severe craniosynostosis, specifically involving the coronal sutures, and variable learning disability are the most characteristic features. The location of the nonsense mutations predicts escape of mutant ZIC1 transcripts from nonsense-mediated decay, which was confirmed in a cell line from an affected individual. Both nonsense and missense mutations are associated with altered and/or enhanced expression of a target gene, engrailed-2, in a Xenopus embryo assay. Analysis of mouse embryos revealed a localized domain of Zic1 expression at embryonic days 11.5-12.5 in a region overlapping the supraorbital regulatory center, which patterns the coronal suture. We conclude that the human mutations uncover a previously unsuspected role for Zic1 in early cranial suture development, potentially by regulating engrailed 1, which was previously shown to be critical for positioning of the murine coronal suture. The diagnosis of a ZIC1 mutation has significant implications for prognosis and we recommend genetic testing when common causes of coronal synostosis have been excluded.
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Codón sin Sentido/genética , Craneosinostosis/genética , Regulación del Desarrollo de la Expresión Génica/genética , Discapacidades para el Aprendizaje/genética , Fenotipo , Factores de Transcripción/genética , Animales , Secuencia de Bases , Clonación Molecular , Femenino , Proteínas de Homeodominio/metabolismo , Humanos , Hibridación in Situ , Cariotipificación , Masculino , Ratones , Datos de Secuencia Molecular , Mutación Missense/genética , Proteínas del Tejido Nervioso/metabolismo , Linaje , Análisis de Secuencia de ADN , Xenopus laevisRESUMEN
BACKGROUND: Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in â¼1 in 2250 births, either in isolation or as part of a syndrome. Mutations in at least 57 genes have been associated with craniosynostosis, but only a minority of these are included in routine laboratory genetic testing. METHODS: We used exome or whole genome sequencing to seek a genetic cause in a cohort of 40 subjects with craniosynostosis, selected by clinical or molecular geneticists as being high-priority cases, and in whom prior clinically driven genetic testing had been negative. RESULTS: We identified likely associated mutations in 15 patients (37.5%), involving 14 different genes. All genes were mutated in single families, except for IL11RA (two families). We classified the other positive diagnoses as follows: commonly mutated craniosynostosis genes with atypical presentation (EFNB1, TWIST1); other core craniosynostosis genes (CDC45, MSX2, ZIC1); genes for which mutations are only rarely associated with craniosynostosis (FBN1, HUWE1, KRAS, STAT3); and known disease genes for which a causal relationship with craniosynostosis is currently unknown (AHDC1, NTRK2). In two further families, likely novel disease genes are currently undergoing functional validation. In 5 of the 15 positive cases, the (previously unanticipated) molecular diagnosis had immediate, actionable consequences for either genetic or medical management (mutations in EFNB1, FBN1, KRAS, NTRK2, STAT3). CONCLUSIONS: This substantial genetic heterogeneity, and the multiple actionable mutations identified, emphasises the benefits of exome/whole genome sequencing to identify causal mutations in craniosynostosis cases for which routine clinical testing has yielded negative results.
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Craneosinostosis/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Neoplasias/genética , Craneosinostosis/diagnóstico , Craneosinostosis/patología , Exoma/genética , Pruebas Genéticas , Humanos , Mutación , Valor Predictivo de las PruebasRESUMEN
Cilia are architecturally complex organelles that protrude from the cell membrane and have signalling, sensory and motility functions that are central to normal tissue development and homeostasis. There are two broad categories of cilia; motile and non-motile, or primary, cilia. The central role of primary cilia in health and disease has become prominent in the past decade with the recognition of a number of human syndromes that result from defects in the formation or function of primary cilia. This rapidly growing class of conditions, now known as ciliopathies, impact the development of a diverse range of tissues including the neural axis, craniofacial structures, skeleton, kidneys, eyes and lungs. The broad impact of cilia dysfunction on development reflects the pivotal position of the primary cilia within a signalling nexus involving a growing number of growth factor systems including Hedgehog, Pdgf, Fgf, Hippo, Notch and both canonical Wnt and planar cell polarity. We have identified a novel ENU mutant allele of Ift140, which causes a mid-gestation embryonic lethal phenotype in homozygous mutant mice. Mutant embryos exhibit a range of phenotypes including exencephaly and spina bifida, craniofacial dysmorphism, digit anomalies, cardiac anomalies and somite patterning defects. A number of these phenotypes can be attributed to alterations in Hedgehog signalling, although additional signalling systems are also likely to be involved. We also report the identification of a homozygous recessive mutation in IFT140 in a Jeune syndrome patient. This ENU-induced Jeune syndrome model will be useful in delineating the origins of dysmorphology in human ciliopathies.
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Proteínas Portadoras/genética , Comunicación Celular/genética , Cilios/patología , Síndrome de Ellis-Van Creveld/genética , Desarrollo Embrionario/genética , Animales , Polaridad Celular , Cilios/genética , Modelos Animales de Enfermedad , Síndrome de Ellis-Van Creveld/patología , Proteínas Hedgehog/genética , Humanos , Ratones , Mutación , Transducción de SeñalRESUMEN
BACKGROUND: The YPEL (Yippee-like) gene family comprises five highly conserved members (YPEL1-5), but their biological function remains largely unknown. Early studies of YPEL1 function suggested that it plays a role in the development of structures derived from the pharyngeal arches. Human YPEL1 localises to distal chromosome 22q11.2 and copy number changes at this locus lead to diverse phenotypes that include facial dysmorphism, facial asymmetry, and palatal anomalies comprising the distal 22q11.2 deletion/duplication syndromes (OMIM 611867). We therefore investigated the role of chick YPEL1 in craniofacial development using ex vivo and in vivo approaches in the avian model. RESULTS: We found that retroviral-mediated in vivo overexpression of YPEL1 causes abnormal mandibular morphogenesis associated with increased apoptosis and involvement of the BMP/MSX pathway. CONCLUSIONS: Our results suggest that YPEL1 expression is regulated by bone morphogenetic protein signaling and suggest a role for YPEL1 in the pathogenesis of the craniofacial abnormalities observed in humans with distal chromosome 22q11.2 deletions or duplications.
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Proteínas Aviares/metabolismo , Huesos Faciales/embriología , Huesos Faciales/metabolismo , Animales , Proteínas Aviares/genética , Pollos , Morfogénesis/genética , Morfogénesis/fisiología , Activación TranscripcionalRESUMEN
We report the identification of three new mouse models, baringo, nice, and stitch, with recessively inherited sensorineural deafness due to novel mutations in the transmembrane channel-like gene 1 (Tmc1). These strains were generated by N-ethyl-N-nitrosourea mutagenesis. DNA sequence analysis revealed changes in c.545A>G, c.1345T>C, and c.1661G>T, causing p.Y182C, p.Y449H, and p.W554L amino acid substitutions in baringo, nice, and stitch mutants, respectively. The mutations affect amino acid residues that are evolutionarily conserved across species. Similar to the previously reported Beethoven Tmc1 mutant, both p.Y182C and p.W554L are located outside a predicted transmembrane domain, whereas the p.Y449H mutation resides in the predicted transmembrane domain 4. Homozygous stitch-mutant mice have severe hearing loss at the age of 4 weeks and are deaf by the age of 8 weeks, whereas both baringo and nice mutants are profoundly deaf at the age of 4 weeks. None of the strains displays signs of vestibular dysfunction. Scanning electron microscopy revealed degeneration of outer hair cells in the basal region of baringo, nice, and stitch mutants. Immunolocalization studies revealed expression of TMC1 protein in the hair cells, spiral ganglion neurons, supporting cells, and stria ligament in the inner ear. Reduced levels of TMC1 protein were observed in the spiral ligament of mutants when compared with wild-type animals. These three allelic mutants provide valuable models for studying nonsyndromic recessive sensorineural hearing loss (DFNB7/11) in humans.
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Pérdida Auditiva Sensorineural/genética , Proteínas de la Membrana/genética , Mutación Puntual , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Cóclea/crecimiento & desarrollo , Cóclea/metabolismo , Cóclea/ultraestructura , Análisis Mutacional de ADN/métodos , Modelos Animales de Enfermedad , Genes Recesivos , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/ultraestructura , Pérdida Auditiva Sensorineural/metabolismo , Pérdida Auditiva Sensorineural/patología , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Mutagénesis , Alineación de Secuencia , Especificidad de la EspecieRESUMEN
Mutations in the human cadherin 23 (CDH23) gene cause deafness, neurosensory, autosomal recessive 12 (DFNB12) nonsyndromic hearing loss or Usher syndrome, type 1D (characterized by hearing impairment, vestibular dysfunction, and visual impairment). Reported waltzer mouse strains each harbor a Cdh23-null mutation and present with hearing loss and vestibular dysfunction. Two additional Cdh23 mouse mutants, salsa and erlong, each carry a homozygous Cdh23 missense mutation and have progressive hearing loss. We report the identification of a novel mouse strain, jera, with inherited hearing loss caused by an N-ethyl-N-nitrosourea-induced c.7079T>A mutation in the Cdh23 gene. The mutation generates a missense change, p.V2360E, in Cdh23. Affected mice have profound sensorineural deafness, with no vestibular dysfunction. The p.V2360E mutation is semidominant because heterozygous mice have milder and more progressive hearing loss in advanced age. The mutation affects a highly conserved Ca(2+)-binding motif in extracellular domain 22, thought to be important for Cdh23 structure and dimerization. Molecular modeling suggests that the Cdh23(V2360E/V2360E) mutation alters the structural conformation of the protein and affects Ca(2+)-binding properties. Similar to salsa mice, but in contrast to waltzer mice, hair bundle development is normal in jera and hearing loss appears to be due to the loss of tip links. Thus, jera is a novel mouse model for DFNB12.
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Cadherinas/fisiología , Pérdida Auditiva Sensorineural/genética , Mutación , Enfermedades Vestibulares/patología , Alelos , Secuencia de Aminoácidos , Animales , Cadherinas/química , Cadherinas/genética , Análisis Mutacional de ADN , Etilnitrosourea/farmacología , Audición , Pérdida Auditiva Sensorineural/congénito , Humanos , Ratones , Ratones Transgénicos , Modelos Moleculares , Datos de Secuencia Molecular , Conformación Proteica , Homología de Secuencia de Aminoácido , Enfermedades Vestibulares/genética , Vestíbulo del Laberinto/patologíaRESUMEN
Multiple cytokines, including interleukin 6 (IL-6), IL-11, IL-27, oncostatin M (OSM), and leukemia inhibitory factor (LIF), signal via the common GP130 cytokine receptor subunit. In this study, we describe a patient with a homozygous mutation of IL6ST (encoding GP130 p.N404Y) who presented with recurrent infections, eczema, bronchiectasis, high IgE, eosinophilia, defective B cell memory, and an impaired acute-phase response, as well as skeletal abnormalities including craniosynostosis. The p.N404Y missense substitution is associated with loss of IL-6, IL-11, IL-27, and OSM signaling but a largely intact LIF response. This study identifies a novel immunodeficiency with phenotypic similarities to STAT3 hyper-IgE syndrome caused by loss of function of GP130.
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Craneosinostosis/genética , Receptor gp130 de Citocinas/genética , Síndromes de Inmunodeficiencia/genética , Mutación Missense/genética , Preescolar , Receptor gp130 de Citocinas/fisiología , Exoma/genética , Femenino , Humanos , Interleucina-11/deficiencia , Interleucina-6/deficiencia , Interleucinas/deficienciaRESUMEN
BACKGROUND: Olfactory receptors were initially believed to be expressed specifically within the olfactory neurons. However, accumulating genome-scale data has recently demonstrated more extensive expression. There are hundreds of olfactory receptor family members and the realisation of their widespread expression provides an opportunity to reveal new biology. However, existing data is predominantly based on RT-PCR, microarray and RNA-seq approaches and the details of tissue and cell-type specific expression are lacking. RESULTS: As a proof of principle, we selected Olfr603 for expression analysis. We generated an antibody against a non-conserved epitope of Olfr603 and characterised its expression in E8.5-E12.5 mouse embryos using immunohistochemistry. This analysis demonstrated a dynamic pattern of expression in diverse cell types within the developing embryo unrelated to the olfactory system. Expression was detected in migrating neural crest, endothelial precursors and vascular endothelium, endocardial cells, smooth muscle, neuroepithelium and within the ocular tissues. This complex distribution does not conform to any apparent germ layer or tissue origin. CONCLUSIONS: This initial characterisation of Olfr603 expression highlights the potential for a broad role for this receptor in the development of many tissues.
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Neuronas Receptoras Olfatorias/metabolismo , Receptores Nucleares Huérfanos/biosíntesis , Receptores Odorantes/biosíntesis , Secuencia de Aminoácidos , Animales , Embrión de Mamíferos , Expresión Génica , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Datos de Secuencia Molecular , Proteínas del Tejido Nervioso/biosíntesis , Sistema Nervioso/metabolismo , Cresta Neural/metabolismo , Tubo Neural/metabolismo , Vías Olfatorias/metabolismoRESUMEN
The 22q11.2 deletion syndrome (22q11DS) is thought to be a contiguous gene syndrome caused by haploinsufficiency for a variable number of genes with overlapping function during the development of the craniofacial, pharyngeal and cardiac structures. The complexity of genetic and developmental anomalies resulting in 22q11DS has made attributing causation to specific genes difficult. The CRKL gene resides within the common 3-Mb region, most frequently affected in 22q11DS, and has been shown to play an essential role in the development of tissues affected in 22q11DS. Here, we report the characterisation of a mouse strain we named 'snoopy', harbouring a novel Crkl splice-site mutation that results in a loss of Crkl expression. The snoopy strain exhibits a variable phenotype that includes micrognathia, pharyngeal occlusion, aglossia and holoprosencephaly, and altered retinoic acid and endothelin signalling. Together, these features are reminiscent of malformations occurring in auriculocondylar syndrome and agnathia-otocephaly complex, 2 conditions not previously associated with the CRKL function. Comparison of the features of a cohort of patients harbouring small 22q11.2 deletions centred over the CRKL gene, but sparing TBX1, highlights the role of CRKL in contributing to the craniofacial features of 22q11DS. These analyses demonstrate the central role of Crkl in regulating signalling events in the developing oropharyngeal complex and its potential to contribute to dysmorphology.
RESUMEN
Myosin VI (Myo6) is known to play an important role in the mammalian auditory and vestibular systems. We have identified a novel N-ethyl-N-nitrosourea mutagenised mouse strain, charlie, carrying an intronic Myo6 splice site mutation. This mutation (IVS5+5G > A) results in skipping of exon 5, and is predicted to cause a frameshift and premature termination of the protein. We detected essentially no Myo6 transcript in tissue from charlie homozygous mutant mice (Myo6(chl/chl)). Myo6(chl/chl) mice exhibit vestibular dysfunction and profound hearing impairment when first tested at four weeks of age. Analysis of vestibular and cochlear hair cells by scanning electron microscopy and immunohistochemistry revealed highly disorganised hair bundles with irregular orientation and kinocilium position at postnatal stage P2-P3. Within a few weeks, the majority of hair cell stereocilia are missing, or fused and elongated, and degeneration of the sensory epithelium occurs. This novel mouse strain will be an important resource in elucidating the role myosin VI plays in the mammalian auditory system, as well as its non-auditory functions.
Asunto(s)
Sordera/congénito , Sordera/fisiopatología , Etilnitrosourea/farmacología , Mutágenos/farmacología , Mutación , Cadenas Pesadas de Miosina/genética , Vestíbulo del Laberinto/fisiopatología , Secuencia de Aminoácidos , Animales , Percepción Auditiva , Secuencia de Bases , Cardiomiopatía Hipertrófica/genética , Cardiomiopatía Hipertrófica/metabolismo , Cardiomiopatía Hipertrófica/patología , Cóclea/metabolismo , Cóclea/fisiopatología , Cóclea/ultraestructura , Sordera/metabolismo , Sordera/patología , Sordera/psicología , Modelos Animales de Enfermedad , Predisposición Genética a la Enfermedad , Pruebas Auditivas , Homocigoto , Inmunohistoquímica , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Microscopía Electrónica de Rastreo , Datos de Secuencia Molecular , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Vestíbulo del Laberinto/metabolismo , Vestíbulo del Laberinto/ultraestructuraRESUMEN
Animal models that recapitulate human disease are proving to be an invaluable tool in the identification of novel disease-associated genes. These models can improve our understanding of the complex genetic mechanisms involved in disease and provide a basis to guide therapeutic strategies to combat these conditions. We have identified a novel mouse model of non-syndromic sensorineural hearing loss with linkage to a region on chromosome 18. Eeyore mutant mice have early onset progressive hearing impairment and show abnormal structure of the sensory epithelium from as early as 4 weeks of age. Ultrastructural and histological analyses show irregular hair cell structure and degeneration of the sensory hair bundles in the cochlea. The identification of new genes involved in hearing is central to understanding the complex genetic pathways involved in the hearing process and the loci at which these pathways are interrupted in people with a genetic hearing loss. We therefore discuss possible candidate genes within the linkage region identified in eeyore that may underlie the deafness phenotype in these mice. Eeyore provides a new model of hereditary sensorineural deafness and will be an important tool in the search for novel deafness genes.
Asunto(s)
Sordera/genética , Modelos Animales de Enfermedad , Enfermedades Genéticas Congénitas/genética , Animales , Sordera/patología , Progresión de la Enfermedad , Oído Medio/patología , Enfermedades Genéticas Congénitas/patología , Ratones , Ratones Mutantes , Microscopía Electrónica de RastreoRESUMEN
Fras1 is an extracellular matrix associated protein with essential roles in adhesion of epithelia and mesenchyme during early embryonic development. The adhesive function of Fras1 is achieved through interaction with a group of related proteins, Frem 1-3, and a cytoplasmic adaptor protein Grip1. Mutation of each of these proteins results in characteristic epithelial blistering and have therefore become known as "blebs" proteins. Human Fraser syndrome presents with a similar phenotype and the blebs mice have been instrumental in identification of the genetic basis of Fraser syndrome. We have identified a new ENU-induced blebs allele resulting from a novel missense mutation in Fras1. The resulting mouse strain, blood filled blisters (bfb), presents with a classic blebs phenotype but does not exhibit embryonic lethality typical of other blebs mutants and in addition, we report novel palate and sternal defects. Analysis of the bfb phenotype confirms the presence of epithelial-mesenchymal adhesion defects but also supports the emerging role of blebs proteins in regulating signalling during organogenesis. The bfb strain provides new opportunities to investigate the role of Fras1 in development.