RESUMEN
Sequence polymorphisms linked to human diseases and phenotypes in genome-wide association studies often affect noncoding regions. A SNP within an intron of the gene encoding Interferon Regulatory Factor 4 (IRF4), a transcription factor with no known role in melanocyte biology, is strongly associated with sensitivity of skin to sun exposure, freckles, blue eyes, and brown hair color. Here, we demonstrate that this SNP lies within an enhancer of IRF4 transcription in melanocytes. The allele associated with this pigmentation phenotype impairs binding of the TFAP2A transcription factor that, together with the melanocyte master regulator MITF, regulates activity of the enhancer. Assays in zebrafish and mice reveal that IRF4 cooperates with MITF to activate expression of Tyrosinase (TYR), an essential enzyme in melanin synthesis. Our findings provide a clear example of a noncoding polymorphism that affects a phenotype by modulating a developmental gene regulatory network.
Asunto(s)
Factores Reguladores del Interferón/metabolismo , Polimorfismo de Nucleótido Simple , Animales , Secuencia de Bases , Elementos de Facilitación Genéticos , Humanos , Factores Reguladores del Interferón/química , Factores Reguladores del Interferón/genética , Melanocitos/metabolismo , Ratones , Datos de Secuencia Molecular , Pigmentación , Transducción de Señal , Factor de Transcripción AP-2/química , Factor de Transcripción AP-2/metabolismo , Pez CebraRESUMEN
Oculocutaneous albinism (OCA) is a rare disorder of pigment production. Affected individuals have variably decreased global pigmentation and visual-developmental changes that lead to low vision. OCA is notable for significant missing heritability, particularly among individuals with residual pigmentation. Tyrosinase (TYR) is the rate-limiting enzyme in melanin pigment biosynthesis and mutations that decrease enzyme function are one of the most common causes of OCA. We present the analysis of high-depth short-read TYR sequencing data for a cohort of 352 OCA probands, â¼50% of whom were previously sequenced without yielding a definitive diagnostic result. Our analysis identified 66 TYR single-nucleotide variants (SNVs) and small insertion/deletions (indels), 3 structural variants, and a rare haplotype comprised of two common frequency variants (p.Ser192Tyr and p.Arg402Gln) in cis-orientation, present in 149/352 OCA probands. We further describe a detailed analysis of the disease-causing haplotype, p.[Ser192Tyr; Arg402Gln] ("cis-YQ"). Haplotype analysis suggests that the cis-YQ allele arose by recombination and that multiple cis-YQ haplotypes are segregating in OCA-affected individuals and control populations. The cis-YQ allele is the most common disease-causing allele in our cohort, representing 19.1% (57/298) of TYR pathogenic alleles in individuals with type 1 (TYR-associated) OCA. Finally, among the 66 TYR variants, we found several additional alleles defined by a cis-oriented combination of minor, potentially hypomorph-producing alleles at common variant sites plus a second, rare pathogenic variant. Together, these results suggest that identification of phased variants for the full TYR locus are required for an exhaustive assessment for potentially disease-causing alleles.
Asunto(s)
Albinismo Oculocutáneo , Humanos , Haplotipos/genética , Albinismo Oculocutáneo/genética , Albinismo Oculocutáneo/diagnóstico , Mutación , AlelosRESUMEN
Starting with the launch of the Human Genome Project three decades ago, and continuing after its completion in 2003, genomics has progressively come to have a central and catalytic role in basic and translational research. In addition, studies increasingly demonstrate how genomic information can be effectively used in clinical care. In the future, the anticipated advances in technology development, biological insights, and clinical applications (among others) will lead to more widespread integration of genomics into almost all areas of biomedical research, the adoption of genomics into mainstream medical and public-health practices, and an increasing relevance of genomics for everyday life. On behalf of the research community, the National Human Genome Research Institute recently completed a multi-year process of strategic engagement to identify future research priorities and opportunities in human genomics, with an emphasis on health applications. Here we describe the highest-priority elements envisioned for the cutting-edge of human genomics going forward-that is, at 'The Forefront of Genomics'.
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Investigación Biomédica/tendencias , Genoma Humano/genética , Genómica/tendencias , Salud Pública/normas , Investigación Biomédica Traslacional/tendencias , Investigación Biomédica/economía , COVID-19/genética , Genómica/economía , Humanos , National Human Genome Research Institute (U.S.)/economía , Cambio Social , Investigación Biomédica Traslacional/economía , Estados UnidosRESUMEN
The rare, fatal neurodegenerative disorder Niemann-Pick disease type C1 (NPC1) arises from lysosomal accumulation of unesterified cholesterol and glycosphingolipids. These subcellular pathologies lead to phenotypes of hepatosplenomegaly, neurological degeneration and premature death. The timing and severity of NPC1 clinical presentation is extremely heterogeneous. This study analyzed RNA-Seq data from 42 NPC1 patient-derived, primary fibroblast cell lines to determine transcriptional changes induced by treatment with 2-hydroxypropyl-ß-cyclodextrin (HPßCD), a compound currently under investigation in clinical trials. A total of 485 HPßCD-responsive genes were identified. Pathway enrichment analysis of these genes showed significant involvement in cholesterol and lipid biosynthesis. Furthermore, immunohistochemistry of the cerebellum as well as measurements of plasma from Npc1m1N null mice treated with HPßCD and adeno-associated virus gene therapy suggests that one of the identified genes, GPNMB, may serve as a useful biomarker of treatment response in NPC1 disease. Overall, this large NPC1 patient-derived dataset provides a comprehensive foundation for understanding the genomic response to HPßCD treatment.
Asunto(s)
Enfermedad de Niemann-Pick Tipo C , 2-Hidroxipropil-beta-Ciclodextrina , Animales , Biomarcadores , Modelos Animales de Enfermedad , Proteínas del Ojo/genética , Humanos , Glicoproteínas de Membrana/genética , Ratones , Ratones Noqueados , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/patología , TranscriptomaRESUMEN
Human skin and hair color are visible traits that can vary dramatically within and across ethnic populations. The genetic makeup of these traits-including polymorphisms in the enzymes and signaling proteins involved in melanogenesis, and the vital role of ion transport mechanisms operating during the maturation and distribution of the melanosome-has provided new insights into the regulation of pigmentation. A large number of novel loci involved in the process have been recently discovered through four large-scale genome-wide association studies in Europeans, two large genetic studies of skin color in Africans, one study in Latin Americans, and functional testing in animal models. The responsible polymorphisms within these pigmentation genes appear at different population frequencies, can be used as ancestry-informative markers, and provide insight into the evolutionary selective forces that have acted to create this human diversity.
Asunto(s)
Color del Ojo/genética , Color del Cabello/genética , Melaninas/biosíntesis , Trastornos de la Pigmentación/genética , Pigmentación/genética , Pigmentación de la Piel/genética , Ojo/metabolismo , Regulación de la Expresión Génica , Cabello/metabolismo , Humanos , Queratinocitos/metabolismo , Queratinocitos/patología , Melaninas/genética , Melanocitos/metabolismo , Melanocitos/patología , Oxigenasas de Función Mixta/genética , Oxigenasas de Función Mixta/metabolismo , Trastornos de la Pigmentación/fisiopatología , Grupos Raciales/genética , Piel/metabolismoRESUMEN
Group 3 innate lymphoid cells (ILC3) are major regulators of inflammation and infection at mucosal barriers. ILC3 development is thought to be programmed, but how ILC3 perceive, integrate and respond to local environmental signals remains unclear. Here we show that ILC3 in mice sense their environment and control gut defence as part of a glialILC3epithelial cell unit orchestrated by neurotrophic factors. We found that enteric ILC3 express the neuroregulatory receptor RET. ILC3-autonomous Ret ablation led to decreased innate interleukin-22 (IL-22), impaired epithelial reactivity, dysbiosis and increased susceptibility to bowel inflammation and infection. Neurotrophic factors directly controlled innate Il22 downstream of the p38 MAPK/ERK-AKT cascade and STAT3 activation. Notably, ILC3 were adjacent to neurotrophic-factor-expressing glial cells that exhibited stellate-shaped projections into ILC3 aggregates. Glial cells sensed microenvironmental cues in a MYD88-dependent manner to control neurotrophic factors and innate IL-22. Accordingly, glial-intrinsic Myd88 deletion led to impaired production of ILC3-derived IL-22 and a pronounced propensity towards gut inflammation and infection. Our work sheds light on a novel multi-tissue defence unit, revealing that glial cells are central hubs of neuron and innate immune regulation by neurotrophic factor signals.
Asunto(s)
Inmunidad Innata , Intestinos/inmunología , Linfocitos/inmunología , Neuroglía/metabolismo , Neurotransmisores/metabolismo , Animales , Microambiente Celular/inmunología , Células Epiteliales/citología , Células Epiteliales/inmunología , Femenino , Microbioma Gastrointestinal/inmunología , Inmunidad Mucosa , Inflamación/inmunología , Inflamación/metabolismo , Interleucinas/biosíntesis , Interleucinas/inmunología , Mucosa Intestinal/citología , Mucosa Intestinal/inmunología , Intestinos/citología , Linfocitos/citología , Linfocitos/metabolismo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Factor 88 de Diferenciación Mieloide/deficiencia , Factor 88 de Diferenciación Mieloide/metabolismo , Neuroglía/inmunología , Neurotransmisores/inmunología , Proteínas Proto-Oncogénicas c-akt/metabolismo , Proteínas Proto-Oncogénicas c-ret/deficiencia , Proteínas Proto-Oncogénicas c-ret/metabolismo , Factor de Transcripción STAT3/metabolismo , Interleucina-22RESUMEN
Oculocutaneous albinism (OCA) is a heritable disorder of pigment production that manifests as hypopigmentation and altered eye development. Exon sequencing of known OCA genes is unsuccessful in producing a complete molecular diagnosis for a significant number of affected individuals. We sequenced the DNA of individuals with OCA using short-read custom capture sequencing that targeted coding, intronic, and noncoding regulatory regions of known OCA genes, and genome-wide association study-associated pigmentation loci. We identified an OCA2 complex structural variant (CxSV), defined by a 143 kb inverted segment reintroduced in intron 1, upstream of the native location. The corresponding CxSV junctions were observed in 11/390 probands screened. The 143 kb CxSV presents in one family as a copy number variant duplication for the 143 kb region. In the remaining 10/11 families, the 143 kb CxSV acquired an additional 184 kb deletion across the same region, restoring exons 3-19 of OCA2 to a copy-number neutral state. Allele-associated haplotype analysis found rare SNVs rs374519281 and rs139696407 are linked with the 143 kb CxSV in both OCA2 alleles. For individuals in which customary molecular evaluation does not reveal a biallelic OCA diagnosis, we recommend preliminary screening for these haplotype-associated rare variants, followed by junction-specific validation for the OCA2 143 kb CxSV.
Asunto(s)
Albinismo Oculocutáneo , Estudio de Asociación del Genoma Completo , Albinismo Oculocutáneo/diagnóstico , Albinismo Oculocutáneo/genética , Alelos , Humanos , Proteínas de Transporte de Membrana/genética , MutaciónRESUMEN
Most expression quantitative trait locus (eQTL) studies to date have been performed in heterogeneous tissues as opposed to specific cell types. To better understand the cell-type-specific regulatory landscape of human melanocytes, which give rise to melanoma but account for <5% of typical human skin biopsies, we performed an eQTL analysis in primary melanocyte cultures from 106 newborn males. We identified 597,335 cis-eQTL SNPs prior to linkage disequilibrium (LD) pruning and 4997 eGenes (FDR < 0.05). Melanocyte eQTLs differed considerably from those identified in the 44 GTEx tissue types, including skin. Over a third of melanocyte eGenes, including key genes in melanin synthesis pathways, were unique to melanocytes compared to those of GTEx skin tissues or TCGA melanomas. The melanocyte data set also identified trans-eQTLs, including those connecting a pigmentation-associated functional SNP with four genes, likely through cis-regulation of IRF4 Melanocyte eQTLs are enriched in cis-regulatory signatures found in melanocytes as well as in melanoma-associated variants identified through genome-wide association studies. Melanocyte eQTLs also colocalized with melanoma GWAS variants in five known loci. Finally, a transcriptome-wide association study using melanocyte eQTLs uncovered four novel susceptibility loci, where imputed expression levels of five genes (ZFP90, HEBP1, MSC, CBWD1, and RP11-383H13.1) were associated with melanoma at genome-wide significant P-values. Our data highlight the utility of lineage-specific eQTL resources for annotating GWAS findings, and present a robust database for genomic research of melanoma risk and melanocyte biology.
Asunto(s)
Predisposición Genética a la Enfermedad , Melanocitos/metabolismo , Melanoma/genética , Sitios de Carácter Cuantitativo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Proteínas Portadoras/genética , Células Cultivadas , Proteínas de Unión al Hemo , Hemoproteínas/genética , Humanos , Factores Reguladores del Interferón/genética , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Proteínas RepresorasRESUMEN
Melanocyte stem cells (McSCs) and mouse models of hair graying serve as useful systems to uncover mechanisms involved in stem cell self-renewal and the maintenance of regenerating tissues. Interested in assessing genetic variants that influence McSC maintenance, we found previously that heterozygosity for the melanogenesis associated transcription factor, Mitf, exacerbates McSC differentiation and hair graying in mice that are predisposed for this phenotype. Based on transcriptome and molecular analyses of Mitfmi-vga9/+ mice, we report a novel role for MITF in the regulation of systemic innate immune gene expression. We also demonstrate that the viral mimic poly(I:C) is sufficient to expose genetic susceptibility to hair graying. These observations point to a critical suppressor of innate immunity, the consequences of innate immune dysregulation on pigmentation, both of which may have implications in the autoimmune, depigmenting disease, vitiligo.
Asunto(s)
Células Madre Adultas , Color del Cabello/inmunología , Inmunidad Innata , Melanocitos , Factor de Transcripción Asociado a Microftalmía/fisiología , Animales , Femenino , Regulación de la Expresión Génica , Predisposición Genética a la Enfermedad , Color del Cabello/genética , Interferón Tipo I/metabolismo , Ratones , Ratones Transgénicos , Poli I-CRESUMEN
Niemann-Pick disease, type C1 (NPC1) is a rare neurodegenerative lysosomal storage disease with a wide spectrum of clinical manifestation. Multiple genetic factors influence the NPC1 mouse phenotype, but very little attention has been given to prenatal environmental factors that might have long-term effects on the neuroinflammatory component of NPC1 pathology. Studies in other mouse models of cerebellar ataxia have shown that developmental exposures lead to Purkinje neuron degeneration later in life, suggesting that environmental exposures during development can impact cerebellar biology. Thus, we evaluated the potential effect of maternal immune activation (MIA) on disease progression in an Npc1 mouse model. The MIA paradigm used mimics viral infection using the toll like receptor 3 agonist polyinosinic-polycytidilic acid during gestation. Through phenotypic and pathologic tests, we measured motor and behavioral changes as well as cerebellar neuroinflammation and neurodegeneration. We observed a gender and genotype dependent effect of MIA on the cerebellum. While the effects of MIA have been previously shown to primarily affect male progeny, we observed increased sensitivity of female mutant progeny to prenatal exposure to treatment with polyinosinic-polycytidilic acid. Specifically, prenatal MIA resulted in female NPC1 mutant progeny with greater motor deficits and a corresponding decrease in cerebellar Purkinje neurons. Our data suggest that prenatal environmental exposures may be one factor contributing to the phenotypic variability observed in individuals with NPC1.
Asunto(s)
Intercambio Materno-Fetal/inmunología , Neuronas/patología , Enfermedad de Niemann-Pick Tipo C/genética , Enfermedad de Niemann-Pick Tipo C/inmunología , Animales , Modelos Animales de Enfermedad , Femenino , Masculino , Intercambio Materno-Fetal/efectos de los fármacos , Ratones , Ratones Endogámicos BALB C , Neuronas/inmunología , Poli I-C/administración & dosificación , Embarazo , Efectos Tardíos de la Exposición Prenatal/fisiopatología , Factores SexualesRESUMEN
Genes and pathways that allow cells to cope with oncogene-induced stress represent selective cancer therapeutic targets that remain largely undiscovered. In this study, we identify a RhoJ signaling pathway that is a selective therapeutic target for BRAF mutant cells. RhoJ deletion in BRAF mutant melanocytes modulates the expression of the pro-apoptotic protein BAD as well as genes involved in cellular metabolism, impairing nevus formation, cellular transformation, and metastasis. Short-term treatment of nascent melanoma tumors with PAK inhibitors that block RhoJ signaling halts the growth of BRAF mutant melanoma tumors in vivo and induces apoptosis in melanoma cells in vitro via a BAD-dependent mechanism. As up to 50% of BRAF mutant human melanomas express high levels of RhoJ, these studies nominate the RhoJ-BAD signaling network as a therapeutic vulnerability for fledgling BRAF mutant human tumors.
Asunto(s)
Melanoma/genética , Proteínas Proto-Oncogénicas B-raf/genética , Proteína Letal Asociada a bcl/biosíntesis , Quinasas p21 Activadas/genética , Proteínas de Unión al GTP rho/genética , Apoptosis/efectos de los fármacos , Apoptosis/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Inhibidores Enzimáticos/administración & dosificación , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Melanocitos/efectos de los fármacos , Melanocitos/patología , Melanoma/tratamiento farmacológico , Melanoma/patología , Mutación , Metástasis de la Neoplasia , Nevo/genética , Nevo/patología , Transducción de Señal/efectos de los fármacos , Proteína Letal Asociada a bcl/genética , Quinasas p21 Activadas/antagonistas & inhibidoresRESUMEN
Mutations in the gene encoding transcription factor TFAP2A result in pigmentation anomalies in model organisms and premature hair graying in humans. However, the pleiotropic functions of TFAP2A and its redundantly-acting paralogs have made the precise contribution of TFAP2-type activity to melanocyte differentiation unclear. Defining this contribution may help to explain why TFAP2A expression is reduced in advanced-stage melanoma compared to benign nevi. To identify genes with TFAP2A-dependent expression in melanocytes, we profile zebrafish tissue and mouse melanocytes deficient in Tfap2a, and find that expression of a small subset of genes underlying pigmentation phenotypes is TFAP2A-dependent, including Dct, Mc1r, Mlph, and Pmel. We then conduct TFAP2A ChIP-seq in mouse and human melanocytes and find that a much larger subset of pigmentation genes is associated with active regulatory elements bound by TFAP2A. These elements are also frequently bound by MITF, which is considered the "master regulator" of melanocyte development. For example, the promoter of TRPM1 is bound by both TFAP2A and MITF, and we show that the activity of a minimal TRPM1 promoter is lost upon deletion of the TFAP2A binding sites. However, the expression of Trpm1 is not TFAP2A-dependent, implying that additional TFAP2 paralogs function redundantly to drive melanocyte differentiation, which is consistent with previous results from zebrafish. Paralogs Tfap2a and Tfap2b are both expressed in mouse melanocytes, and we show that mouse embryos with Wnt1-Cre-mediated deletion of Tfap2a and Tfap2b in the neural crest almost completely lack melanocytes but retain neural crest-derived sensory ganglia. These results suggest that TFAP2 paralogs, like MITF, are also necessary for induction of the melanocyte lineage. Finally, we observe a genetic interaction between tfap2a and mitfa in zebrafish, but find that artificially elevating expression of tfap2a does not increase levels of melanin in mitfa hypomorphic or loss-of-function mutants. Collectively, these results show that TFAP2 paralogs, operating alongside lineage-specific transcription factors such as MITF, directly regulate effectors of terminal differentiation in melanocytes. In addition, they suggest that TFAP2A activity, like MITF activity, has the potential to modulate the phenotype of melanoma cells.
Asunto(s)
Diferenciación Celular/genética , Melanocitos/metabolismo , Factor de Transcripción Asociado a Microftalmía/genética , Factor de Transcripción AP-2/genética , Animales , Secuencia de Bases , Sitios de Unión/genética , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones Noqueados , Factor de Transcripción Asociado a Microftalmía/metabolismo , Microscopía Confocal , Mutación , Pigmentación/genética , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Homología de Secuencia de Ácido Nucleico , Factor de Transcripción AP-2/metabolismo , Pez Cebra , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Niemann-Pick disease, type C1 (NPC1) is a heritable lysosomal storage disease characterized by a progressive neurological degeneration that causes disability and premature death. A murine model of NPC1 disease (Npc1-/-) displays a rapidly progressing form of NPC1 disease which is characterized by weight loss, ataxia, increased cholesterol storage, loss of cerebellar Purkinje neurons and early lethality. To test the potential efficacy of gene therapy for NPC1, we constructed adeno-associated virus serotype 9 (AAV9) vectors to deliver the NPC1 gene under the transcriptional control of the neuronal-specific (CamKII) or a ubiquitous (EF1a) promoter. The Npc1-/- mice that received a single dose of AAV9-CamKII-NPC1 as neonates (2.6 × 1011GC) or at weaning (1.3 × 1012GC), and the mice that received a single dose of AAV9-EF1a-NPC1 at weaning (1.2 × 1012GC), exhibited an increased life span, characterized by delayed weight loss and diminished motor decline. Cholesterol storage and Purkinje neuron loss were also reduced in the central nervous system of AAV9 treated Npc1-/- mice. Treatment with AAV9-EF1a-NPC1, as compared to AAV9-CamKII-NPC1, resulted in significantly increased survival (mean survival increased from 69 days to 166 and 97 days, respectively) and growth, and reduced hepatic-cholesterol accumulation. Our results provide the first demonstration that gene therapy may represent a therapeutic option for NPC1 patients and suggest that extraneuronal NPC1 expression can further augment the lifespan of the Npc1-/- mice after systemic AAV gene delivery.
Asunto(s)
Dependovirus/genética , Terapia Genética , Vectores Genéticos/administración & dosificación , Longevidad/genética , Enfermedad de Niemann-Pick Tipo C/terapia , Proteínas/genética , Animales , Cerebelo/metabolismo , Cerebelo/patología , Colesterol/metabolismo , Modelos Animales de Enfermedad , Femenino , Péptidos y Proteínas de Señalización Intracelular , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Neuronas/metabolismo , Neuronas/patología , Proteína Niemann-Pick C1 , Enfermedad de Niemann-Pick Tipo C/genética , Células de Purkinje/metabolismo , Células de Purkinje/patologíaRESUMEN
BACKGROUND: Association studies in recently admixed populations are extremely useful to identify the genetic architecture of pigmentation, due to their high genotypic and phenotypic variation. However, to date only four Genome-Wide Association Studies (GWAS) have been carried out in these populations. RESULTS: We present a GWAS of skin pigmentation in an admixed sample from Cuba (N = 762). Additionally, we conducted a meta-analysis including the Cuban sample, and admixed samples from Cape Verde, Puerto Rico and African-Americans from San Francisco. This meta-analysis is one of the largest efforts so far to characterize the genetic basis of skin pigmentation in admixed populations (N = 2,104). We identified five genome-wide significant regions in the meta-analysis, and explored if the markers observed in these regions are associated with the expression of relevant pigmentary genes in human melanocyte cultures. In three of the regions identified in the meta-analysis (SLC24A5, SLC45A2, and GRM5/TYR), the association seems to be driven by non-synonymous variants (rs1426654, rs16891982, and rs1042602, respectively). The rs16891982 polymorphism is strongly associated with the expression of the SLC45A2 gene. In the GRM5/TYR region, in addition to the rs1042602 non-synonymous SNP located on the TYR gene, variants located in the nearby GRM5 gene have an independent effect on pigmentation, possibly through regulation of gene expression of the TYR gene. We also replicated an association recently described near the MFSD12 gene on chromosome 19 (lead variant rs112332856). Additionally, our analyses support the presence of multiple signals in the OCA2/HERC2/APBA2 region on chromosome 15. A clear causal candidate is the HERC2 intronic variant rs12913832, which has a profound influence on OCA2 expression. This variant has pleiotropic effects on eye, hair, and skin pigmentation. However, conditional and haplotype-based analyses indicate the presence of other variants with independent effects on melanin levels in OCA2 and APBA2. Finally, a follow-up of genome-wide signals identified in a recent GWAS for tanning response indicates that there is a substantial overlap in the genetic factors influencing skin pigmentation and tanning response. CONCLUSIONS: Our meta-analysis of skin pigmentation GWAS in recently admixed populations provides new insights about the genetic architecture of this complex trait.
Asunto(s)
Genética de Población , Estudio de Asociación del Genoma Completo , Pigmentación de la Piel/genética , Alelos , Genotipo , Humanos , Desequilibrio de Ligamiento , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.
Asunto(s)
Diferenciación Celular , ADN Helicasas/metabolismo , Melanocitos/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción SOXE/metabolismo , Factores de Transcripción/metabolismo , Animales , Línea Celular , Elementos de Facilitación Genéticos , Expresión Génica , Regulación de la Expresión Génica , Melaninas/biosíntesis , Glicoproteínas de Membrana/genética , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Transgénicos , Oxidorreductasas/genéticaRESUMEN
BACKGROUND: Niemann-Pick disease, type C1 (NPC1) is a lysosomal storage disorder characterised by progressive neurodegeneration. In preclinical testing, 2-hydroxypropyl-ß-cyclodextrins (HPßCD) significantly delayed cerebellar Purkinje cell loss, slowed progression of neurological manifestations, and increased lifespan in mouse and cat models of NPC1. The aim of this study was to assess the safety and efficacy of lumbar intrathecal HPßCD. METHODS: In this open-label, dose-escalation phase 1-2a study, we gave monthly intrathecal HPßCD to participants with NPC1 with neurological manifestation at the National Institutes of Health (NIH), Bethesda, MD, USA. To explore the potential effect of 2-week dosing, three additional participants were enrolled in a parallel study at Rush University Medical Center (RUMC), Chicago, IL, USA. Participants from the NIH were non-randomly, sequentially assigned in cohorts of three to receive monthly initial intrathecal HPßCD at doses of 50, 200, 300, or 400 mg per month. A fifth cohort of two participants received initial doses of 900 mg. Participants from RUMC initially received 200 or 400 mg every 2 weeks. The dose was escalated based on tolerance or safety data from higher dose cohorts. Serum and CSF 24(S)-hydroxycholesterol (24[S]-HC), which serves as a biomarker of target engagement, and CSF protein biomarkers were evaluated. NPC Neurological Severity Scores (NNSS) were used to compare disease progression in HPßCD-treated participants relative to a historical comparison cohort of 21 NPC1 participants of similar age range. FINDINGS: Between Sept 21, 2013, and Jan 19, 2015, 32 participants with NPC1 were assessed for eligibility at the National Institutes of Health. 18 patients were excluded due to inclusion criteria not met (six patients), declined to participate (three patients), pursued independent expanded access and obtained the drug outside of the study (three patients), enrolled in the RUMC cohort (one patient), or too late for the trial enrolment (five patients). 14 patients were enrolled and sequentially assigned to receive intrathecal HPßCD at a starting dose of 50 mg per month (three patients), 200 mg per month (three patients), 300 mg per month (three patients), 400 mg per month (three patients), or 900 mg per month (two patients). During the first year, two patients had treatment interrupted for one dose, based on grade 1 ototoxicity. All 14 patients were assessed at 12 months. Between 12 and 18 months, one participant had treatment interrupted at 17 months due to hepatocellular carcinoma, one patient had dose interruption for 2 doses based on caregiver hardship and one patient had treatment interrupted for 1 dose for mastoiditis. 11 patients were assessed at 18 months. Between Dec 11, 2013, and June 25, 2014, three participants were assessed for eligibility and enrolled at RUMC, and were assigned to receive intrathecal HPßCD at a starting dose of 200 mg every 2 weeks (two patients), or 400 mg every two weeks (one patient). There were no dropouts in this group and all 3 patients were assessed at 18 months. Biomarker studies were consistent with improved neuronal cholesterol homoeostasis and decreased neuronal pathology. Post-drug plasma 24(S)-HC area under the curve (AUC8-72) values, an indicator of neuronal cholesterol homoeostasis, were significantly higher than post-saline plasma 24(S)-HC AUC8-72 after doses of 900 mg (p=0·0063) and 1200 mg (p=0·0037). CSF 24(S)-HC concentrations in three participants given either 600 or 900 mg of HPßCD were increased about two fold (p=0·0032) after drug administration. No drug-related serious adverse events were observed. Mid-frequency to high-frequency hearing loss, an expected adverse event, was documented in all participants. When managed with hearing aids, this did not have an appreciable effect on daily communication. The NNSS for the 14 participants treated monthly increased at a rate of 1·22, SEM 0·34 points per year compared with 2·92, SEM 0·27 points per year (p=0·0002) for the 21 patient comparison group. Decreased progression was observed for NNSS domains of ambulation (p=0·0622), cognition (p=0·0040) and speech (p=0·0423). INTERPRETATION: Patients with NPC1 treated with intrathecal HPßCD had slowed disease progression with an acceptable safety profile. These data support the initiation of a multinational, randomised, controlled trial of intrathecal HPßCD. FUNDING: National Institutes of Health, Dana's Angels Research Trust, Ara Parseghian Medical Research Foundation, Hope for Haley, Samantha's Search for the Cure Foundation, National Niemann-Pick Disease Foundation, Support of Accelerated Research for NPC Disease, Vtesse, Janssen Research and Development, a Johnson & Johnson company, and Johnson & Johnson.
Asunto(s)
2-Hidroxipropil-beta-Ciclodextrina/administración & dosificación , Progresión de la Enfermedad , Enfermedad de Niemann-Pick Tipo C/tratamiento farmacológico , 2-Hidroxipropil-beta-Ciclodextrina/efectos adversos , Adolescente , Biomarcadores/sangre , Biomarcadores/líquido cefalorraquídeo , Calbindinas/líquido cefalorraquídeo , Niño , Preescolar , Relación Dosis-Respuesta a Droga , Proteína 3 de Unión a Ácidos Grasos/líquido cefalorraquídeo , Femenino , Pérdida Auditiva de Alta Frecuencia/inducido químicamente , Humanos , Hidroxicolesteroles/sangre , Hidroxicolesteroles/líquido cefalorraquídeo , Inyecciones Espinales , Masculino , Enfermedad de Niemann-Pick Tipo C/sangre , Enfermedad de Niemann-Pick Tipo C/líquido cefalorraquídeo , Enfermedades Raras/tratamiento farmacológico , Adulto JovenRESUMEN
SOX10 is required for melanocyte development and maintenance, and has been linked to melanoma initiation and progression. However, the molecular mechanisms by which SOX10 guides the appropriate gene expression programs necessary to promote the melanocyte lineage are not fully understood. Here we employ genetic and epigenomic analysis approaches to uncover novel genomic targets and previously unappreciated molecular roles of SOX10 in melanocytes. Through global analysis of SOX10-binding sites and epigenetic characteristics of chromatin states, we uncover an extensive catalog of SOX10 targets genome-wide. Our findings reveal that SOX10 predominantly engages 'open' chromatin regions and binds to distal regulatory elements, including novel and previously known melanocyte enhancers. Integrated chromatin occupancy and transcriptome analysis suggest a role for SOX10 in both transcriptional activation and repression to regulate functionally distinct classes of genes. We demonstrate that distinct epigenetic signatures and cis-regulatory sequence motifs predicted to bind putative co-regulatory transcription factors define SOX10-activated and SOX10-repressed target genes. Collectively, these findings uncover a central role of SOX10 as a global regulator of gene expression in the melanocyte lineage by targeting diverse regulatory pathways.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Redes Reguladoras de Genes , Melanocitos/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Factores de Transcripción SOXE/metabolismo , Animales , Sitios de Unión , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Epigenómica/métodos , Melanocitos/citología , Ratones , Factores de Transcripción SOXE/química , Factores de Transcripción SOXE/genéticaRESUMEN
Melanocytes, the pigment-producing cells, arise from multipotent neural crest (NC) cells during embryogenesis. Many genes required for melanocyte development were identified using mouse pigmentation mutants. The variable spotting mouse pigmentation mutant arose spontaneously at the Jackson Laboratory. We identified a G-to-A nucleotide transition in exon 3 of the Ets1 gene in variable spotting, which results in a missense G102E mutation. Homozygous variable spotting mice exhibit sporadic white spotting. Similarly, mice carrying a targeted deletion of Ets1 exhibit hypopigmentation; nevertheless, the function of Ets1 in melanocyte development is unknown. The transcription factor Ets1 is widely expressed in developing organs and tissues, including the NC. In the chick, Ets1 is required for the expression of Sox10, a transcription factor critical for the development of various NC derivatives, including melanocytes. We show that Ets1 is required early for murine NC cell and melanocyte precursor survival in vivo. Given the importance of Ets1 for Sox10 expression in the chick, we investigated a potential genetic interaction between these genes by comparing the hypopigmentation phenotypes of single and double heterozygous mice. The incidence of hypopigmentation in double heterozygotes was significantly greater than in single heterozygotes. The area of hypopigmentation in double heterozygotes was significantly larger than would be expected from the addition of the areas of hypopigmentation of single heterozygotes, suggesting that Ets1 and Sox10 interact synergistically in melanocyte development. Since Sox10 is also essential for enteric ganglia development, we examined the distal colons of Ets1 null mutants and found a significant decrease in enteric innervation, which was exacerbated by Sox10 heterozygosity. At the molecular level, Ets1 was found to activate an enhancer critical for Sox10 expression in NC-derived structures. Furthermore, enhancer activation was significantly inhibited by the variable spotting mutation. Together, these results suggest that Ets1 and Sox10 interact to promote proper melanocyte and enteric ganglia development from the NC.
Asunto(s)
Melanocitos/citología , Melanocitos/metabolismo , Proteína Proto-Oncogénica c-ets-1/metabolismo , Factores de Transcripción SOXE/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Tipificación del Cuerpo , Recuento de Células , Línea Celular Tumoral , Linaje de la Célula , Supervivencia Celular , Embrión de Mamíferos/metabolismo , Elementos de Facilitación Genéticos/genética , Epigénesis Genética , Ganglios/embriología , Ganglios/metabolismo , Ratones Endogámicos C57BL , Ratones Mutantes , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación Missense/genética , Cresta Neural/citología , Unión Proteica , Proteína Proto-Oncogénica c-ets-1/química , Proteína Proto-Oncogénica c-ets-1/genética , Activación Transcripcional/genéticaRESUMEN
During embryogenesis, the transcription factor, Sox10, drives the survival and differentiation of the melanocyte lineage. However, the role that Sox10 plays in postnatal melanocytes is not established. We show in vivo that melanocyte stem cells (McSCs) and more differentiated melanocytes express SOX10 but that McSCs remain undifferentiated. Sox10 knockout (Sox10(fl); Tg(Tyr::CreER)) results in loss of both McSCs and differentiated melanocytes, while overexpression of Sox10 (Tg(DctSox10)) causes premature differentiation and loss of McSCs, leading to hair graying. This suggests that levels of SOX10 are key to normal McSC function and Sox10 must be downregulated for McSC establishment and maintenance. We examined whether the mechanism of Tg(DctSox10) hair graying is through increased expression of Mitf, a target of SOX10, by asking if haploinsufficiency for Mitf (Mitf(vga9) ) can rescue hair graying in Tg(DctSox10) animals. Surprisingly, Mitf(vga9) does not mitigate but exacerbates Tg(DctSox10) hair graying suggesting that MITF participates in the negative regulation of Sox10 in McSCs. These observations demonstrate that while SOX10 is necessary to maintain the postnatal melanocyte lineage it is simultaneously prevented from driving differentiation in the McSCs. This data illustrates how tissue-specific stem cells can arise from lineage-specified precursors through the regulation of the very transcription factors important in defining that lineage.
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Desarrollo Embrionario/genética , Melanocitos/citología , Factores de Transcripción SOXE/genética , Células Madre/citología , Animales , Diferenciación Celular/genética , Linaje de la Célula , Color del Cabello/genética , Melanocitos/metabolismo , Ratones , Ratones Noqueados , Factores de Transcripción SOXE/metabolismo , Células Madre/metabolismoRESUMEN
The ribosome is an evolutionarily conserved organelle essential for cellular function. Ribosome construction requires assembly of approximately 80 different ribosomal proteins (RPs) and four different species of rRNA. As RPs co-assemble into one multi-subunit complex, mutation of the genes that encode RPs might be expected to give rise to phenocopies, in which the same phenotype is associated with loss-of-function of each individual gene. However, a more complex picture is emerging in which, in addition to a group of shared phenotypes, diverse RP gene-specific phenotypes are observed. Here we report the first two mouse mutations (Rps7(Mtu) and Rps7(Zma)) of ribosomal protein S7 (Rps7), a gene that has been implicated in Diamond-Blackfan anemia. Rps7 disruption results in decreased body size, abnormal skeletal morphology, mid-ventral white spotting, and eye malformations. These phenotypes are reported in other murine RP mutants and, as demonstrated for some other RP mutations, are ameliorated by Trp53 deficiency. Interestingly, Rps7 mutants have additional overt malformations of the developing central nervous system and deficits in working memory, phenotypes that are not reported in murine or human RP gene mutants. Conversely, Rps7 mouse mutants show no anemia or hyperpigmentation, phenotypes associated with mutation of human RPS7 and other murine RPs, respectively. We provide two novel RP mouse models and expand the repertoire of potential phenotypes that should be examined in RP mutants to further explore the concept of RP gene-specific phenotypes.