Metilación del ADN. Hábitos saludables.

 Hábitos saludables epigenómicos

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Metilación del ADN.

Se ha descubierto que en eucariotas, a la base citosina se le añade un grupo metilo el cual permite la conformación cerrada de la cromatina. 

Parece ser  que un alto grado de metilación se asocia con el silenciamiento de genes. 

Una forma de controlar el grado de metilación es por medio de acción de efectos ambientales. En los mamíferos se ha visto que la  S-adenosil metionina, la colina, el ácido fólico y las piridoxinas (que son sustancias provenientes de la dieta, aunque   existan medicamentos donadores de grupos metilo que fueron perseguidos inmisericordemente por la Administración diciendo que eran in

utiles, hasta agotar económicamente a  laboratorio que producía la S-adenosil metionina) , y estos productos y suplementos nutricionales ) tienen como función la adición de grupos metilos. 

Por lo general la metilación se da en mayor grado en las islas CpG (regiones con alta concentración de citosina y guanina) las cuales forman parte de la región promotora de los genes. Para que la metilación se produzca de forma adecuada necesita del ADN metiltransferasa, la cual se encarga de establecer y mantener los patrones de metilación y necesita de las proteínas de unión metil-CpG las cuales están involucradas en hacer las marcas de metilación.

Con una técnica llamada Bead Chip, podemos  ver la metilación en el genoma humano. Estos  métodos de  alta densidad Bead Chip  puede analizar 480.000 sitios CpG  s  y analizar 12 muestras  al mismo tiempo., cubriendo el 99 % de  los genes RefSeq.

¿Que son las CpG islands ? Click para  respuesta, cortesía de la infame Wikipedia.

Una técnica que se emplea es el uso de  bisulfite sequencing  y también les aconsejamos   leer el apartado DNA methylation . 

Por hoy, suficiente, que me he hipermetilado.

IIntroduction DNA methylation plays an important and dynamic role in regulating gene expression. It allows cells to become specialized and stably maintain those unique characteristics throughout the life of the organism, suppresses the deleterious expression of viral genes and other non-host DNA elements, and provides a mechanism for response to environmental stimuli. Aberrant DNA methylation (hyperor hypomethylation) and its impact on gene expression have been implicated in many disease processes, including cancer1 . To enable cost-effective DNA methylation analysis for a variety of applications, Illumina offers a robust methylation profiling platform consisting of proven chemistries and the iScan and HiScan®SQ systems. The HumanMethylation450 BeadChip (Figure 1) offers a unique combination of comprehensive, expert-selected coverage and high throughput at a low price, making it ideal for screening large sample populations such as those used in genome-wide association study (GWAS) cohorts. By providing quantitative methylation measurement at the single-CpG–site level for normal and formalin-fixed parafin-embedded (FFPE) samples, this assay offers powerful resolution for understanding epigenetic changes. Comprehensive Genome-Wide Coverage The Infinium HumanMethylation450 BeadChip provides unparalleled, genome-wide coverage featuring comprehensive gene region and CpG island coverage, plus additional high-value content selected with the guidance of methylation experts. Infinium HD technology enables content selection independent of bias-associated limitations often associated with methylated DNA capture methods. As a result, 99% of RefSeq genes are covered, including those in regions of low CpG island density and at risk for being missed by commonly used capture methods. Importantly, coverage was targeted across gene regions with sites in the promoter region, 5'UTR, first exon, gene body, and 3'UTR in order to provide the broadest, most comprehensive view of methylation state possible (Figure 2). This multiple-site approach was extended to CpG islands/CpG island regions for which 96% of islands were covered overall, with multiple sites within islands and island shores, as well as those regions flanking island shores (island shelves). Beyond gene and CpG island regions, multiple additional content categories requested by methylation experts were also included: • CpG sites outside of CpG islands • Non-CpG methylated sites identified in human stem cells • Differentially methylated sites identified in tumor versus normal (multiple forms of cancer) and across several tissue types • FANTOM 4 promoters • DNase hypersensitive sites • miRNA promoter regions • ~ 90% of content contained on the Illumina HumanMethylation27 BeadChip Streamlined Workflow The HumanMethylation450 BeadChip follows a user-friendly, streamlined workflow that does not require PCR. Its low sample input requirement (as low as 500 ng), enables analysis of valuable samples Infinium® HumanMethylation450 BeadChip The ideal solution for affordable, large sample–size genome-wide DNA methylation studies. Figure 1: Infinium HumanMethylation450 BeadChip The Infinium HumanMethylation450 BeadChip features more than 450,000 methylation sites, within and outside of CpG islands. 4305493023 ® Data Sheet: Epigenetics derived from limited DNA sources. HumanMethylation450 BeadChip kits contain all required reagents for performing methylation analyses (except for the bisulfite conversion kit, which is available separately). Data Integration Of all the genes represented on the HumanMethylation450 BeadChip, more than 20,000 are also present on the HumanHT-12 v4 Expression BeadChip2 , permitting combined analysis of global methylation status and gene expression levels. In addition, investigators may integrate methylation data with genotyping data from GWAS studies to better understand the interplay between genotype and methylation state in driving phenotypes of interest. High-Quality Data The HumanMethylation450 BeadChip applies both Infinium I and II assay chemistry technologies (Figure 3) to enhance the depth of coverage for methylation analysis. The addition of the Infinium II design allows use of degenerate oligonucleotide probes for a single bead type, enabling each of up to three underlying CpG sites to be either methylated or unmethylated with no impact on the result for the queried site. Illumina scientists rigorously test every product to ensure strong and reproducible performance, enabling researchers to achieve industryleading data quality. Precision and Accuracy Reproducibility has been determined based on the correlation of results generated from technical replicates. The HumanMethylation450 BeadChip showed strong correlation between replicates (r>0.98), as well as with the HumanMethylation27 BeadChip and whole-genome bisulfite sequencing (Figure 4). Sensitivity By comparing the results of replicate experiments (duplicates of eight biological samples), Illumina scientists have shown that the HumanMethylation450 BeadChip reliably detects a delta-beta value of 0.2 with a lower than 1% false positive rate. Internal Quality Controls Infinium HD–based assays possess several sample-dependent and sample-independent controls so researchers have confidence in producing the highest quality data. The HumanMethylation450 BeadChip includes 600 negative controls, which are particularly important in methylation analysis assays since sequence complexity is decreased after bisulfite conversion. The GenomeStudio® Methylation Module Software has an integrated Controls Dashboard where the performance of all controls can be easily monitored. Figure 3: Broader Coverage Using Infinium I and II Assay Designs The HumanMethylation450 BeadChip employs both Infinium I and Infinium II assays, enhancing its breadth of coverage. Infinium I assay design employs two bead types per CpG locus, one each for the methylated and unmethylated states. The Infinium II design uses one bead type, with the methylated state determined at the single base extension step after hybridization. Unmethylated locus Infinium I Infinium II Methylated locus Unmethylated locus Methylated locus Unmethylated bead type Methylated bead type CpG locus Bisulfite converted DNA CA GT CG GC CAx GC CGx GT U U M M M Single bead type CpG locus Bisulfite converted DNA U A G C T A G C T A G C T A G C T CG GC CA GT A G CT A G CT 5’ 5’ 5’ 5’ 5’ 5’ Figure 2: HumanMethylation450 BeadChip Provides Coverage Throughout Gene Regions TSS1500 TSS200 5’ UTR 1st exon Gene body 3’ UTR Feature Type Genes Mapped Percent Genes Covered Numberof Loci on Array NM_TSS200 14895 0.79 2.56 NM_TS1500 17820 0.94 3.41 NM_5'UTR 13865 0.78 3.34 NM_1stExon 15127 0.80 1.62 NM_3'UTR 13042 0.72 1.02 NM_GeneBody 17071 0.97 8.97 NR_TSS200 1967 0.65 1.84 NR_TSS1500 2672 0.88 2.92 NR_GeneBody 2345 0.77 5.34 N Shelf N Shore CpG Island S Shore S Shelf Feature Type Islands Mapped Percent Islands Covered Average Numberof Loci on Array Island 26153 0.94 5.08 N_Shore 25770 0.93 2.74 S_Shore 25614 0.92 2.66 N_Shelf 23896 0.86 1.97 S_Shelf 23968 0.86 1.94 The HumanMethylation450 BeadChip offers broad coverage across gene regions, as well as CpG islands/CPG island regions, shelves, and shores for the most comprehensive view of methylation state. Data Sheet: Epigenetics Figure 4: High Assay Reproducibility A: HumanMethylation450 Replicate Correlation R2 = 0.9969 HumanMethylation450 BeadChip HumanMethylation450 BeadChip B: HumanMethylation27 vs. HumanMethylation450 Correlation HumanMethylation450 BeadChip HumanMethylation27 BeadChip C. HumanMethylation450 vs. Whole-Genome Bisulfite Sequencing Array Array Sequencing Sequencing Lung Normal Lung Tumor R2= 0.92 R2= 0.93 Using the HumanMethylation450 BeadChip, users can be confident of obtaining consistent, robust data. Representative plots from internal testing show strong replicate correlation (A), as well as strong correlation with the HumanMethylation27 BeadChip (B) and whole-genome bisulfite sequencing (C). Figure 5: Integrated Data Analysis with Illumina GenomeStudio Software H U Normal Cancer M GenomeStudio software supports DNA methylation analysis on any platform. Data are displayed in intuitive graphics. Gene expression data can be easily integrated with methylation projects (plotted on right). Table 1: Comparative Infinium HumanMethylation450 Data Quality Metrics—Standard vs. FFPE HumanMethylation450 BeadChip Standard Protocol FFPE Protocol Reproducibility (Technical replicates) r2 ≥ 98% r2 ≥ 98% Number of sites detected* ≥ 99% ≥ 95% *Based on non-cancer samples, recommended sample input amounts of high-quality DNA as confirmed by PicoGreen and following all other Illumina recommendations as per respective User Guides. Integrated Analysis Software HumanMethylation450 BeadChip data analysis is supported by the powerful and intuitive GenomeStudio Methylation Module, enabling researchers to effortlessly perform differential methylation analysis (Figure 5). The GenomeStudio software features advanced visualization tools that enable researchers to view vast amounts of data in a single graph, such as heat maps, scatter plots, and line plots. These tools and the GenomeStudio Genome Browser display valuable information such as chromosomal coordinates, percent GC, location in a CpG Island, and methylation β values. Data generated by the Infinium HD methylation assay are easily compatible with data from other Illumina applications, including gene expression profiling. This enables researchers to perform crossapplication analysis such as the integration of gene expression data with HumanMethylation450 BeadChip methylation data. Methylation Studies with FFPE Samples Researchers can perform methylation studies on FFPE samples by using a special, modified version of the Infinium HumanMethylation450 BeadChip protocol3 that leverages the easy-to-use Infinium FFPE DNA Restoration Solution4, to produce robust, highly reproducible results (Table 1). The FFPE DNA Restoration Solution includes the Illumina FFPE QC and the Infinium HD FFPE DNA Restore Kits. Please note that while the FFPE DNA Restoration Solution and HumanMethylation450 BeadChip kits are the same for normal and FFPE samples, investigators running FFPE samples should only follow the workflow described in the Infinium HD FFPE Methylation Assay protocol (manual or automated)5,6, as it includes important changes to the standard protocols for each kit. Data Sheet: Epigenetics Illumina • +1.800.809.4566 toll-free • 1.858.202.4566 tel • techsupport@illumina.com • www.illumina.com For research use only © 2012 Illumina, Inc. All rights reserved. Illumina, illuminaDx, BaseSpace, BeadArray, BeadXpress, cBot, CSPro, DASL, DesignStudio, Eco, GAIIx, Genetic Energy, Genome Analyzer, GenomeStudio, GoldenGate, HiScan, HiSeq, Infinium, iSelect, MiSeq, Nextera, Sentrix, SeqMonitor, Solexa, TruSeq, VeraCode, the pumpkin orange color, and the Genetic Energy streaming bases design are trademarks or registered trademarks of Illumina, Inc. All other brands and names contained herein are the property of their respective owners. Pub. No. 270-2010-001 Current as of 09 March 2012 Summary The HumanMethylation450 BeadChip’s unique combination of comprehensive, expert-selected coverage, high sample throughput capacity, and affordable price makes it an ideal solution for large sample–size, genome-wide DNA methylation studies. References 1. Portela A, Esteller M (2010) Epigenetic modifications and human disease. Nat Biotechnology 28: 1057–1068. 2. http://www.illumina.com/products/humanht_12_expression_beadchip_kits_ v4.ilmn 3. Infinium HD FFPE DNA Restoration Protocol 4. http://www.illumina.com/products/infinium_ffpe_dna_restoration_solution. ilmn 5. Infinium HD FFPE Methylation Assay, Manual Protocol 6. Infinium HD FFPE Methylation Assay, Automated Protocol 7. Illumina FFPE QC Assay Protocol Ordering Information Catalog No. Product Description WG-314-1003 Infinium HumanMethylation450 BeadChip Kit (24 samples) Each package contains two BeadChips and reagents for analyzing DNA methylation in 24 human DNA samples. WG-314-1001 Infinium HumanMethylation450 BeadChip Kit (48 samples) Each package contains four BeadChips and reagents for analyzing DNA methylation in 48 human DNA samples. WG-314-1002 Infinium HumanMethylation450 BeadChip Kit (96 samples) Each package contains eight BeadChips and reagents for analyzing DNA methylation in 96 human DNA samples. Each HumanMethylation450 BeadChip can process 12 samples in parallel and assay >450,000 methylation sitesmpronta genómica y herencia epigenética

Impronta genómica

Los procesos de metilación juegan un papel importante en la acción de la impronta genómica. En los vertebrados solo se ha descubierto este mecanismo en los mamíferos. Según el origen parental los genes pueden ser activados o silenciados. La impronta afecta el crecimiento prenatal y se ha establecido su importancia en la generación de enfermedades. Durante la gametogénesis se inicia la impronta genómica y por lo tanto esta es heredada durante la fusión de los gametos. Durante la formación del cigoto la impronta es reprogramada en el nuevo individuo. El ejemplo más claro de este mecanismo se da en la regulación de la dosis compensatoria del cromosoma X. Esta reprogramación juega un papel importante en la expresión de los genes de tejidos específicos que si llegan a ser modificados pueden tener consecuencias en el desarrollo adecuado del organismo. Por lo tanto, con un mejor entendimiento de cómo ocurren estos procesos y como son regulados, se puede llegar a entender enfermedades como la preeclampsia, las pérdidas durante la gestación, los fallos que se dan en la reproducción asistida, los problemas asociados con la infertilidad y el cáncer entre otros.

Herencia epigenética

La herencia epigenética resulta de la transmisión de información que no depende de secuencias de las bases nitrogenadas del ADN a través de la meiosis o mitosis. La información epigenética modula, por tanto, la expresión de los genes sin alterar la secuencia de ADN. Los patrones de metilación de ADN son los mejor estudiados y entendidos como marcadores de fenómenos epigenéticos.

El epigenoma es la información epigenética global de un organismo.

Los tres principales tipos de información epigenética son:

• Metilación de la citosina del ADN: es un cambio en el ADN, en la que un grupo metilo es transferido desde S-adenosilmetionina a una posición C-5 de citosina por una ADN-5 metiltrasferasa. La metilación del ADN ocurre, casi exclusivamente, en dinucleótidos CpG, teniendo un importante papel en la regulación de la expresión del gen.
• Impronta genética: la impronta se manifiesta solo en organismos superiores (enlace roto disponible en Internet Archive; véase el historial y la última versión). Cuando hablamos de "imprinting", nos referimos a genes que pueden modificar su funcionamiento sin necesidad de un cambio en la secuencia del ADN. Este cambio en su forma de manifestarse que tienen los genes "imprintados" está generalmente ligado a su origen parental. Un gen imprintado se manifiesta de una manera cuando su origen es paterno y de otra cuando proviene del gameto materno. Parece ser que existe un mecanismo celular que de algún modo "marca" o deja una impronta sobre todos los genes "imprintables" de acuerdo al sexo del individuo.
• Modificación de histonas: incluye acetilación, metilación y fosforilación.

También hay que indicar que la célula no puede sintetizar los orgánulos "de novo"; por ello, además de la información que contiene el ADN, una célula necesita información epigenética en forma de al menos una proteína característica en la membrana del orgánulo que se quiera sintetizar. Esta información se transmite desde la membrana del padre a la de la progenie en forma del propio orgánulo.

Sin embargo, al nombrar estos mecanismos, hay que recordar que "indirectamente", al analizar el origen de cada proceso en sí mismo, aún están involucrados los genes (como por ejemplo los genes de la enzima ADN-metiltransferasa, histonas, etcétera) y, por ende, también indirectamente están involucrados los cambios genéticos (como mutaciones) que puedan sufrir estos genes, o sobre los genes en que actúan. Del mismo modo, aunque las modificaciones epigenéticas no implican en el proceso un cambio en la secuencia de nucleótidos del ADN, sino que consisten en un cambio en la expresión de los genes, la selección natural igualmente, a partir del resultado biológico de dicha expresión de genes, actuará sobre el proceso epigenético y sobre el organismo que lo manifiesta.