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1.
Bioinformatics ; 2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31350555

RESUMO

SUMMARY: amplimap is a command-line tool to automate the processing and analysis of data from targeted next-generation sequencing (NGS) 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 (UMIs) 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.

2.
Am J Hum Genet ; 102(6): 1195-1203, 2018 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-29861108

RESUMO

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.

3.
J Exp Med ; 214(9): 2547-2562, 2017 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-28747427

RESUMO

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.


Assuntos
Craniossinostoses/genética , Receptor gp130 de Citocina/genética , Síndromes de Imunodeficiência/genética , Mutação de Sentido Incorreto/genética , Pré-Escolar , Receptor gp130 de Citocina/fisiologia , Exoma/genética , Feminino , Humanos , Interleucina-11/deficiência , Interleucina-6/deficiência , Interleucinas/deficiência
4.
J Med Genet ; 54(4): 260-268, 2017 04.
Artigo em Inglês | MEDLINE | ID: mdl-27884935

RESUMO

BACKGROUND: Craniosynostosis, the premature fusion of one or more cranial sutures, occurs in ∼1 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.


Assuntos
Craniossinostoses/genética , Genoma Humano , Sequenciamento de Nucleotídeos em Larga Escala , Proteínas de Neoplasias/genética , Craniossinostoses/diagnóstico , Craniossinostoses/patologia , Exoma/genética , Testes Genéticos , Humanos , Mutação , Valor Preditivo dos Testes
5.
Am J Hum Genet ; 99(1): 125-38, 2016 07 07.
Artigo em Inglês | MEDLINE | ID: mdl-27374770

RESUMO

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.


Assuntos
Proteínas de Ciclo Celular/genética , Microtia Congênita/genética , Craniossinostoses/genética , Transtornos do Crescimento/genética , Micrognatismo/genética , Mutação , Patela/anormalidades , Adolescente , Adulto , Alelos , Processamento Alternativo/genética , Sequência de Aminoácidos , Âmnio/citologia , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/deficiência , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Células Cultivadas , Criança , Pré-Escolar , Análise Mutacional de DNA , Replicação do DNA , Exoma/genética , Éxons/genética , Feminino , Estudos de Associação Genética , Humanos , Masculino , Modelos Moleculares , Conformação Proteica , Síndrome , Adulto Jovem
6.
Am J Hum Genet ; 98(6): 1256-1265, 2016 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-27236920

RESUMO

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.


Assuntos
Anormalidades Craniofaciais/etiologia , Intestinos/anormalidades , Mutação/genética , Anormalidades da Pele/etiologia , Receptor Smoothened/genética , Sindactilia/etiologia , Pré-Escolar , Anormalidades Craniofaciais/patologia , Feminino , Humanos , Lactente , Recém-Nascido , Intestinos/patologia , Masculino , Transdução de Sinais , Anormalidades da Pele/patologia , Sindactilia/patologia
7.
Am J Hum Genet ; 97(3): 378-88, 2015 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-26340333

RESUMO

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.


Assuntos
Códon sem Sentido/genética , Craniossinostoses/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Transtornos de Aprendizagem/genética , Fenótipo , Fatores de Transcrição/genética , Animais , Sequência de Bases , Clonagem Molecular , Feminino , Proteínas de Homeodomínio/metabolismo , Humanos , Hibridização In Situ , Cariotipagem , Masculino , Camundongos , Dados de Sequência Molecular , Mutação de Sentido Incorreto/genética , Proteínas do Tecido Nervoso/metabolismo , Linhagem , Análise de Sequência de DNA , Xenopus laevis
8.
Dev Dyn ; 244(8): 1022-30, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26061551

RESUMO

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.


Assuntos
Proteínas Aviárias/metabolismo , Ossos Faciais/embriologia , Ossos Faciais/metabolismo , Animais , Proteínas Aviárias/genética , Galinhas , Morfogênese/genética , Morfogênese/fisiologia , Ativação Transcricional
9.
Gene Expr Patterns ; 19(1-2): 30-5, 2015 Sep-Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26116001

RESUMO

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.


Assuntos
Neurônios Receptores Olfatórios/metabolismo , Receptores Nucleares Órfãos/biossíntese , Receptores Odorantes/biossíntese , Sequência de Aminoácidos , Animais , Embrião de Mamíferos , Expressão Gênica , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Dados de Sequência Molecular , Proteínas do Tecido Nervoso/biossíntese , Sistema Nervoso/metabolismo , Crista Neural/metabolismo , Tubo Neural/metabolismo , Condutos Olfatórios/metabolismo
10.
Mol Syndromol ; 5(6): 276-86, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-25565927

RESUMO

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.

11.
PLoS One ; 8(10): e76342, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24143185

RESUMO

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.


Assuntos
Proteínas da Matriz Extracelular/genética , Mutagênese , Mutação de Sentido Incorreto , Alelos , Sequência de Aminoácidos , Animais , Proteínas da Matriz Extracelular/química , Feminino , Humanos , Camundongos , Dados de Sequência Molecular , Organogênese/genética , Fenótipo
12.
PLoS One ; 8(9): e74243, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-24086324

RESUMO

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.


Assuntos
Surdez/genética , Modelos Animais de Doenças , Doenças Genéticas Inatas/genética , Animais , Surdez/patologia , Progressão da Doença , Orelha Média/patologia , Doenças Genéticas Inatas/patologia , Camundongos , Camundongos Mutantes , Microscopia Eletrônica de Varredura
13.
PLoS Genet ; 9(8): e1003746, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24009529

RESUMO

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.


Assuntos
Proteínas de Transporte/genética , Comunicação Celular/genética , Cílios/patologia , Síndrome de Ellis-Van Creveld/genética , Desenvolvimento Embrionário/genética , Animais , Polaridade Celular , Cílios/genética , Modelos Animais de Doenças , Síndrome de Ellis-Van Creveld/patologia , Proteínas Hedgehog/genética , Humanos , Camundongos , Mutação , Transdução de Sinais
14.
Hear Res ; 299: 53-62, 2013 May.
Artigo em Inglês | MEDLINE | ID: mdl-23485424

RESUMO

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.


Assuntos
Surdez/congênito , Surdez/fisiopatologia , Etilnitrosoureia/farmacologia , Mutagênicos/farmacologia , Mutação , Cadeias Pesadas de Miosina/genética , Vestíbulo do Labirinto/fisiopatologia , Sequência de Aminoácidos , Animais , Percepção Auditiva , Sequência de Bases , Cardiomiopatia Hipertrófica/genética , Cardiomiopatia Hipertrófica/metabolismo , Cardiomiopatia Hipertrófica/patologia , Cóclea/metabolismo , Cóclea/fisiopatologia , Cóclea/ultraestrutura , Surdez/metabolismo , Surdez/patologia , Surdez/psicologia , Modelos Animais de Doenças , Predisposição Genética para Doença , Testes Auditivos , Homozigoto , Imuno-Histoquímica , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Cadeias Pesadas de Miosina/metabolismo , Fenótipo , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/ultraestrutura
15.
PLoS One ; 8(3): e55429, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23469164

RESUMO

BACKGROUND: Mice harbouring gene mutations that cause phenotypic abnormalities during organogenesis are invaluable tools for linking gene function to normal development and human disorders. To generate mouse models harbouring novel alleles that are involved in organogenesis we conducted a phenotype-driven, genome-wide mutagenesis screen in mice using the mutagen N-ethyl-N-nitrosourea (ENU). METHODOLOGY/PRINCIPAL FINDINGS: ENU was injected into male C57BL/6 mice and the mutations transmitted through the germ-line. ENU-induced mutations were bred to homozygosity and G3 embryos screened at embryonic day (E) 13.5 and E18.5 for abnormalities in limb and craniofacial structures, skin, blood, vasculature, lungs, gut, kidneys, ureters and gonads. From 52 pedigrees screened 15 were detected with anomalies in one or more of the structures/organs screened. Using single nucleotide polymorphism (SNP)-based linkage analysis in conjunction with candidate gene or next-generation sequencing (NGS) we identified novel recessive alleles for Fras1, Ift140 and Lig1. CONCLUSIONS/SIGNIFICANCE: In this study we have generated mouse models in which the anomalies closely mimic those seen in human disorders. The association between novel mutant alleles and phenotypes will lead to a better understanding of gene function in normal development and establish how their dysfunction causes human anomalies and disease.


Assuntos
Anormalidades Congênitas/genética , Modelos Animais de Doenças , Etilnitrosoureia/toxicidade , Exoma/efeitos dos fármacos , Camundongos Endogâmicos C57BL/genética , Mutagênicos/toxicidade , Polimorfismo de Nucleotídeo Único , Alelos , Animais , Anormalidades Congênitas/embriologia , Anormalidades Congênitas/etiologia , DNA Ligase Dependente de ATP , DNA Ligases/genética , Proteínas da Matriz Extracelular/genética , Feminino , Estudo de Associação Genômica Ampla , Genótipo , Mutação em Linhagem Germinativa/efeitos dos fármacos , Sequenciamento de Nucleotídeos em Larga Escala , Homozigoto , Fatores de Determinação Direita-Esquerda/genética , Masculino , Camundongos , Mutagênese , Fenótipo
16.
PLoS One ; 7(12): e51284, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23251483

RESUMO

Human MYO7A mutations can cause a variety of conditions involving the inner ear. These include dominant and recessive non-syndromic hearing loss and syndromic conditions such as Usher syndrome. Mouse models of deafness allow us to investigate functional pathways involved in normal and abnormal hearing processes. We present two novel mouse models with mutations in the Myo7a gene with distinct phenotypes. The mutation in Myo7a(I487N/I487N) ewaso is located within the head motor domain of Myo7a. Mice exhibit a profound hearing loss and manifest behaviour associated with a vestibular defect. A mutation located in the linker region between the coiled-coil and the first MyTH4 domains of the protein is responsible in Myo7a(F947I/F947I) dumbo. These mice show a less severe hearing loss than in Myo7a(I487N/I487N) ewaso; their hearing loss threshold is elevated at 4 weeks old, and progressively worsens with age. These mice show no obvious signs of vestibular dysfunction, although scanning electron microscopy reveals a mild phenotype in vestibular stereocilia bundles. The Myo7a(F947I/F947I) dumbo strain is therefore the first reported Myo7a mouse model without an overt vestibular phenotype; a possible model for human DFNB2 deafness. Understanding the molecular basis of these newly identified mutations will provide knowledge into the complex genetic pathways involved in the maintenance of hearing, and will provide insight into recessively inherited sensorineural hearing loss in humans.


Assuntos
Surdez/genética , Modelos Animais de Doenças , Orelha Interna/anatomia & histologia , Genes Recessivos , Sequência de Aminoácidos , Animais , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Miosinas/química , Miosinas/genética , Homologia de Sequência de Aminoácidos
17.
Am J Pathol ; 180(4): 1560-9, 2012 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-22330676

RESUMO

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.


Assuntos
Perda Auditiva Neurossensorial/genética , Proteínas de Membrana/genética , Mutação Puntual , Sequência de Aminoácidos , Animais , Sequência de Bases , Cóclea/crescimento & desenvolvimento , Cóclea/metabolismo , Cóclea/ultraestrutura , Análise Mutacional de DNA/métodos , Modelos Animais de Doenças , Genes Recessivos , Células Ciliadas Auditivas Externas/metabolismo , Células Ciliadas Auditivas Externas/ultraestrutura , Perda Auditiva Neurossensorial/metabolismo , Perda Auditiva Neurossensorial/patologia , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microscopia Eletrônica de Varredura , Dados de Sequência Molecular , Mutagênese , Alinhamento de Sequência , Especificidade da Espécie
18.
Am J Pathol ; 179(2): 903-14, 2011 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-21689626

RESUMO

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.


Assuntos
Caderinas/fisiologia , Perda Auditiva Neurossensorial/genética , Mutação , Doenças Vestibulares/patologia , Alelos , Sequência de Aminoácidos , Animais , Caderinas/química , Caderinas/genética , Análise Mutacional de DNA , Etilnitrosoureia/farmacologia , Audição , Perda Auditiva Neurossensorial/congênito , Humanos , Camundongos , Camundongos Transgênicos , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Doenças Vestibulares/genética , Vestíbulo do Labirinto/patologia
19.
PLoS One ; 6(3): e17607, 2011 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-21423608

RESUMO

BACKGROUND: Hearing impairment is the most common sensory impairment in humans, affecting 1:1,000 births. We have identified an ENU generated mouse mutant, Mozart, with recessively inherited, non-syndromic progressive hearing loss caused by a mutation in the synaptojanin 2 (Synj2), a central regulatory enzyme in the phosphoinositide-signaling cascade. METHODOLOGY/PRINCIPAL FINDINGS: The hearing loss in Mozart is caused by a p.Asn538Lys mutation in the catalytic domain of the inositol polyphosphate 5-phosphatase synaptojanin 2. Within the cochlea, Synj2 mRNA expression was detected in the inner and outer hair cells but not in the spiral ganglion. Synj2(N538K) mutant protein showed loss of lipid phosphatase activity, and was unable to degrade phosphoinositide signaling molecules. Mutant Mozart mice (Synj2(N538K/N538K)) exhibited progressive hearing loss and showed signs of hair cell degeneration as early as two weeks of age, with fusion of stereocilia followed by complete loss of hair bundles and ultimately loss of hair cells. No changes in vestibular or neurological function, or other clinical or behavioral manifestations were apparent. CONCLUSIONS/SIGNIFICANCE: Phosphoinositides are membrane associated signaling molecules that regulate many cellular processes including cell death, proliferation, actin polymerization and ion channel activity. These results reveal Synj2 as a critical regulator of hair cell survival that is essential for hair cell maintenance and hearing function.


Assuntos
Perda Auditiva Neurossensorial/enzimologia , Perda Auditiva Neurossensorial/genética , Mutagênese/genética , Mutação/genética , Proteínas do Tecido Nervoso/genética , Monoéster Fosfórico Hidrolases/genética , Sequência de Aminoácidos , Substituição de Aminoácidos/genética , Animais , Comportamento Animal , Etilnitrosoureia , Células Ciliadas Auditivas/metabolismo , Células Ciliadas Auditivas/patologia , Perda Auditiva Neurossensorial/fisiopatologia , Inositol Polifosfato 5-Fosfatases , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Dados de Sequência Molecular , Fibras Nervosas/metabolismo , Fibras Nervosas/patologia , Proteínas do Tecido Nervoso/química , Condução Nervosa/fisiologia , Monoéster Fosfórico Hidrolases/química , Gânglio Espiral da Cóclea/metabolismo , Gânglio Espiral da Cóclea/patologia , Vestíbulo do Labirinto/metabolismo , Vestíbulo do Labirinto/patologia , Vestíbulo do Labirinto/fisiopatologia
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