RESUMO
Cas9 transgenes can be employed for genome editing in mouse zygotes. However, using transgenic instead of exogenous Cas9 to produce gene-edited animals creates unique issues including ill-defined transgene integration sites, the potential for prolonged Cas9 expression in transgenic embryos, and increased genotyping burden. To overcome these issues, we generated mice harboring an oocyte-specific, Gdf9 promoter driven, Cas9 transgene (Gdf9-Cas9) targeted as a single copy into the Hprt1 locus. The X-linked Hprt1 locus was selected because it is a defined integration site that does not influence transgene expression, and breeding of transgenic males generates obligate transgenic females to serve as embryo donors. Using microinjections and electroporation to introduce sgRNAs into zygotes derived from transgenic dams, we demonstrate that Gdf9-Cas9 mediates genome editing as efficiently as exogenous Cas9 at several loci. We show that genome editing efficiency is independent of transgene inheritance, verifying that maternally derived Cas9 facilitates genome editing. We also show that paternal inheritance of Gdf9-Cas9 does not mediate genome editing, confirming that Gdf9-Cas9 is not expressed in embryos. Finally, we demonstrate that off-target mutagenesis is equally rare when using transgenic or exogenous Cas9. Together, these results show that the Gdf9-Cas9 transgene is a viable alternative to exogenous Cas9.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Feminino , Masculino , Camundongos , Animais , Edição de Genes/métodos , RNA Guia de Sistemas CRISPR-Cas , Mutação , Zigoto/metabolismo , Animais Geneticamente Modificados , OócitosRESUMO
Coatomer complexes function in the sorting and trafficking of proteins between subcellular organelles. Pathogenic variants in coatomer subunits or associated factors have been reported in multi-systemic disorders, i.e., coatopathies, that can affect the skeletal and central nervous systems. We have identified loss-of-function variants in COPB2, a component of the coatomer complex I (COPI), in individuals presenting with osteoporosis, fractures, and developmental delay of variable severity. Electron microscopy of COPB2-deficient subjects' fibroblasts showed dilated endoplasmic reticulum (ER) with granular material, prominent rough ER, and vacuoles, consistent with an intracellular trafficking defect. We studied the effect of COPB2 deficiency on collagen trafficking because of the critical role of collagen secretion in bone biology. COPB2 siRNA-treated fibroblasts showed delayed collagen secretion with retention of type I collagen in the ER and Golgi and altered distribution of Golgi markers. copb2-null zebrafish embryos showed retention of type II collagen, disorganization of the ER and Golgi, and early larval lethality. Copb2+/- mice exhibited low bone mass, and consistent with the findings in human cells and zebrafish, studies in Copb2+/- mouse fibroblasts suggest ER stress and a Golgi defect. Interestingly, ascorbic acid treatment partially rescued the zebrafish developmental phenotype and the cellular phenotype in Copb2+/- mouse fibroblasts. This work identifies a form of coatopathy due to COPB2 haploinsufficiency, explores a potential therapeutic approach for this disorder, and highlights the role of the COPI complex as a regulator of skeletal homeostasis.
Assuntos
Osso e Ossos/metabolismo , Complexo I de Proteína do Envoltório/genética , Proteína Coatomer/genética , Deficiências do Desenvolvimento/genética , Deficiência Intelectual/genética , Osteoporose/genética , Animais , Ácido Ascórbico/farmacologia , Osso e Ossos/efeitos dos fármacos , Osso e Ossos/patologia , Encéfalo/diagnóstico por imagem , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encéfalo/patologia , Criança , Pré-Escolar , Complexo I de Proteína do Envoltório/deficiência , Proteína Coatomer/química , Proteína Coatomer/deficiência , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Deficiências do Desenvolvimento/diagnóstico por imagem , Deficiências do Desenvolvimento/metabolismo , Deficiências do Desenvolvimento/patologia , Embrião não Mamífero , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/patologia , Feminino , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica no Desenvolvimento , Complexo de Golgi , Haploinsuficiência , Humanos , Deficiência Intelectual/diagnóstico por imagem , Deficiência Intelectual/metabolismo , Deficiência Intelectual/patologia , Masculino , Camundongos , Osteoporose/tratamento farmacológico , Osteoporose/metabolismo , Osteoporose/patologia , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Índice de Gravidade de Doença , Peixe-ZebraRESUMO
CD4(+) interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are instrumental in the immune response to pathogens. However, an overactive T(H)17 response results in tissue inflammation and autoimmunity, and therefore it is important to identify the molecular mechanisms that control the development of T(H)17 cells. IL-2 suppresses such development, but how IL-2 production is actively suppressed during T(H)7 differentiation is not understood. Here we report that under T(H)17-polarizing conditions, the transcription factors STAT3 and AhR upregulated the expression of Aiolos, a member of the Ikaros family of transcription factors. Using Aiolos-deficient mice, we demonstrated that Aiolos silenced the Il2 locus, promoting T(H)17 differentiation in vitro and in vivo. Thus, we have identified a module in the transcriptional program of T(H)17 cells that actively limits IL-2 production and promotes their differentiation.
Assuntos
Interleucina-2/biossíntese , Ativação Linfocitária , Células Th17/metabolismo , Transativadores/metabolismo , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Células Cultivadas , Colite/imunologia , Regulação da Expressão Gênica , Fator de Transcrição Ikaros , Interferon gama/biossíntese , Interferon gama/imunologia , Interleucina-2/genética , Interleucina-2/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Hidrocarboneto Arílico/metabolismo , Fator de Transcrição STAT3/metabolismo , Células Th17/citologia , Células Th17/imunologia , Transativadores/deficiência , Transativadores/genéticaRESUMO
Cell fate depends on the interplay between chromatin regulators and transcription factors. Here we show that activity of the Mi-2ß nucleosome-remodeling and histone-deacetylase (NuRD) complex was controlled by the Ikaros family of lymphoid lineage-determining proteins. Ikaros, an integral component of the NuRD complex in lymphocytes, tethered this complex to active genes encoding molecules involved in lymphoid differentiation. Loss of Ikaros DNA-binding activity caused a local increase in chromatin remodeling and histone deacetylation and suppression of lymphoid cell-specific gene expression. Without Ikaros, the NuRD complex also redistributed to transcriptionally poised genes that were not targets of Ikaros (encoding molecules involved in proliferation and metabolism), which induced their reactivation. Thus, release of NuRD from Ikaros regulation blocks lymphocyte maturation and mediates progression to a leukemic state by engaging functionally opposing epigenetic and genetic networks.
Assuntos
Linfócitos/enzimologia , Complexo Mi-2 de Remodelação de Nucleossomo e Desacetilase/metabolismo , Animais , Sequência de Bases , Diferenciação Celular/genética , Montagem e Desmontagem da Cromatina , Perfilação da Expressão Gênica , Redes Reguladoras de Genes , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Leucemia/genética , Linfócitos/imunologia , Camundongos , Motivos de Nucleotídeos , Ligação Proteica , Timócitos/metabolismoRESUMO
This corrects the article DOI: 10.1038/nature19356.
RESUMO
The genetic landscape of diseases associated with changes in bone mineral density (BMD), such as osteoporosis, is only partially understood. Here, we explored data from 3,823 mutant mouse strains for BMD, a measure that is frequently altered in a range of bone pathologies, including osteoporosis. A total of 200 genes were found to significantly affect BMD. This pool of BMD genes comprised 141 genes with previously unknown functions in bone biology and was complementary to pools derived from recent human studies. Nineteen of the 141 genes also caused skeletal abnormalities. Examination of the BMD genes in osteoclasts and osteoblasts underscored BMD pathways, including vesicle transport, in these cells and together with in silico bone turnover studies resulted in the prioritization of candidate genes for further investigation. Overall, the results add novel pathophysiological and molecular insight into bone health and disease.
Assuntos
Densidade Óssea/genética , Regulação da Expressão Gênica/genética , Osteoblastos/metabolismo , Osteoclastos/metabolismo , Osteoporose/genética , Animais , Feminino , Ontologia Genética , Pleiotropia Genética , Estudo de Associação Genômica Ampla , Genótipo , Masculino , Camundongos , Camundongos Transgênicos , Mutação , Osteoblastos/patologia , Osteoclastos/patologia , Osteoporose/metabolismo , Fenótipo , Regiões Promotoras Genéticas , Mapas de Interação de Proteínas , Caracteres Sexuais , TranscriptomaRESUMO
Lysinuric protein intolerance (LPI) is an inborn error of cationic amino acid (arginine, lysine, ornithine) transport caused by biallelic pathogenic variants in SLC7A7, which encodes the light subunit of the y+LAT1 transporter. Treatments for the complications of LPI, including growth failure, renal disease, pulmonary alveolar proteinosis, autoimmune disorders and osteoporosis, are limited. Given the early lethality of the only published global Slc7a7 knockout mouse model, a viable animal model to investigate global SLC7A7 deficiency is needed. Hence, we generated two mouse models with global Slc7a7 deficiency (Slc7a7em1Lbu/em1Lbu; Slc7a7Lbu/Lbu and Slc7a7em1(IMPC)Bay/em1(IMPC)Bay; Slc7a7Bay/Bay) using CRISPR/Cas9 technology by introducing a deletion of exons 3 and 4. Perinatal lethality was observed in Slc7a7Lbu/Lbu and Slc7a7Bay/Bay mice on the C57BL/6 and C57BL/6NJ inbred genetic backgrounds, respectively. We noted improved survival of Slc7a7Lbu/Lbu mice on the 129 Sv/Ev × C57BL/6 F2 background, but postnatal growth failure occurred. Consistent with human LPI, these Slc7a7Lbu/Lbu mice exhibited reduced plasma and increased urinary concentrations of the cationic amino acids. Histopathological assessment revealed loss of brush border and lipid vacuolation in the renal cortex of Slc7a7Lbu/Lbu mice, which combined with aminoaciduria suggests proximal tubular dysfunction. Micro-computed tomography of L4 vertebrae and skeletal radiographs showed delayed skeletal development and suggested decreased mineralization in Slc7a7Lbu/Lbu mice, respectively. In addition to delayed skeletal development and delayed development in the kidneys, the lungs and liver were observed based on histopathological assessment. Overall, our Slc7a7Lbu/Lbu mouse model on the F2 mixed background recapitulates multiple human LPI phenotypes and may be useful for future studies of LPI pathology.
Assuntos
Erros Inatos do Metabolismo dos Aminoácidos/genética , Sistema y+L de Transporte de Aminoácidos/genética , Rim/metabolismo , Erros Inatos do Metabolismo dos Aminoácidos/diagnóstico por imagem , Erros Inatos do Metabolismo dos Aminoácidos/patologia , Sistema y+L de Transporte de Aminoácidos/deficiência , Aminoácidos/genética , Animais , Modelos Animais de Doenças , Éxons/genética , Humanos , Rim/patologia , Camundongos , Camundongos Knockout , Fenótipo , Microtomografia por Raio-XRESUMO
SPONASTRIME dysplasia is an autosomal-recessive spondyloepimetaphyseal dysplasia characterized by spine (spondylar) abnormalities, midface hypoplasia with a depressed nasal bridge, metaphyseal striations, and disproportionate short stature. Scoliosis, coxa vara, childhood cataracts, short dental roots, and hypogammaglobulinemia have also been reported in this disorder. Although an autosomal-recessive inheritance pattern has been hypothesized, pathogenic variants in a specific gene have not been discovered in individuals with SPONASTRIME dysplasia. Here, we identified bi-allelic variants in TONSL, which encodes the Tonsoku-like DNA repair protein, in nine subjects (from eight families) with SPONASTRIME dysplasia, and four subjects (from three families) with short stature of varied severity and spondylometaphyseal dysplasia with or without immunologic and hematologic abnormalities, but no definitive metaphyseal striations at diagnosis. The finding of early embryonic lethality in a Tonsl-/- murine model and the discovery of reduced length, spinal abnormalities, reduced numbers of neutrophils, and early lethality in a tonsl-/- zebrafish model both support the hypomorphic nature of the identified TONSL variants. Moreover, functional studies revealed increased amounts of spontaneous replication fork stalling and chromosomal aberrations, as well as fewer camptothecin (CPT)-induced RAD51 foci in subject-derived cell lines. Importantly, these cellular defects were rescued upon re-expression of wild-type (WT) TONSL; this rescue is consistent with the hypothesis that hypomorphic TONSL variants are pathogenic. Overall, our studies in humans, mice, zebrafish, and subject-derived cell lines confirm that pathogenic variants in TONSL impair DNA replication and homologous recombination-dependent repair processes, and they lead to a spectrum of skeletal dysplasia phenotypes with numerous extra-skeletal manifestations.
Assuntos
Instabilidade Cromossômica , Dano ao DNA , Variação Genética , Anormalidades Musculoesqueléticas/patologia , NF-kappa B/genética , Osteocondrodisplasias/patologia , Adolescente , Adulto , Alelos , Animais , Células Cultivadas , Criança , Pré-Escolar , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Estudos de Associação Genética , Humanos , Camundongos , Camundongos Knockout , Anormalidades Musculoesqueléticas/genética , Osteocondrodisplasias/genética , Sequenciamento do Exoma , Adulto Jovem , Peixe-ZebraRESUMO
Approximately one-third of all mammalian genes are essential for life. Phenotypes resulting from knockouts of these genes in mice have provided tremendous insight into gene function and congenital disorders. As part of the International Mouse Phenotyping Consortium effort to generate and phenotypically characterize 5,000 knockout mouse lines, here we identify 410 lethal genes during the production of the first 1,751 unique gene knockouts. Using a standardized phenotyping platform that incorporates high-resolution 3D imaging, we identify phenotypes at multiple time points for previously uncharacterized genes and additional phenotypes for genes with previously reported mutant phenotypes. Unexpectedly, our analysis reveals that incomplete penetrance and variable expressivity are common even on a defined genetic background. In addition, we show that human disease genes are enriched for essential genes, thus providing a dataset that facilitates the prioritization and validation of mutations identified in clinical sequencing efforts.
Assuntos
Embrião de Mamíferos/embriologia , Embrião de Mamíferos/metabolismo , Genes Essenciais/genética , Genes Letais/genética , Mutação/genética , Fenótipo , Animais , Sequência Conservada/genética , Doença , Estudo de Associação Genômica Ampla , Ensaios de Triagem em Larga Escala , Humanos , Imageamento Tridimensional , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Penetrância , Polimorfismo de Nucleotídeo Único/genética , Homologia de SequênciaRESUMO
MOTIVATION: High-throughput phenomic projects generate complex data from small treatment and large control groups that increase the power of the analyses but introduce variation over time. A method is needed to utlize a set of temporally local controls that maximizes analytic power while minimizing noise from unspecified environmental factors. RESULTS: Here we introduce 'soft windowing', a methodological approach that selects a window of time that includes the most appropriate controls for analysis. Using phenotype data from the International Mouse Phenotyping Consortium (IMPC), adaptive windows were applied such that control data collected proximally to mutants were assigned the maximal weight, while data collected earlier or later had less weight. We applied this method to IMPC data and compared the results with those obtained from a standard non-windowed approach. Validation was performed using a resampling approach in which we demonstrate a 10% reduction of false positives from 2.5 million analyses. We applied the method to our production analysis pipeline that establishes genotype-phenotype associations by comparing mutant versus control data. We report an increase of 30% in significant P-values, as well as linkage to 106 versus 99 disease models via phenotype overlap with the soft-windowed and non-windowed approaches, respectively, from a set of 2082 mutant mouse lines. Our method is generalizable and can benefit large-scale human phenomic projects such as the UK Biobank and the All of Us resources. AVAILABILITY AND IMPLEMENTATION: The method is freely available in the R package SmoothWin, available on CRAN http://CRAN.R-project.org/package=SmoothWin. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Assuntos
Saúde da População , Software , Animais , Estudos de Associação Genética , Humanos , Camundongos , FenótipoRESUMO
Ubiquitination is a posttranslational modification that regulates many cellular processes including protein degradation, intracellular trafficking, cell signaling, and protein-protein interactions. Deubiquitinating enzymes (DUBs), which reverse the process of ubiquitination, are important regulators of the ubiquitin system. OTUD6B encodes a member of the ovarian tumor domain (OTU)-containing subfamily of deubiquitinating enzymes. Herein, we report biallelic pathogenic variants in OTUD6B in 12 individuals from 6 independent families with an intellectual disability syndrome associated with seizures and dysmorphic features. In subjects with predicted loss-of-function alleles, additional features include global developmental delay, microcephaly, absent speech, hypotonia, growth retardation with prenatal onset, feeding difficulties, structural brain abnormalities, congenital malformations including congenital heart disease, and musculoskeletal features. Homozygous Otud6b knockout mice were subviable, smaller in size, and had congenital heart defects, consistent with the severity of loss-of-function variants in humans. Analysis of peripheral blood mononuclear cells from an affected subject showed reduced incorporation of 19S subunits into 26S proteasomes, decreased chymotrypsin-like activity, and accumulation of ubiquitin-protein conjugates. Our findings suggest a role for OTUD6B in proteasome function, establish that defective OTUD6B function underlies a multisystemic human disorder, and provide additional evidence for the emerging relationship between the ubiquitin system and human disease.
Assuntos
Anormalidades Múltiplas/genética , Endopeptidases/genética , Deficiência Intelectual/genética , Adolescente , Animais , Criança , Pré-Escolar , Modelos Animais de Doenças , Feminino , Deleção de Genes , Humanos , Masculino , Camundongos , Linhagem , Complexo de Endopeptidases do Proteassoma/genética , Complexo de Endopeptidases do Proteassoma/metabolismo , Convulsões/genéticaRESUMO
BACKGROUND: The International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs). RESULTS: Using pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected. CONCLUSIONS: Our studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.
Assuntos
Sistemas CRISPR-Cas/genética , DNA de Cadeia Simples/genética , Edição de Genes , Mutação com Perda de Função , Camundongos Knockout/genética , RNA Guia de Cinetoplastídeos/genética , Alelos , Animais , Éxons , CamundongosRESUMO
The evolutionarily conserved protein kinase p38 mediates innate resistance to environmental stress and microbial infection. Four p38 isoforms exist in mammals and may have been co-opted for new roles in adaptive immunity. Murine T cells deficient in p38α, the ubiquitously expressed p38 isoform, showed no readily apparent cell-autonomous defects while expressing elevated amounts of another isoform, p38ß. Mice with T cells simultaneously lacking p38α and p38ß displayed lymphoid atrophy and elevated Foxp3+ regulatory T cell frequencies. Double deficiency of p38α and p38ß in naïve CD4+ T cells resulted in an attenuation of MAPK-activated protein kinase (MK)-dependent mTOR signaling after T cell receptor engagement, and enhanced their differentiation into regulatory T cells under appropriate inducing conditions. Pharmacological inhibition of the p38-MK-mTOR signaling module produced similar effects, revealing potential for therapeutic applications.
Assuntos
Sistema de Sinalização das MAP Quinases/imunologia , Proteína Quinase 11 Ativada por Mitógeno/imunologia , Proteína Quinase 14 Ativada por Mitógeno/imunologia , Receptores de Antígenos de Linfócitos T/imunologia , Linfócitos T Reguladores/imunologia , Animais , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/imunologia , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Camundongos Knockout , Proteína Quinase 11 Ativada por Mitógeno/genética , Proteína Quinase 14 Ativada por Mitógeno/genética , Receptores de Antígenos de Linfócitos T/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologiaRESUMO
The International Mouse Phenotyping Consortium (IMPC) is building a catalogue of mammalian gene function by producing and phenotyping a knockout mouse line for every protein-coding gene. To date, the IMPC has generated and characterised 5186 mutant lines. One-third of the lines have been found to be non-viable and over 300 new mouse models of human disease have been identified thus far. While current bioinformatics efforts are focused on translating results to better understand human disease processes, IMPC data also aids understanding genetic function and processes in other species. Here we show, using gorilla genomic data, how genes essential to development in mice can be used to help assess the potentially deleterious impact of gene variants in other species. This type of analyses could be used to select optimal breeders in endangered species to maintain or increase fitness and avoid variants associated to impaired-health phenotypes or loss-of-function mutations in genes of critical importance. We also show, using selected examples from various mammal species, how IMPC data can aid in the identification of candidate genes for studying a condition of interest, deliver information about the mechanisms involved, or support predictions for the function of genes that may play a role in adaptation. With genotyping costs decreasing and the continued improvements of bioinformatics tools, the analyses we demonstrate can be routinely applied.
RESUMO
In this work, we report the use of iodine-contrast microCT to perform high-throughput 3D morphological analysis of mouse embryos and neonates between embryonic day 8.5 to postnatal day 3, with high spatial resolution up to 3µm/voxel. We show that mouse embryos at early stages can be imaged either within extra embryonic tissues such as the yolk sac or the decidua without physically disturbing the embryos. This method enables a full, undisturbed analysis of embryo turning, allantois development, vitelline vessels remodeling, yolk sac and early placenta development, which provides increased insights into early embryonic lethality in mutant lines. Moreover, these methods are inexpensive, simple to learn and do not require substantial processing time, making them ideal for high throughput analysis of mouse mutants with embryonic and early postnatal lethality.
Assuntos
Desenvolvimento Embrionário , Imageamento Tridimensional/métodos , Camundongos/embriologia , Microtomografia por Raio-X/métodos , Animais , Animais Recém-Nascidos , Meios de Contraste , Decídua/ultraestrutura , Feminino , Genes Letais , Estudos de Associação Genética , Idade Gestacional , Hidrogéis , Iodo , Fenótipo , Coloração e Rotulagem/métodos , Saco Vitelino/ultraestruturaRESUMO
Mutations in CRIPT encoding cysteine-rich PDZ domain-binding protein are rare, and to date have been reported in only two patients with autosomal recessive primordial dwarfism and distinctive facies. Here, we describe a female with biallelic mutations in CRIPT presenting with postnatal growth retardation, global developmental delay, and dysmorphic features including frontal bossing, high forehead, and sparse hair and eyebrows. Additional clinical features included high myopia, admixed hyper- and hypopigmented macules primarily on the face, arms, and legs, and syndactyly of 4-5 toes bilaterally. Using whole exome sequencing (WES) and chromosomal microarray analysis (CMA), we detected a c.8G>A (p.C3Y) missense variant in exon 1 of the CRIPT gene inherited from the mother and a 1,331 bp deletion encompassing exon 1, inherited from the father. The c.8G>A (p.C3Y) missense variant in CRIPT was apparently homozygous in the proband due to the exon 1 deletion. Our findings illustrate the clinical utility of combining WES with copy number variant (CNV) analysis to provide a molecular diagnosis to patients with rare Mendelian disorders. Our findings also illustrate the clinical spectrum of CRIPT related mutations. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Éxons , Estudos de Associação Genética , Mutação de Sentido Incorreto , Fenótipo , Deleção de Sequência , Anormalidades Múltiplas/diagnóstico , Anormalidades Múltiplas/genética , Alelos , Substituição de Aminoácidos , Pré-Escolar , Análise Mutacional de DNA , Nanismo/diagnóstico , Nanismo/genética , Fácies , Feminino , Humanos , Microcefalia/diagnóstico , Microcefalia/genética , LinhagemRESUMO
Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.
Assuntos
Sistemas CRISPR-Cas , Edição de Genes , Camundongos , Animais , Genoma , Mutação , MutagêneseRESUMO
SLC7A7 deficiency, or lysinuric protein intolerance (LPI), causes loss of function of the y+LAT1 transporter critical for efflux of arginine, lysine and ornithine in certain cells. LPI is characterized by urea cycle dysfunction, renal disease, immune dysregulation, growth failure, delayed bone age and osteoporosis. We previously reported that Slc7a7 knockout mice (C57BL/6×129/SvEv F2) recapitulate LPI phenotypes, including growth failure. Our main objective in this study was to characterize the skeletal phenotype in these mice. Compared to wild-type littermates, juvenile Slc7a7 knockout mice demonstrated 70% lower body weights, 87% lower plasma IGF-1 concentrations and delayed skeletal development. Because poor survival prevents evaluation of mature knockout mice, we generated a conditional Slc7a7 deletion in mature osteoblasts or mesenchymal cells of the osteo-chondroprogenitor lineage, but no differences in bone architecture were observed. Overall, global Slc7a7 deficiency caused growth failure with low plasma IGF-1 concentrations and delayed skeletal development, but Slc7a7 deficiency in the osteoblastic lineage was not a major contributor to these phenotypes. Future studies utilizing additional tissue-specific Slc7a7 knockout models may help dissect cell-autonomous and non-cell-autonomous mechanisms underlying phenotypes in LPI.
Assuntos
Fator de Crescimento Insulin-Like I , Animais , Camundongos , Sistema y+L de Transporte de Aminoácidos , Modelos Animais de Doenças , Camundongos Endogâmicos C57BL , Camundongos KnockoutRESUMO
Though biallelic variants in SLC13A5 are known to cause severe encephalopathy, the mechanism of this disease is poorly understood. SLC13A5 protein deficiency reduces citrate transport into the cell. Downstream abnormalities in fatty acid synthesis and energy generation have been described, though biochemical signs of these perturbations are inconsistent across SLC13A5 deficiency patients. To investigate SLC13A5-related disorders, we performed untargeted metabolic analyses on the liver, brain, and serum from a Slc13a5-deficient mouse model. Metabolomic data were analyzed using the connect-the-dots (CTD) methodology and were compared to plasma and CSF metabolomics from SLC13A5-deficient patients. Mice homozygous for the Slc13a5tm1b/tm1b null allele had perturbations in fatty acids, bile acids, and energy metabolites in all tissues examined. Further analyses demonstrated that for several of these molecules, the ratio of their relative tissue concentrations differed widely in the knockout mouse, suggesting that deficiency of Slc13a5 impacts the biosynthesis and flux of metabolites between tissues. Similar findings were observed in patient biofluids, indicating altered transport and/or flux of molecules involved in energy, fatty acid, nucleotide, and bile acid metabolism. Deficiency of SLC13A5 likely causes a broader state of metabolic dysregulation than previously recognized, particularly regarding lipid synthesis, storage, and metabolism, supporting SLC13A5 deficiency as a lipid disorder.