RESUMO
Genome-wide association studies (GWASs) have successfully identified numerous non-coding genetic variants for type 2 diabetes (T2D), but the functional roles underlying these non-coding variants remain largely unknown. The effects of T2D GWAS lead SNPs on transcriptional factors binding motifs were firstly analyzed via JASPAR, followed by functional validations including dual-luciferase reporter assays, biotin-based DNA pull-down assays, real-time quantitative PCR, and western blotting. The results showed that GWAS SNP rs4430796 conferred T allele specific transcriptional enhancer activity via a PAX6 binding element, and upregulated the expression of HNF1B. GWAS SNP rs4607103 showed a bidirectional modulation of ADAMTS9-AS2 and ADAMTS9 by TCF7L2 in a T allele-specific manner. GWAS SNP rs849135 conferred C allele-specific bidirectional transcriptional enhancer activity via a CREB1 binding element. Our findings have uncovered the functional mechanisms of three T2D GWAS SNPs via affecting the binding of transcription factors, providing new insights into the genetics and molecular pathogenesis of T2D.
Assuntos
Diabetes Mellitus Tipo 2/genética , Polimorfismo de Nucleotídeo Único , Proteína ADAMTS9/genética , Proteína ADAMTS9/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Predisposição Genética para Doença , Estudo de Associação Genômica Ampla , Células HEK293 , Fator 1-beta Nuclear de Hepatócito/genética , Fator 1-beta Nuclear de Hepatócito/metabolismo , Humanos , Fator de Transcrição PAX6/genética , Fator de Transcrição PAX6/metabolismo , Ligação Proteica , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
OBJECTIVES: Genome-wide association studies (GWASs) have discovered associations of numerous SNPs and genes with obesity. However, the underlying molecular mechanisms through which these SNPs and genes affect the predisposition to obesity remain not fully understood. Aims of our study are to comprehensively characterize obesity GWAS SNPs and genes through computational approaches. METHODS: For obesity GWAS identified SNPs, functional annotation, effects on miRNAs binding and impact on protein phosphorylation were performed via RegulomeDB and 3DSNP, miRNASNP, and the PhosSNP 1.0 database, respectively. For obesity associated genes, protein-protein interaction network construction, gene ontology and pathway enrichment analyses were performed by STRING, PANTHER and STRING, respectively. RESULTS: A total of 445 SNPs are significantly associated with obesity related phenotypes at threshold P < 5×10-8. A number of SNPs were eQTLs for obesity associated genes, some SNPs located at binding sites of obesity related transcription factors. SNPs that might affect miRNAs binding and protein phosphorylation were identified. Protein-protein interaction network analysis identified the highly-interconnected "hub" genes. Obesity associated genes mainly involved in metabolic process and catalytic activity, and significantly enriched in 15 signal pathways. CONCLUSIONS: Our results provided the targets for follow-up experimental testing and further shed new light on obesity pathophysiology.
Assuntos
Estudo de Associação Genômica Ampla , Genômica , Obesidade/genética , Ontologia Genética , Predisposição Genética para Doença/genética , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/metabolismo , Fosforilação/genética , Polimorfismo de Nucleotídeo Único , Mapas de Interação de Proteínas/genéticaRESUMO
BACKGROUND: Genome-wide association studies (GWAS) of type 2 diabetes (T2D) have discovered a number of loci that contribute to susceptibility to the disease. Future challenges include elucidation of functional mechanisms through which these GWAS-identified loci modulate T2D disease risk. The aim of the present study was to comprehensively characterize T2D associated single nucleotide polymorphisms (SNPs) and genes through computational approaches. METHODS: Computational biology approaches used in the present study included comparative genomic analyses and functional annotation using GWAS3D and RegulomeDB, investigation of the effects of T2D-associated SNPs on miRNA binding and protein phosphorylation, and gene ontology, pathway enrichment, protein-protein interaction (PPI) networks and functional module analysis of T2D-associated genes from previously published GWAS. RESULTS: Computational analysis identified a number of T2D GWAS-associated SNPs that were located at protein binding sites, including CCCTC-binding factor (CTCF), E1A binding protein p300 (EP300), hepatocyte nuclear factor 4alpha (HNF4A), transcription factor 7 like 2 (TCF7L2), forkhead box A1 (FOXA1) and A2 (FOXA2), and potentially affected the binding of miRNAs and protein phosphorylation. Pathway enrichment analysis confirmed two well-known maturity onset diabetes of the young and T2D pathways, whereas PPI network analysis identified highly interconnected "hub" genes, such as TCF7L2, melatonin receptor 1B (MTNR1B), and solute carrier family 30 (zinc transporter), member 8 (SLC30A8), that created two tight subnetworks. CONCLUSIONS: The results provide objectives and clues for future experimental studies and further insights into the molecular pathogenesis of T2D.
Assuntos
Biologia Computacional/métodos , Diabetes Mellitus Tipo 2/genética , Predisposição Genética para Doença/genética , Estudo de Associação Genômica Ampla/métodos , Fator de Ligação a CCCTC , Proteína p300 Associada a E1A/genética , Proteína p300 Associada a E1A/metabolismo , Fator 3-alfa Nuclear de Hepatócito/genética , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Fator 3-beta Nuclear de Hepatócito/genética , Fator 3-beta Nuclear de Hepatócito/metabolismo , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Humanos , MicroRNAs/genética , MicroRNAs/metabolismo , Modelos Genéticos , Fosforilação , Polimorfismo de Nucleotídeo Único , Ligação Proteica , Mapas de Interação de Proteínas/genética , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteína 2 Semelhante ao Fator 7 de Transcrição/genética , Proteína 2 Semelhante ao Fator 7 de Transcrição/metabolismoRESUMO
Type 2 diabetes mellitus (T2DM) is a complex disease characterized by hyperglycemia, insulin resistance, and impaired insulin secretion. T2DM is under strong genetic control. Identification and characterization of genes involved in determining T2DM will contribute to a greater understanding of the pathogenesis of T2DM, and ultimately might lead to the development of better diagnosis, prevention and treatment strategies. Efforts to identify T2DM susceptibility genes have focused on candidate gene approach (association studies) and genome-wide scans (linkage analyses). In this article, we review the current status for mapping and identification of genes for T2DM, with a focus on some promising regions (or genes) and future prospects.