RESUMEN
BACKGROUND: Germline mutations in the coding sequence of the tumour suppressor APC gene give rise to familial adenomatous polyposis (which leads to colorectal cancer) and are associated with many other oncopathologies. The loss of APC function because of deletion of putative promoter 1A or 1B also results in the development of colorectal cancer. Since the regions of promoters 1A and 1B contain many single nucleotide polymorphisms (SNPs), the aim of this study was to perform functional analysis of some of these SNPs by means of an electrophoretic mobility shift assay (EMSA) and a luciferase reporter assay. RESULTS: First, it was shown that both putative promoters of APC (1A and 1B) drive transcription in an in vitro reporter experiment. From eleven randomly selected SNPs of promoter 1A and four SNPs of promoter 1B, nine and two respectively showed differential patterns of binding of nuclear proteins to oligonucleotide probes corresponding to alternative alleles. The luciferase reporter assay showed that among the six SNPs tested, the rs75612255 C allele and rs113017087 C allele in promoter 1A as well as the rs138386816 T allele and rs115658307 T allele in promoter 1B significantly increased luciferase activity in the human erythromyeloblastoid leukaemia cell line K562. In human colorectal cancer HCT-116 cells, none of the substitutions under study had any effect, with the exception of minor allele G of rs79896135 in promoter 1B. This allele significantly decreased the luciferase reporter's activity CONCLUSION: Our results indicate that many SNPs in APC promoters 1A and 1B are functionally relevant and that allele G of rs79896135 may be associated with the predisposition to colorectal cancer.
Asunto(s)
Proteína de la Poliposis Adenomatosa del Colon/genética , Regiones no Traducidas 5' , Poliposis Adenomatosa del Colon/genética , Proteína de la Poliposis Adenomatosa del Colon/metabolismo , Alelos , Secuencia de Bases , Línea Celular Tumoral , Ensayo de Cambio de Movilidad Electroforética , Genes Reporteros , Células HCT116 , Humanos , Células K562 , Mutagénesis Sitio-Dirigida , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Sondas de Oligonucleótidos/química , Sondas de Oligonucleótidos/metabolismo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Unión Proteica , Transcripción GenéticaRESUMEN
BACKGROUND: Aggressiveness in humans is a hereditary behavioral trait that mobilizes all systems of the body-first of all, the nervous and endocrine systems, and then the respiratory, vascular, muscular, and others-e.g., for the defense of oneself, children, family, shelter, territory, and other possessions as well as personal interests. The level of aggressiveness of a person determines many other characteristics of quality of life and lifespan, acting as a stress factor. Aggressive behavior depends on many parameters such as age, gender, diseases and treatment, diet, and environmental conditions. Among them, genetic factors are believed to be the main parameters that are well-studied at the factual level, but in actuality, genome-wide studies of aggressive behavior appeared relatively recently. One of the biggest projects of the modern science-1000 Genomes-involves identification of single nucleotide polymorphisms (SNPs), i.e., differences of individual genomes from the reference genome. SNPs can be associated with hereditary diseases, their complications, comorbidities, and responses to stress or a drug. Clinical comparisons between cohorts of patients and healthy volunteers (as a control) allow for identifying SNPs whose allele frequencies significantly separate them from one another as markers of the above conditions. Computer-based preliminary analysis of millions of SNPs detected by the 1000 Genomes project can accelerate clinical search for SNP markers due to preliminary whole-genome search for the most meaningful candidate SNP markers and discarding of neutral and poorly substantiated SNPs. RESULTS: Here, we combine two computer-based search methods for SNPs (that alter gene expression) {i} Web service SNP_TATA_Comparator (DNA sequence analysis) and {ii} PubMed-based manual search for articles on aggressiveness using heuristic keywords. Near the known binding sites for TATA-binding protein (TBP) in human gene promoters, we found aggressiveness-related candidate SNP markers, including rs1143627 (associated with higher aggressiveness in patients undergoing cytokine immunotherapy), rs544850971 (higher aggressiveness in old women taking lipid-lowering medication), and rs10895068 (childhood aggressiveness-related obesity in adolescence with cardiovascular complications in adulthood). CONCLUSIONS: After validation of these candidate markers by clinical protocols, these SNPs may become useful for physicians (may help to improve treatment of patients) and for the general population (a lifestyle choice preventing aggressiveness-related complications).
Asunto(s)
Enfermedades Genéticas Congénitas/genética , Enfermedades Genéticas Congénitas/metabolismo , Polimorfismo de Nucleótido Simple , Regiones Promotoras Genéticas , Proteína de Unión a TATA-Box/metabolismo , Alelos , Progresión de la Enfermedad , Femenino , Estudios de Asociación Genética , Enfermedades Genéticas Congénitas/complicaciones , Enfermedades Genéticas Congénitas/patología , Marcadores Genéticos , Predisposición Genética a la Enfermedad , Humanos , Masculino , Obesidad/complicaciones , Obesidad/genética , Fenotipo , Pronóstico , Unión Proteica , Resultado del TratamientoRESUMEN
There are two regulatory single nucleotide polymorphisms (rSNPs) at the beginning of the second intron of the mouse K-ras gene that are strongly associated with lung cancer susceptibility. We performed functional analysis of three SNPs (rs12228277: T greater than A, rs12226937: G greater than A, and rs61761074: T greater than G) located in the same region of human KRAS. We found that rs12228277 and rs61761074 result in differential binding patterns of lung nuclear proteins to oligonucleotide probes corresponding two alternative alleles; in both cases, the transcription factor NF-Y is involved. G greater than A substitution (rs12226937) had no effect on the binding of lung nuclear proteins. However, all the nucleotide substitutions under study showed functional effects in a luciferase reporter assay. Among them, rs61761074 demonstrated a significant correlation with allele frequency in non-small-cell lung cancer (NSCLC). Taken together, the results of our study suggest that a T greater than G substitution at nucleotide position 615 in the second intron of the KRAS gene (rs61761074) may represent a promising genetic marker of NSCLC.
Asunto(s)
Genes ras , Neoplasias Pulmonares/genética , Polimorfismo de Nucleótido Simple , Adenocarcinoma/genética , Adenocarcinoma del Pulmón , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/genética , Estudios de Casos y Controles , Factor de Transcripción GATA6/genética , Factor de Transcripción GATA6/metabolismo , Frecuencia de los Genes , Marcadores Genéticos , Humanos , Intrones , Neoplasias de Células Escamosas/genética , Siberia , Proteínas ras/genética , Proteínas ras/metabolismoRESUMEN
A vast amount of SNPs derived from genome-wide association studies are represented by non-coding ones, therefore exacerbating the need for effective identification of regulatory SNPs (rSNPs) among them. However, this task remains challenging since the regulatory part of the human genome is annotated much poorly as opposed to coding regions. Here we describe an approach aggregating the whole set of ENCODE ChIP-seq data in order to search for rSNPs, and provide the experimental evidence of its efficiency. Its algorithm is based on the assumption that the enrichment of a genomic region with transcription factor binding loci (ChIP-seq peaks) indicates its regulatory function, and thereby SNPs located in this region are more likely to influence transcription regulation. To ensure that the approach preferably selects functionally meaningful SNPs, we performed enrichment analysis of several human SNP datasets associated with phenotypic manifestations. It was shown that all samples are significantly enriched with SNPs falling into the regions of multiple ChIP-seq peaks as compared with the randomly selected SNPs. For experimental verification, 40 SNPs falling into overlapping regions of at least 7 TF binding loci were selected from OMIM. The effect of SNPs on the binding of the DNA fragments containing them to the nuclear proteins from four human cell lines (HepG2, HeLaS3, HCT-116, and K562) has been tested by EMSA. A radical change in the binding pattern has been observed for 29 SNPs, besides, 6 more SNPs also demonstrated less pronounced changes. Taken together, the results demonstrate the effective way to search for potential rSNPs with the aid of ChIP-seq data provided by ENCODE project.