RESUMEN
The discovery of rare genetic variants is accelerating, and clear guidelines for distinguishing disease-causing sequence variants from the many potentially functional variants present in any human genome are urgently needed. Without rigorous standards we risk an acceleration of false-positive reports of causality, which would impede the translation of genomic research findings into the clinical diagnostic setting and hinder biological understanding of disease. Here we discuss the key challenges of assessing sequence variants in human disease, integrating both gene-level and variant-level support for causality. We propose guidelines for summarizing confidence in variant pathogenicity and highlight several areas that require further resource development.
Asunto(s)
Enfermedad , Predisposición Genética a la Enfermedad/genética , Variación Genética/genética , Guías como Asunto , Reacciones Falso Positivas , Genes/genética , Humanos , Difusión de la Información , Edición , Reproducibilidad de los Resultados , Proyectos de Investigación , Investigación Biomédica Traslacional/normasRESUMEN
Functional genomic quantities such as histone modifications, chromatin accessibility, and evolutionary constraint can now be measured in a nearly continuous fashion across the genome. The genome is highly heterogeneous, and the relationships between different functional annotations may be fluid. Here we present an approach for visualizing, quantifying, and determining the statistical significance of local and regional correlations between high-density continuous genomic datasets. We use wavelets to generate a multi-scale view of each component data set and calculate correlations between data types as a function of genome position over a continuous range of scales in sliding window fashion. We determine the statistical significance of correlations using a non-parametric sampling approach. We apply the wavelet correlation method to histone modification and chromatin accessibility (DNasel sensitivity) data from the NHGRI ENCODE project. We show that DNaseI sensitivity is broadly correlated (though to differing degrees) with a number of different activating histone modifications. We examine the continuous relationship between the repressive histone modification H3K27me3 and the activating mark H3K4me2, and find these modifications to display significant duality, with both significant positively and negatively correlated genomic territories. While the former appear to recapitulate in definitive cells the so-called "bi-valent" pattern originally proposed as a signature of pluripotency, the presence of negatively correlated regions suggests that the regulatory events that underlie the observed modification patterns are complex and highly regionalized in the genome.
Asunto(s)
Genómica/estadística & datos numéricos , Animales , Cromatina/genética , Cromatina/metabolismo , Biología Computacional , Interpretación Estadística de Datos , Bases de Datos Genéticas , Desoxirribonucleasa I , Histonas/genética , Histonas/metabolismo , Humanos , Metilación , RatonesRESUMEN
Integration position-independent expression of human globin transgenes in transgenic mice requires the presence of regulatory elements from the beta-globin locus control region (LCR) in the transgene construct. However, several recent studies have suggested that, while clearly necessary, such elements are not by themselves sufficient to realize this effect. In the case of the human fetal gamma-globin genes, previous results have indicated that additional regulatory information required for sheltering of gamma-globin transgene expression from position effects may reside downstream from the A gamma gene. To investigate this possibility, we established 17 lines of transgenic mice carrying constructs comprising a micro-LCR (microLCR) element, an A gamma-globin gene fragment, and a variable length of 3' sequence information beyond the A gamma 3' HindIII site. gamma-Globin expression during development was studied in 170 individual F2 progeny from these lines. We find that gamma-globin expression becomes sheltered from position effects when the normally position-sensitive microLCR-A gamma construct is extended by 600 bp beyond the 3' HindIII site to include a previously identified regulatory sequence (the A gamma-globin enhancer), the functional significance of which in vivo had heretofore been unclear. The results suggest that the mechanism whereby an upstream LCR achieves sheltering of globin gene expression from position effects involves cooperation with a gene-proximal regulatory element distinct from the promoter region.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/genética , Globinas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Dosificación de Gen , Humanos , Ratones , Ratones Transgénicos , ARN Mensajero/análisis , Transgenes/genéticaRESUMEN
The beta-like globin genes require the upstream locus control region (LCR) for proper expression. The active elements of the LCR coincide with strong erythroid-specific DNase I-hypersensitive sites (HSs). We have used 5' HS4 as a model to study the formation of these HSs. Previously, we identified a 101 bp element that is required for the formation of this HS. This element binds six proteins in vitro. We now report a mutational analysis of the HS4 HS-forming element (HSFE). This analysis indicates that binding sites for the hematopoietic transcription factors NF-E2 and GATA-1 are required for the formation of the characteristic chromatin structure of the HS following stable transfection into murine erythroleukemia cells. Similarly arranged NF-E2 and GATA binding sites are present in the other HSs of the human LCR, as well as in the homologous mouse and goat sequences and the chicken beta-globin enhancer. A combination of DNase I and micrococcal nuclease sensitivity assays indicates that the characteristic erythroid-specific hypersensitivity of HS4 to DNase I is the result of tissue-specific alterations in both nucleosome positioning and tertiary DNA structure.
Asunto(s)
Cromatina/metabolismo , Proteínas de Unión al ADN/metabolismo , Globinas/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Factores de Transcripción/metabolismo , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Desoxirribonucleasa I/metabolismo , Factores de Unión al ADN Específico de las Células Eritroides , Factor de Transcripción GATA1 , Regulación de la Expresión Génica , Hematopoyesis/genética , Humanos , Leucemia Eritroblástica Aguda , Ratones , Modelos Genéticos , Datos de Secuencia Molecular , Factor de Transcripción NF-E2 , Subunidad p45 del Factor de Transcripción NF-E2 , Nucleosomas/metabolismo , Unión Proteica , Homología de Secuencia de Ácido Nucleico , Células Tumorales CultivadasRESUMEN
We have analyzed the expression of human gamma-globin genes during development in F2 progeny of transgenic mice carrying two types of constructs. In the first type, gamma-globin genes were linked individually to large (approximately 4-kb) sequence fragments spanning locus control region (LCR) hypersensitive site 2 (HS2) or HS3. These LCR fragments contained not only the core HS elements but also extensive evolutionarily conserved flanking sequences. The second type of construct contained tandem gamma- and beta-globin genes linked to identical HS2 or HS3 fragments. We show that gamma-globin expression in transgenic mice carrying HS2 gamma or HS3 gamma constructs is highly sensitive to position effects and that such effects override the cis regulatory elements present in these constructs to produce markedly different developmental patterns of gamma-globin expression in lines carrying the same transgene. In contrast, gamma-globin expression in both HS2 gamma beta and HS3 gamma beta mice is sheltered from position effects and the developmental patterns of gamma-globin expression in lines carrying the same transgene are identical and display stage-specific regulation. The results suggest that cis regulatory sequences required for proper developmental control of fetal globin expression in the presence of an LCR element reside downstream from the gamma genes.
Asunto(s)
Globinas/genética , Secuencias Reguladoras de Ácidos Nucleicos , Factores de Edad , Animales , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Transgénicos , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
Strategies for the treatment of sickle cell anemia and beta-thalassemia are founded on the knowledge that these disorders result from structural or functional defects in an adult gene for which an intact fetal counterpart exists. During the past decade, several pharmacologic agents have been investigated for their potential to ameliorate sickle cell anemia and beta-thalassemia by increasing the synthesis of fetal hemoglobin in adults. Progress in understanding globin gene regulation is now being combined with advances in retrovirus-mediated gene transfer, and the once-distant goal of providing gene therapy for hemoglobinopathies is rapidly approaching reality.
Asunto(s)
Anemia de Células Falciformes/terapia , Terapia Genética , Talasemia beta/terapia , Adulto , Anemia de Células Falciformes/genética , Anemia de Células Falciformes/metabolismo , Hemoglobina Fetal/biosíntesis , Hemoglobina Fetal/genética , Vectores Genéticos , Hemoglobinas/genética , Humanos , Plásmidos , Retroviridae/genética , Proteínas de los Retroviridae/uso terapéutico , Transfección , Talasemia beta/genética , Talasemia beta/metabolismoRESUMEN
The past decade has witnessed profound increases in knowledge of the structure, function, and developmental regulation of the human globin genes. This information has deepened our understanding of the molecular and cellular mechanisms underlying inherited disorders affecting hemoglobin, and it has provided a new perspective for attaining meaningful increases in fetal hemoglobin synthesis in the management of sickle cell anemia and beta thalassemia. Efforts to provide therapy for these disorders are based on three factors: an understanding of their pathophysiology; the potential for fetal hemoglobin to alter its manifestation; and the concept that developmental changes in globin gene expression might be reversed by manipulating cellular and molecular regulatory mechanisms. In this review we discuss these topics and examine critically recent efforts to apply various pharmacological agents to in vitro, animal, and human models with the goal of increasing HbF synthesis. Several agents have demonstrated activity in patients with hemoglobin disorders. One such agent, hydroxyurea, has been shown to be potentially efficacious in phase II clinical trials in patients with sickle cell anemia and awaits testing in a placebo-controlled phase III study.