RESUMEN
Cilia (flagella) are important eukaryotic organelles, present in the Last Eukaryotic Common Ancestor, and are involved in cell motility and integration of extracellular signals. Ciliary dysfunction causes a class of genetic diseases, known as ciliopathies, however current knowledge of the underlying mechanisms is still limited and a better characterization of genes is needed. As cilia have been lost independently several times during evolution and they are subject to important functional variation between species, ciliary genes can be investigated through comparative genomics. We performed phylogenetic profiling by predicting orthologs of human protein-coding genes in 100 eukaryotic species. The analysis integrated three independent methods to predict a consensus set of 274 ciliary genes, including 87 new promising candidates. A fine-grained analysis of the phylogenetic profiles allowed a partitioning of ciliary genes into modules with distinct evolutionary histories and ciliary functions (assembly, movement, centriole, etc.) and thus propagation of potential annotations to previously undocumented genes. The cilia/basal body localization was experimentally confirmed for five of these previously unannotated proteins (LRRC23, LRRC34, TEX9, WDR27, and BIVM), validating the relevance of our approach. Furthermore, our multi-level analysis sheds light on the core gene sets retained in gamete-only flagellates or Ecdysozoa for instance. By combining gene-centric and species-oriented analyses, this work reveals new ciliary and ciliopathy gene candidates and provides clues about the evolution of ciliary processes in the eukaryotic domain. Additionally, the positive and negative reference gene sets and the phylogenetic profile of human genes constructed during this study can be exploited in future work.
Asunto(s)
Cilios/genética , Ciliopatías/genética , Animales , Movimiento Celular/genética , Cilios/metabolismo , Ciliopatías/metabolismo , Bases de Datos de Ácidos Nucleicos , Eucariontes , Células Eucariotas , Evolución Molecular , Flagelos/genética , Flagelos/metabolismo , Genómica , Humanos , Filogenia , Análisis de Secuencia de ADN/métodosRESUMEN
Inherited dental malformations constitute a clinically and genetically heterogeneous group of disorders. Here, we report on four families, three of them consanguineous, with an identical phenotype, characterized by significant short stature with brachyolmia and hypoplastic amelogenesis imperfecta (AI) with almost absent enamel. This phenotype was first described in 1996 by Verloes et al. as an autosomal recessive form of brachyolmia associated with AI. Whole-exome sequencing resulted in the identification of recessive hypomorphic mutations including deletion, nonsense and splice mutations, in the LTBP3 gene, which is involved in the TGF-beta signaling pathway. We further investigated gene expression during mouse development and tooth formation. Differentiated ameloblasts synthesizing enamel matrix proteins and odontoblasts expressed the gene. Study of an available knockout mouse model showed that the mutant mice displayed very thin to absent enamel in both incisors and molars, hereby recapitulating the AI phenotype in the human disorder.
Asunto(s)
Amelogénesis Imperfecta/genética , Proteínas de Unión a TGF-beta Latente/genética , Osteocondrodisplasias/genética , Adolescente , Amelogénesis Imperfecta/diagnóstico por imagen , Animales , Secuencia de Bases , Niño , Consanguinidad , Análisis Mutacional de ADN , Femenino , Mutación del Sistema de Lectura , Estudios de Asociación Genética , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación Missense , Osteocondrodisplasias/diagnóstico por imagen , Linaje , Radiografía , Eliminación de SecuenciaRESUMEN
BACKGROUND: Orodental diseases include several clinically and genetically heterogeneous disorders that can present in isolation or as part of a genetic syndrome. Due to the vast number of genes implicated in these disorders, establishing a molecular diagnosis can be challenging. We aimed to develop a targeted next-generation sequencing (NGS) assay to diagnose mutations and potentially identify novel genes mutated in this group of disorders. METHODS: We designed an NGS gene panel that targets 585 known and candidate genes in orodental disease. We screened a cohort of 101 unrelated patients without a molecular diagnosis referred to the Reference Centre for Oro-Dental Manifestations of Rare Diseases, Strasbourg, France, for a variety of orodental disorders including isolated and syndromic amelogenesis imperfecta (AI), isolated and syndromic selective tooth agenesis (STHAG), isolated and syndromic dentinogenesis imperfecta, isolated dentin dysplasia, otodental dysplasia and primary failure of tooth eruption. RESULTS: We discovered 21 novel pathogenic variants and identified the causative mutation in 39 unrelated patients in known genes (overall diagnostic rate: 39%). Among the largest subcohorts of patients with isolated AI (50 unrelated patients) and isolated STHAG (21 unrelated patients), we had a definitive diagnosis in 14 (27%) and 15 cases (71%), respectively. Surprisingly, COL17A1 mutations accounted for the majority of autosomal-dominant AI cases. CONCLUSIONS: We have developed a novel targeted NGS assay for the efficient molecular diagnosis of a wide variety of orodental diseases. Furthermore, our panel will contribute to better understanding the contribution of these genes to orodental disease. TRIAL REGISTRATION NUMBERS: NCT01746121 and NCT02397824.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Anomalías Dentarias/genética , Amelogénesis Imperfecta/genética , Autoantígenos/genética , Deleción Cromosómica , Trastornos de los Cromosomas/genética , Cromosomas Humanos Par 11/genética , Estudios de Cohortes , Coloboma/genética , Displasia de la Dentina/genética , Francia , Pérdida Auditiva Sensorineural/genética , Humanos , Colágenos no Fibrilares/genética , Reproducibilidad de los Resultados , Colágeno Tipo XVIIRESUMEN
Bardet-Biedl syndrome (BBS; MIM 209900) is a recessive heterogeneous ciliopathy characterized by retinitis pigmentosa (RP), postaxial polydactyly, obesity, hypogonadism, cognitive impairment and kidney dysfunction. So far, 20 BBS genes have been identified, with the last reported ones being found in one or very few families. Whole-exome sequencing was performed in a consanguineous family in which two affected children presented typical BBS features (retinitis pigmentosa, postaxial polydactyly, obesity, hypogonadism and cognitive impairment) without any mutation identified in known BBS genes at the time of the study. We identified a homozygous splice-site mutation (NM_015662.2: c.4428+3A>G) in both affected siblings in the last reported BBS gene, namely, Intraflagellar Transport 172 Homolog (IFT172). Familial mutation segregation was consistent with autosomal recessive inheritance. IFT172 mutations were initially reported in Jeune and Mainzer-Saldino syndromes. Recently, mutations have also been found in isolated RP and Bardet-Biedl-like ciliopathy. This is the second report of IFT172 mutations in BBS patients validating IFT172 as the twentieth BBS gene (BBS20). Moreover, another IFT gene, IFT27, was already associated with BBS, confirming the implication of IFT genes in the pathogenesis of BBS.
Asunto(s)
Síndrome de Bardet-Biedl/diagnóstico , Síndrome de Bardet-Biedl/genética , Proteínas Portadoras/genética , Mutación , Proteínas Adaptadoras Transductoras de Señales , Niño , Preescolar , Biología Computacional/métodos , Proteínas del Citoesqueleto , Exoma , Secuenciación de Nucleótidos de Alto Rendimiento , Homocigoto , Humanos , Masculino , Linaje , Fenotipo , Esqueleto/diagnóstico por imagen , Esqueleto/patologíaRESUMEN
BACKGROUND: Oliver-McFarlane syndrome is characterised by trichomegaly, congenital hypopituitarism and retinal degeneration with choroidal atrophy. Laurence-Moon syndrome presents similarly, though with progressive spinocerebellar ataxia and spastic paraplegia and without trichomegaly. Both recessively inherited disorders have no known genetic cause. METHODS: Whole-exome sequencing was performed to identify the genetic causes of these disorders. Mutations were functionally validated in zebrafish pnpla6 morphants. Embryonic expression was evaluated via in situ hybridisation in human embryonic sections. Human neurohistopathology was performed to characterise cerebellar degeneration. Enzymatic activities were measured in patient-derived fibroblast cell lines. RESULTS: Eight mutations in six families with Oliver-McFarlane or Laurence-Moon syndrome were identified in the PNPLA6 gene, which encodes neuropathy target esterase (NTE). PNPLA6 expression was found in the developing human eye, pituitary and brain. In zebrafish, the pnpla6 curly-tailed morphant phenotype was fully rescued by wild-type human PNPLA6 mRNA and not by mutation-harbouring mRNAs. NTE enzymatic activity was significantly reduced in fibroblast cells derived from individuals with Oliver-McFarlane syndrome. Intriguingly, adult brain histology from a patient with highly overlapping features of Oliver-McFarlane and Laurence-Moon syndromes revealed extensive cerebellar degeneration and atrophy. CONCLUSIONS: Previously, PNPLA6 mutations have been associated with spastic paraplegia type 39, Gordon-Holmes syndrome and Boucher-Neuhäuser syndromes. Discovery of these additional PNPLA6-opathies further elucidates a spectrum of neurodevelopmental and neurodegenerative disorders associated with NTE impairment and suggests a unifying mechanism with diagnostic and prognostic importance.
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Blefaroptosis/enzimología , Blefaroptosis/genética , Hidrolasas de Éster Carboxílico/genética , Enanismo/enzimología , Enanismo/genética , Predisposición Genética a la Enfermedad , Hipertricosis/enzimología , Hipertricosis/genética , Discapacidad Intelectual/enzimología , Discapacidad Intelectual/genética , Síndrome de Laurence-Moon/enzimología , Síndrome de Laurence-Moon/genética , Retinitis Pigmentosa/enzimología , Retinitis Pigmentosa/genética , Alelos , Secuencia de Aminoácidos , Animales , Hidrolasas de Éster Carboxílico/química , Sistema Nervioso Central/patología , Discapacidades del Desarrollo/enzimología , Discapacidades del Desarrollo/genética , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Datos de Secuencia Molecular , Mutación/genética , Fenotipo , Fosfolipasas/química , Fosfolipasas/genética , Estructura Terciaria de Proteína , Retina/patología , Pez Cebra/embriologíaRESUMEN
The transcription factor SOX10 has essential roles in neural crest-derived cell populations, including myelinating Schwann cells-specialized glial cells responsible for ensheathing axons in the peripheral nervous system. Importantly, SOX10 directly regulates the expression of genes essential for proper myelin function. To date, only a handful of SOX10 target loci have been characterized in Schwann cells. Addressing this lack of knowledge will provide a better understanding of Schwann cell biology and candidate loci for relevant diseases such as demyelinating peripheral neuropathies. We have identified a highly-conserved SOX10 binding site within an alternative promoter at the SH3-domain kinase binding protein 1 (Sh3kbp1) locus. The genomic segment identified at Sh3kbp1 binds to SOX10 and displays strong promoter activity in Schwann cells in vitro and in vivo. Mutation of the SOX10 binding site ablates promoter activity, and ectopic expression of SOX10 in SOX10-negative cells promotes the expression of endogenous Sh3kbp1. Combined, these data reveal Sh3kbp1 as a novel target of SOX10 and raise important questions regarding the function of SH3KBP1 isoforms in Schwann cells.
Asunto(s)
Regulación de la Expresión Génica , Proteínas de Neoplasias/genética , Proteínas del Tejido Nervioso/genética , Regiones Promotoras Genéticas/genética , Factores de Transcripción SOXE/metabolismo , Células de Schwann/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Secuencia de Aminoácidos , Animales , Sitios de Unión/genética , Sitios Genéticos , Humanos , Ratones , Datos de Secuencia Molecular , Mutación/genética , Ratas , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/fisiología , Pez Cebra/genética , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
Vertebrate sensory organs originate from both cranial neural crest cells (CNCCs) and placodes. Previously, we have shown that the olfactory placode (OP) forms from a large field of cells extending caudally to the premigratory neural crest domain, and that OPs form through cell movements and not cell division. Concurrent with OP formation, CNCCs migrate rostrally to populate the frontal mass. However, little is known about the interactions between CNCCs and the placodes that form the olfactory sensory system. Previous reports suggest that the OP can generate cell types more typical of neural crest lineages such as neuroendocrine cells and glia, thus marking the OP as an unusual sensory placode. One possible explanation for this exception is that the neural crest origin of glia and neurons has been overlooked due to the intimate association of these two fields during migration. Using molecular markers and live imaging, we followed the development of OP precursors and of dorsally migrating CNCCs in zebrafish embryos. We generated a six4b:mCherry line (OP precursors) that, with a sox10:EGFP line (CNCCs), was used to follow cell migration. Our analyses showed that CNCCs associate with and eventually surround the forming OP with limited cell mixing occurring during this process.
Asunto(s)
Vías Olfatorias/citología , Animales , Animales Modificados Genéticamente , Embrión no Mamífero/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Inmunohistoquímica , Hibridación in Situ , Cresta Neural/citología , Cresta Neural/metabolismo , Vías Olfatorias/metabolismo , Factores de Transcripción SOXE/genética , Factores de Transcripción SOXE/metabolismo , Transactivadores/genética , Transactivadores/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Over the last decade, CRISPR has revolutionized drug development due to its potential to cure genetic diseases that currently do not have any treatment. CRISPR was adapted from bacteria for gene editing in human cells in 2012 and, remarkably, only 11 years later has seen it's very first approval as a medicine for the treatment of sickle cell disease and transfusion-dependent beta-thalassemia. However, the application of CRISPR systems is associated with unintended off-target and on-target alterations (including small indels, and structural variations such as translocations, inversions and large deletions), which are a source of risk for patients and a vital concern for the development of safe therapies. In recent years, a wide range of methods has been developed to detect unwanted effects of CRISPR-Cas nuclease activity. In this review, we summarize the different methods for off-target assessment, discuss their strengths and limitations, and highlight strategies to improve the safety of CRISPR systems. Finally, we discuss their relevance and application for the pre-clinical risk assessment of CRISPR therapeutics within the current regulatory context.
RESUMEN
Single-cell and single-nucleus RNA sequencing have become common laboratory applications due to the wealth of transcriptomic information that they provide. Single nucleus RNA sequencing, particularly, is useful for investigating gene expression in difficult-to-dissociate tissues. Furthermore, this approach is also compatible with frozen (archival) material. Here, we describe a protocol to isolate high-quality single nuclei from frozen mammalian tissues for downstream single nucleus RNA sequencing in a partially-automated manner using commercially available instruments and reagents. Specifically, a robotic dissociator is used to automate and standardize tissue homogenization, followed by an optimized chemical gradient to filter the nuclei. Lastly, we accurately and automatically count the nuclei using an automated fluorescent cell counter. The performance of this protocol is demonstrated on mouse brain, rat kidney, and cynomolgus liver and spleen tissue. This protocol is straightforward, rapid, and readily adaptable to various mammalian tissues without requiring extensive optimization and provides good quality nuclei for downstream single nuclei RNA sequencing.
Asunto(s)
Núcleo Celular , Perfilación de la Expresión Génica , Ratas , Ratones , Animales , Núcleo Celular/metabolismo , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos , Transcriptoma , Indicadores y Reactivos/metabolismo , Mamíferos/genéticaRESUMEN
BACKGROUND: The ERBB3 gene is essential for the proper development of the neural crest (NC) and its derivative populations such as Schwann cells. As with all cell fate decisions, transcriptional regulatory control plays a significant role in the progressive restriction and specification of NC derived lineages during development. However, little is known about the sequences mediating transcriptional regulation of ERBB3 or the factors that bind them. RESULTS: In this study we identified three transcriptional enhancers at the ERBB3 locus and evaluated their regulatory potential in vitro in NC-derived cell types and in vivo in transgenic zebrafish. One enhancer, termed ERBB3_MCS6, which lies within the first intron of ERBB3, directs the highest reporter expression in vitro and also demonstrates epigenetic marks consistent with enhancer activity. We identify a consensus SOX10 binding site within ERBB3_MCS6 and demonstrate, in vitro, its necessity and sufficiency for the activity of this enhancer. Additionally, we demonstrate that transcription from the endogenous Erbb3 locus is dependent on Sox10. Further we demonstrate in vitro that Sox10 physically interacts with that ERBB3_MCS6. Consistent with its in vitro activity, we also show that ERBB3_MCS6 drives reporter expression in NC cells and a subset of its derivative lineages in vivo in zebrafish in a manner consistent with erbb3b expression. We also demonstrate, using morpholino analysis, that Sox10 is necessary for ERBB3_MCS6 expression in vivo in zebrafish. CONCLUSIONS: Taken collectively, our data suggest that ERBB3 may be directly regulated by SOX10, and that this control may in part be facilitated by ERBB3_MCS6.
Asunto(s)
Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Intrones , Cresta Neural/fisiología , Receptor ErbB-3/metabolismo , Factores de Transcripción SOXE/metabolismo , Transcripción Genética , Animales , Animales Modificados Genéticamente , Sitios de Unión , Epigénesis Genética , Genes Reporteros , Humanos , Ratones , Células 3T3 NIH , Cresta Neural/citología , Unión Proteica , Receptor ErbB-3/genética , Factores de Transcripción SOXE/genética , Pez Cebra/anatomía & histología , Pez Cebra/fisiologíaRESUMEN
OCT3/4 is a POU domain transcription factor that is critical for maintenance of pluripotency and self-renewal by embryonic stem (ES) cells and cells of the early mammalian embryo. It has been demonstrated to bind and regulate a number of genes, often in conjunction with the transcription factors SOX2 and NANOG. In an effort to further understand this regulatory network, chromatin immunoprecipitation was used to prepare a library of DNA segments specifically bound by OCT3/4 in undifferentiated mouse ES (mES) cell chromatin. One segment corresponds to a region within the first intron of the gene encoding histone deacetylase 4 (Hdac4), a Class II histone deacetylase. This region acts as a transcriptional repressor and contains at least two functional sites that are specifically bound by OCT3/4. HDAC4 is not expressed in the nuclei of OCT3/4+ mES cells and is upregulated upon differentiation. These findings demonstrate the participation of OCT3/4 in the repression of Hdac4 in ES cells.
Asunto(s)
Células Madre Embrionarias/metabolismo , Histona Desacetilasas/genética , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Transcripción Genética , Animales , Sitios de Unión , Cromatina , ADN/metabolismo , Regulación de la Expresión Génica/genética , Redes Reguladoras de Genes , Ratones , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores de TranscripciónRESUMEN
PBP (peroxisome-proliferator-activated receptor-binding protein) [Med1 (mediator 1)/TRAP220 (thyroid-hormone-receptor-associated protein 220)] is essential for mammary gland development. We established a mammary epithelial cell line with a genotype of PBPLoxP/LoxP by expressing an active form of Notch4. Null mutation of PBP caused severe growth inhibition of the Notch4-immortalized mammary cells. We found that truncated PBP without the two LXXLL motifs could reverse the growth inhibition due to the deficiency of endogenous PBP, indicating that signalling through nuclear receptors is unlikely to be responsible for the growth inhibition as the result of PBP deficiency. Loss of PBP expression was shown to completely ablate the expression of SOX10 [Sry-related HMG (high-mobility group) box gene 10]. The re-expression of SOX10 was capable of reversing the growth inhibition due to PBP deficiency, whereas suppressed expression of SOX10 inhibited the growth of Notch4-immortalized mammary cells. Further studies revealed PBP is directly recruited to the enhancer of the SOX10 gene, indicating that SOX10 is a direct target gene of PBP. We conclude that PBP is essential for the growth of Notch4-immortalized mammary cells by activating SOX10 expression, providing a potential molecular mechanism through which PBP regulates the growth of mammary stem/progenitor cells.
Asunto(s)
Proliferación Celular , Células Epiteliales/citología , Glándulas Mamarias Animales/citología , Subunidad 1 del Complejo Mediador/fisiología , Proteínas Proto-Oncogénicas , Receptores Notch , Factores de Transcripción SOXE/genética , Activación Transcripcional/fisiología , Animales , Línea Celular , Subunidad 1 del Complejo Mediador/deficiencia , Ratones , Receptor Notch4 , Células Madre/citologíaRESUMEN
Microglia are key in the homeostatic well-being of the brain and microglial dysfunction has been implicated in neurodegenerative disorders such as Alzheimer's disease (AD). Due to the many limitations to study microglia in situ or isolated for large scale drug discovery applications, there is a high need to develop robust and scalable human cellular models of microglia with reliable translatability to the disease. Here, we describe the generation of microglia-like cells from human induced pluripotent stem cells (iPSC) with distinct phenotypes for mechanistic studies in AD. We started out from an established differentiation protocol to generate primitive macrophage precursors mimicking the yolk sac ontogeny of microglia. Subsequently, we tested 36 differentiation conditions for the cells in monoculture where we exposed them to various combinations of media, morphogens, and extracellular matrices. The optimized protocol generated robustly ramified cells expressing key microglial markers. Bulk mRNA sequencing expression profiles revealed that compared to cells obtained in co-culture with neurons, microglia-like cells derived from a monoculture condition upregulate mRNA levels for Triggering Receptor Expressed On Myeloid Cells 2 (TREM2), which is reminiscent to the previously described disease-associated microglia. TREM2 is a risk gene for AD and an important regulator of microglia. The regulatory function of TREM2 in these cells was confirmed by comparing wild type with isogenic TREM2 knock-out iPSC microglia. The TREM2-deficient cells presented with stronger increase in free cytosolic calcium upon stimulation with ATP and ADP, as well as stronger migration towards complement C5a, compared to TREM2 expressing cells. The functional differences were associated with gene expression modulation of key regulators of microglia. In conclusion, we have established and validated a work stream to generate functional human iPSC-derived microglia-like cells by applying a directed and neuronal co-culture independent differentiation towards functional phenotypes in the context of AD. These cells can now be applied to study AD-related disease settings and to perform compound screening and testing for drug discovery.
Asunto(s)
Enfermedad de Alzheimer , Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes Inducidas/metabolismo , Glicoproteínas de Membrana/metabolismo , Microglía/metabolismo , Receptores Inmunológicos/metabolismo , Diferenciación Celular , Células Cultivadas , Predisposición Genética a la Enfermedad , Humanos , Glicoproteínas de Membrana/genética , Receptores Inmunológicos/genéticaRESUMEN
For most complex traits, the majority of SNPs identified through genome-wide association studies (GWAS) reside within noncoding regions that have no known function. However, these regions are enriched for the regulatory enhancers specific to the cells relevant to the specific trait. Indeed, many of the GWAS loci that have been functionally characterized lie within enhancers that regulate expression levels of key genes. In order to identify polymorphisms with potential allele-specific regulatory effects, we developed a bioinformatics pipeline that harnesses epigenetic signatures as well as transcription factor (TF) binding motifs to identify putative enhancers containing a SNP with potential allele-specific TF binding in linkage disequilibrium (LD) with a GWAS-identified SNP. We applied the approach to GWAS findings for blood lipids, revealing 7 putative enhancers harboring associated SNPs, 3 of which lie within the introns of LCAT and ABCA1, genes that play crucial roles in cholesterol biogenesis and lipoprotein metabolism. All 3 enhancers demonstrated allele-specific in vitro regulatory activity in liver-derived cell lines. We demonstrated that these putative enhancers are in close physical proximity to the promoters of their respective genes, in situ, likely through chromatin looping. In addition, the associated alleles altered the likelihood of transcription activator STAT3 binding. Our results demonstrate that through our approach, the LD blocks that contain GWAS signals, often hundreds of kilobases in size with multiple SNPs serving as statistical proxies to the true functional site, can provide an experimentally testable hypothesis for the underlying regulatory mechanism linking genetic variants to complex traits.
Asunto(s)
Transportador 1 de Casete de Unión a ATP/genética , Alelos , HDL-Colesterol/metabolismo , Elementos de Facilitación Genéticos , Fosfatidilcolina-Esterol O-Aciltransferasa/genética , Secuencia de Bases , Línea Celular , Cromatina/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Hígado/metabolismo , Polimorfismo de Nucleótido Simple/genética , Regiones Promotoras Genéticas , Unión Proteica , Elementos de Respuesta/genética , Factor de Transcripción STAT3/metabolismoRESUMEN
Rare genetic disorders are often challenging to diagnose. Anomalies of tooth number, shape, size, mineralized tissue structure, eruption, and resorption may exist as isolated symptoms or diseases but are often part of the clinical synopsis of numerous syndromes (Bloch-Zupan A, Sedano H, Scully C. Dento/oro/craniofacial anomalies and genetics, 1st edn. Elsevier, Boston, MA, 2012). Concerning amelogenesis imperfecta (AI), for example, mutations in a number of genes have been reported to cause isolated AI, including AMELX, ENAM, KLK4, MMP20, FAM83H, WDR72, C4orf26, SLC24A4, and LAMB3. In addition, many other genes such as DLX3, CNNM4, ROGDI, FAM20A, STIM1, ORAI1, and LTBP3 have been shown to be involved in developmental syndromes with enamel defects. The clinical presentation of the enamel phenotype (hypoplastic, hypomineralized, hypomature, or a combination of severities) alone does not allow a reliable prediction of possible causative genetic mutations. Understanding the potential genetic cause(s) of rare diseases is critical for overall health management of affected patient. One effective strategy to reach a genetic diagnosis is to sequence a selected gene panel chosen for a determined range of phenotypes. Here we describe a laboratory protocol to set up a specific gene panel for orodental diseases.
Asunto(s)
Anomalías Craneofaciales/genética , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Enfermedades Raras/genética , Anomalías Dentarias/genética , Amelogénesis Imperfecta/diagnóstico , Amelogénesis Imperfecta/genética , Anomalías Craneofaciales/diagnóstico , ADN/genética , Diseño de Equipo , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Humanos , Enfermedades Raras/diagnóstico , Anomalías Dentarias/diagnósticoRESUMEN
Amelogenesis imperfecta (AI) designates a group of genetic diseases characterized by a large range of enamel disorders causing important social and health problems. These defects can result from mutations in enamel matrix proteins or protease encoding genes. A range of mutations in the enamel cleavage enzyme matrix metalloproteinase-20 gene (MMP20) produce enamel defects of varying severity. To address how various alterations produce a range of AI phenotypes, we performed a targeted analysis to find MMP20 mutations in French patients diagnosed with non-syndromic AI. Genomic DNA was isolated from saliva and MMP20 exons and exon-intron boundaries sequenced. We identified several homozygous or heterozygous mutations, putatively involved in the AI phenotypes. To validate missense mutations and predict sensitive positions in the MMP20 sequence, we evolutionarily compared 75 sequences extracted from the public databases using the Datamonkey webserver. These sequences were representative of mammalian lineages, covering more than 150 million years of evolution. This analysis allowed us to find 324 sensitive positions (out of the 483 MMP20 residues), pinpoint functionally important domains, and build an evolutionary chart of important conserved MMP20 regions. This is an efficient tool to identify new- and previously-identified mutations. We thus identified six functional MMP20 mutations in unrelated families, finding two novel mutated sites. The genotypes and phenotypes of these six mutations are described and compared. To date, 13 MMP20 mutations causing AI have been reported, making these genotypes and associated hypomature enamel phenotypes the most frequent in AI.
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Células Madre Adultas/fisiología , Fibroblastos/citología , Células Madre Pluripotentes/citología , Factores de Transcripción/fisiología , Animales , Investigación Biomédica , Células Cultivadas , Células Madre Embrionarias , Proteínas F-Box/genética , Regulación de la Expresión Génica , Genes myc/fisiología , Humanos , Ratones , Factores de Transcripción/genéticaRESUMEN
Although variants in many genes have previously been shown to be associated with blood pressure (BP) levels, the molecular mechanism underlying these associations are mostly unknown. We identified a multi-allelic T-rich sequence (TRS) in the 3'UTR of ATP1B1 that varies in length and sequence composition (T22-27 and T12GT 3GT6). The 3'UTR of ATP1B1 contains 2 functional polyadenylation signals and the TRS is downstream of the proximal polyadenylation site (A2). Therefore, we hypothesized that alleles of this TRS might influence ATP1B1 expression by regulating alternative polyadenylation. In vitro, the T12GT 3GT6 allele increases polyadenylation at the A2 polyadenylation site as compared to the T23 allele. Consistent with our hypothesis, the relative abundance of the A2-polyadenylated ATP1B1 mRNA was higher in human kidneys with at least one copy of the T12GT 3GT6 allele than in those lacking this allele. The T12GT 3GT6 allele is also associated with higher systolic BP (beta = 3.3 mmHg, p = 0.014) and diastolic BP (beta = 2.4 mmHg, p = 0.003) in a European-American population. Therefore, we have identified a novel multi-allelic TRS in the 3'UTR of ATP1B1 that is associated with higher BP and may mediate its effect by regulating the polyadenylation of the ATP1B1 mRNA.
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Regiones no Traducidas 3' , Presión Sanguínea/genética , Poliadenilación , Polimorfismo Genético , ATPasa Intercambiadora de Sodio-Potasio/genética , Adolescente , Adulto , Alelos , Estudios de Asociación Genética , Humanos , Persona de Mediana Edad , Motivos de Nucleótidos , ARN Mensajero/genética , Secuencias Reguladoras de Ácidos Nucleicos , Adulto JovenRESUMEN
Expression profile analysis clusters Gpnmb with known pigment genes, Tyrp1, Dct, and Si. During development, Gpnmb is expressed in a pattern similar to Mitf, Dct and Si with expression vastly reduced in Mitf mutant animals. Unlike Dct and Si, Gpnmb remains expressed in a discrete population of caudal melanoblasts in Sox10-deficient embryos. To understand the transcriptional regulation of Gpnmb we performed a whole genome annotation of 2,460,048 consensus MITF binding sites, and cross-referenced this with evolutionarily conserved genomic sequences at the GPNMB locus. One conserved element, GPNMB-MCS3, contained two MITF consensus sites, significantly increased luciferase activity in melanocytes and was sufficient to drive expression in melanoblasts in vivo. Deletion of the 5'-most MITF consensus site dramatically reduced enhancer activity indicating a significant role for this site in Gpnmb transcriptional regulation. Future analysis of the Gpnmb locus will provide insight into the transcriptional regulation of melanocytes, and Gpnmb expression can be used as a marker for analyzing melanocyte development and disease progression.