Human Genetics: International Projects and Personalized Medicine

Drug Metabol Pers Ther 2016; 31(1): 3–8

Mini Review

Maria Apellaniz-Ruiza, Cristina Gallegoa, Sara Ruiz-Pintoa, Angel Carracedo and Cristina Rodríguez-Antona* Human genetics: international projects and personalized medicine

DOI 10.1515/dmpt-2015-0032 Received August 31, 2015; accepted October 19, 2015; previously Introduction published online November 18, 2015 Genetic variation databases describe naturally occur- Abstract: In this article, we present the progress driven ring genetic differences among individuals of the same by the recent technological advances and new revolu- species. This variation, accounting for 0.1% of our DNA tionary massive sequencing technologies in the field of [1], permits the flexibility and survival of a population human genetics. We discuss this knowledge in relation in the face of changing environmental circumstances, with drug response prediction, from the germline genetic but it also influences how people differ in their risk of variation compiled in the 1000 Genomes Project or in the disease or their response to drugs. It is well known that Genotype-Tissue Expression project, to the phenome- variability in response to drug therapy is the rule rather genome archives, the international cancer projects, such than the exception for most drugs, and these differences as The Cancer Genome Atlas or the International Cancer are among the major challenges in current clinical prac- Genome Consortium, and the epigenetic variation and its tice, drug development, and drug regulation [2, 3]. Thus, influence in gene expression, including the regulation of rather than accepting the “one drug fits all” approach, drug metabolism. This review is based on the lectures pre- researchers envision that drugs need to be tailored to fit sented by the speakers of the Symposium “Human Genet- the profile of each individual patient. Therefore, discover- ics: International Projects & New Technologies” from the ing the DNA sequence variants that contribute to common VII Conference of the Spanish Pharmacogenetics and disease risk and drug toxicity offers one of the best oppor- Pharmacogenomics Society, held on the 20th and 21st of tunities for understanding the complex causes of disease April 2015. in humans and learning how to better treat them. Over the last decade, many efforts have been put to accomplish Keywords: epigenetics; genetic variation; human genome; this goal and to provide a detailed picture of human differ- pharmacogenomics. ences and similarities at the genetic level. The first human genome sequence was completed in 2003 using first-generation sequencing technolo- aMaria Apellaniz-Ruiz, Cristina Gallego and Sara Ruiz-Pinto contributed equally to this work. gies (i.e. Sanger sequencing) [4, 5]. Many previous steps *Corresponding author: Cristina Rodríguez-Antona, Hereditary were crucial to this success, such as the discovery of the Endocrine Cancer Group, Spanish National Cancer Research Center double helical structure of the DNA molecule in 1953 by (CNIO), Madrid, Spain, Phone: +34 917328000, Francis Crick and James Watson [6], the development of Fax: +34 912246972, E-mail: [email protected]; and ISCIII Center DNA sequencing performed by Edward Sanger in the mid- for Biomedical Research on Rare Diseases (CIBERER), Madrid, Spain 1970s [7], and finally the automation of DNA sequencing Maria Apellaniz-Ruiz: Hereditary Endocrine Cancer Group, Spanish National Cancer Research Centre (CNIO), Madrid, Spain in the 1980s. Since then, the demand for cheaper and Cristina Gallego: Stroke pharmacogenomics and genetics, Fundació faster sequencing methods has driven the development Docència i Recerca Mutua Terrassa, Hospital Universitari Mútua de of second-generation sequencing methods, also called Terrassa, Terrassa (Barcelona), Spain next-generation sequencing (NGS). This massively par- Sara Ruiz-Pinto: Human Genotyping-CEGEN Unit, Spanish National allel sequencing technology facilitates high-throughput Cancer Research Centre (CNIO), Madrid, Spain Angel Carracedo: Fundación Pública de Medicina Xenómica- sequencing, allowing an entire genome to be sequenced SERGAS. Grupo de Medicina Xenómica, CIBERER, IDIS, Santiago de in < 1 day. This new availability of large number of complete Compostela, Spain human genome sequences is boosting the identification of 4 Apellaniz-Ruiz et al.: Human genetics: international projects and personalized medicine genetic variants responsible for the pathogenesis of dis- drug metabolism, drug transport, and drug targets, which eases and drug therapy response. could affect either efficacy or drug toxicity, and (ii) the After the publication of the human genome, it became disease heterogeneity that mainly affects drug efficacy. In clear that the DNA sequence changes were not the only recent years, many international efforts are being put into factor shaping gene expression and phenotypes. This unveiling the genetic variability among the population opened the door to epigenetics, a new field still in its early that could explain these differences. Therefore, there has development. Epigenetics refers to nongenetic factors that been a proliferation of both public and private projects have a key role in gene expression and regulation. This that make genomic information widely and rapidly avail- new field explains why different cell types, which have able for the scientific community. the same genome, have different phenotypes [8]. Epige- Known genome browsers for the retrieval of genomic netics also provides an explanation for differences in information exist. Among them, Ensembl (http://www. traits, such as disease discordance, observed in monozy- ensembl.org) is a joint scientific project between the gotic twins [9, 10]. NGS technologies had contributed to European Bioinformatics Institute and the Wellcome the rapid increase of epigenetic studies, adding one more Trust Sanger Institute, which includes genomes of several layer to the genetic information. Therefore, identifying species and different genomic and epigenomic informa- epigenetic patterns associated with complex diseases and tion. Similar databases and browsers are found at the drug response is another step toward the personalization National Center for Biotechnology Information (http:// of medicine. www.ncbi.nlm.nih.gov/), a branch of the National Insti- Many national and international projects focus on tutes of Health, which houses a series of databases rele- unveiling human genetic and epigenetic variation and vant to biotechnology and biomedicine (GenBank, dbSNP, their contribution to the heterogeneity of diseases. This PubMed, or OMIM). The University of California Santa information will be critical to individualize medicine, Cruz browser (https://genome.ucsc.edu/) allows to have making it the norm rather than the exception, maximizing a rapid display of any requested portion of the genome drug efficacy and minimizing drug toxicity. In this review, at any scale, together with dozens of aligned annotation we will describe major international projects studying tracks (known genes, predicted genes, ESTs, mRNAs, CpG genetic variation (e.g. HapMap, the 1000 Genomes islands, mouse homologies, etc.). Project, The Cancer Genome Atlas [TCGA], and the Inter- Concerning germline genetic variation, the Inter- national Cancer Genome Consortium [ICGC]), gene national HapMap Project (http://hapmap.ncbi.nlm.nih. expression and regulation across multiple human tissues gov/) was created to determine the common patterns of (the Genotype-Tissue Expression [GTEx] project) and pro- DNA sequence variation in the human genome by deter- viding phenotype-genotype associations (the European mining the genotypes of more than one million sequence Genome-Phenome Archive [EGA], a repository for all types variants, their frequencies, and the degree of association of genotype experiments, including case control, popula- between them in DNA samples from populations with tion, and family studies, and phenotypic data). Going a ancestry from parts of Africa, Asia, and Europe. step further, we will also discuss gene regulation through Not only projects compiling interindividual genetic epigenetics and how it can modify disease risks and drug variability have been developed, but also databases to responses. This new genetic and epigenetic knowledge unveil disease heterogeneity. This is the case of cancer, will constitute the basis for a more efficient personal- in which the identification of genes mutated and driving ized medicine. In this contribution, we review the latest oncogenesis has been a central aim. Some of the major developments in this field presented in the Symposium cancer databases are the ICGC (https://icgc.org/) and “Human Genetics: International Projects & New Technolo- TCGA (http://cancergenome.nih.gov/). They are compre- gies” from the VII Conference of the Spanish Pharmacoge- hensive catalogues of genomic and molecular abnormali- netics and Pharmacogenomics Society (SEFF), on the 20th ties (somatic mutations, abnormal expression of genes, and 21st of April 2015. and epigenetic modifications) in tumors from different cancer types and/or subtypes and are of clinical and societal importance across the globe. In addition, the Wellcome Trust Sanger Institute’s Cancer Genome Project Advances in human genetics (https://www.sanger.ac.uk/research/projects/cancergenome/) is using high-throughput techniques to identify Two major factors have to be taken into account to per- tumor-acquired sequence variants/mutations and hence sonalize medicine: (i) the interindividual variability in is identifying genes critical for the development of human Apellaniz-Ruiz et al.: Human genetics: international projects and personalized medicine 5 cancer. The project includes resources such as the “Cancer 2015 SEFF VII conference, in which he presented the large Gene Census” (a catalogue of genes for which mutations international collaborative project in human genetics. have been causally implicated in cancer), “COSMIC” (a catalogue of somatic mutations in cancer), and “Cancer Cell Line Project” (a genomic characterization of cancer cell lines). The Memorial Sloan-Kettering Cancer Center The European Genome-Phenome has developed cBioPortal (http://www.cbioportal.org/), a database integrating data from the large-scale cancer Archive, a key tool for biomedical genomic projects and making the multidimensional research cancer genomics data easily and directly available (it includes data from more than 5000 tumor samples from The EGA (https://www.ebi.ac.uk/ega/home) is a public 20 cancer studies). The Cancer Cell Line Encyclopedia repository of all types of genomic and phenotypic data project (http://www.broadinstitute.org/ccle/home), a col- available, jointly coordinated by the European Bioin- laboration between Broad Institute and Novartis, conducts formatics Institute and, since 2013, by the Centre for a detailed genetic and pharmacologic characterization Genomic Regulation. The EGA arises not only as an of 1000 human cancer cell lines. This allows to perform archive but also as a permanent service designed to integrated computational analyses that link distinct phar- facilitate accessibility and sharing of data among the macologic vulnerabilities to genomic patterns and to biomedical community across de globe. Data deposited translate cell line integrative genomics into cancer patient at the EGA are derived from more than 100,000 people, stratification. Regarding the European Union, Horizon including clinically healthy individuals and patients 2020 is the biggest European Research and Innovation diagnosed with a variety of pathologies, mainly, complex Programme, bringing the promise of breakthroughs, dis- diseases. At the moment, it archives information from coveries, and world-firsts by taking great ideas from the more than 700 biomedical research studies focused on a laboratory to the market. One of the financed projects is broad range of diseases, including cancer (with 21 kinds the Breast Cancer Stratification (B-CAST; http://cordis. of tumors included), cardiovascular disease (such as europa.eu/project/rcn/193256_en.html), which aims to hypercholesterolemia, hypertension, and coronary alter- understand the determinants of risk and prognosis of ations), neurological disorders (including emotional and molecular breast cancer subtypes analyzing over 20,000 behavior difficulties and neurodegenerative conditions), breast tumors. and a large group of inflammatory diseases (rheuma- Not only international but also national projects such toid arthritis, multiple sclerosis, asthma, etc.), among as the UK initiative 100,000 Genome Project (http://www. others. These more than 700 studies cover several types genomicsengland.co.uk/the-100000-genomes-project/) of genotyping, epigenetic, sequencing, and transcrip- (Genomics England) are being carried out. This project tomic investigations, including genome-wide associa- will focus on patients with rare diseases and patients with tion studies (GWAS), population, and family studies. The cancer (they aim to provide with data from 100,000 whole EGA stores several levels of data obtained from differ- genomes by 2017). A similar initiative is being conducted ent technologies and platforms, including the raw data, by the United States, namely, the Precision Medicine Ini- the final genomic variants, and the phenotypic data. All tiative (http://www.nih.gov/precisionmedicine/). In this this information is submitted by investigators across project, more than a million Americans will be studied to more than 150 research centers, institutes, universities, understand how diseases can be prevented and treated, and international consortia (e.g. Wellcome Trust Sanger taking into account people’s individual variations in Institute, St Jude Children’s Research Hospital, ICGC), genes, environment, and lifestyle. and the data are available to researchers and clinicians These national and international projects are signifi- worldwide. As a repository of not only genomic but also cantly lowering the barriers between complex genomic clinical information, the EGA provides a controlled data and researchers who want rapid, intuitive, and high- access to data to maintain privacy and patient confiden- quality access to molecular profiles and clinical attributes tiality. To ensure data are safely and securely managed, from large-scale genomics projects. Empowering research- stored, and shared, data access decisions are made not ers to translate these rich data sets into biologic insights by the EGA but by the appropriate and independent data and clinical applications is the challenge, but it holds access committee, normally from the same institution the promise of improving our health and well-being. This submitting the data with individuals involved in data information is based on Angel Carracedo’s lecture at the collection and analysis of the initial study. 6 Apellaniz-Ruiz et al.: Human genetics: international projects and personalized medicine

Another important purpose of the EGA is to make the them (~95%) in the testis. When comparing gene expres- stored data readily accessible to the entire scientific com- sion across individuals, Melé et al. [12] found that only munity. Over the last year, information held in the EGA 4% of the variation in gene expression can be explained was distributed more than 20,000 times to nearly 5000 by variation among individuals (compared with 47% researchers. Although the estimated initial EGA database in tissues), with long noncoding RNAs (lncRNAs) and size was 10 TB, at the moment information archived occu- protein coding genes expressing similarly. However, genes pies 1 PB, and it is growing every day. In fact, through- associated with gender, ethnicity, and age showed a high out 2013, the EGA archive experienced an increase of 50% interindividual variation. Among genes with sex-biased in the number of research studies submitted and 70% expression, they found XIST and JPX and two lncRNAs increase in the number of files stored, and this growth is (RP11-309M23.1 and RP13-216E22.4); all of them may par- expected to be even greater in the coming years. ticipate in the X-chromosome inactivation phenomenon. The lecture given by Arcadi Navarro at the 2015 SEFF MMP3, an autosomal gene previously associated with sus- Conference served as the basis to present the EGA here. ceptibility to coronary heart disease, showed the strong- est difference in expression between males and females, being predominantly expressed in males. They also found an enrichment in lncRNAs for genes showing differential The GTEx project and the new expression by ethnicity, contrary to those observed for genes with a variable expression according to age. Among challenges to decipher the human genes with a decreased expression with age, they found genome genes associated to neurodegenerative diseases such as Parkinson’s and Alzheimer’s diseases. However, these The GTEx project (http://www.gtexportal.org/) compiles sex- and ethnicity-biased expressions were found not only new data on human transcriptome across tissues and indi- between individuals but also across tissues, especially in viduals. The aim of this project was to elaborate an exten- breast and skin, respectively. sive public atlas of human variation and its relationship In contrast to gene expression, variation in splicing to gene expression and regulation across multiple tissues, was found to be very similar between tissues and between helping to decipher the genetic basis of human disease. individuals. Among tissues, brain was distinguished as a During the GTEx pilot phase, DNA and RNA from 237 post- tissue having unusually high levels of splicing events, on mortem donors have been extensively sequenced, and the basis of preferential gene expression of RNA-binding the first results from this pilot phase have been recently proteins involved in splicing and exon inclusion. Among published, consisting on one main GTEx article [11] and genes, those coding ribosomal proteins and involved in 5 companion publications [12–16]. Melé et al. [12] used translation or protein biosynthesis showed the highest RNA sequencing data from 1641 samples covering 43 body interindividual variability. Finally, by comparing the con- sites from 175 donors to investigate the pattern of tran- tribution of gene expression to variation in isoform abun- scriptome variation across tissues and individuals. Impor- dance between individuals and between tissues, they tantly, postmortem and antemortem samples were found found that a large proportion of variation across tissues to have similar expression profiles. When they evaluated (84%) can be explained by gene expression, suggesting a variation in gene expression, they found that tissues are complementary role for splicing. characterized by a unique transcriptional signature, with The information provided here summarizes Roderic solid and hematologic (blood and Epstein-Barr virus- Guigó’s lecture at the 2015 SEFF meeting where he pre- transformed lymphocyte cell lines) tissues clustering sep- sented the GTEx project. arately. Among solid tissues, brain samples showed the largest expression differences. Gene contribution to tissue-specific transcriptional profile derived from this pilot study showed that, for most Epigenetics: new advances tissues, transcription is determined by very few genes (i.e. three hemoglobin genes accounted for 60% of the total and challenges in personalized transcription in blood), and in many of them, transcrip- medicine tion has a mitochondrial (i.e. kidney, heart, and brain) or nuclear (i.e. blood) origin. In addition, fewer than 200 The term epigenetics was coined by the biologist and genes had an exclusive tissue expression, the majority of geneticist Conrad H. Waddington in 1942 to describe how Apellaniz-Ruiz et al.: Human genetics: international projects and personalized medicine 7 genes might interact with the environment. Nowadays, manner, are having a key role in the quick development epigenetics refers to DNA modifications affecting gene of this field. Epigenetic alterations have been related to expression and chromatin structure that are not caused aging and age-related diseases [22, 23]; many types of by changes in DNA sequence [17]. Epigenetic modifica- cancer and tumorigenic process, tobacco smoking, and tions can be stable, heritable, and reversible and can several complex diseases as type II diabetes. In cancer, be modulated by many factors, including physiological a global hypomethylation is a common feature that leads and pathological conditions, and by the environment to genomics instability, whereas hypermethylation of [8, 17]. Epigenetic differences appear during the lifetime specific promoters can change the expression of relevant of monozygotic twins [9] and of monozygotic twins with genes. Gene methylation can also serve as a biomarker discordant diseases [10]. Epigenetics is essential in gene (e.g. GSTP1 in bladder tumors, BRCA1 in breast tumors, expression and regulation and is involved in numerous MGMT in brain tumors, GM-CSF in colorectal tumors). cellular processes such as cellular development and dif- These findings open the door to new approaches in phar- ferentiation. It has also been related to some monogenic macogenetics and personalized medicine. Standard ther- and complex human diseases. The most important epige- apies are given to patients; however, only a fraction of netic mechanisms are DNA methylation, covalent histone them respond optimally to the drugs, in some cases due modifications, and noncoding RNAs [17]. to epigenetic alterations. To accomplish the promises of As many other biomedical sciences, pharmacogenet- personalized medicine, both genetic and epigenetic diag- ics has focused on identifying the genes that contribute nostic testing will be required. to a particular trait or phenotype (i.e. drug response). The data presented herein is based on the lecture that Approaches such as GWAS aim to identify variations Mario Fraga gave on DNA methylation and its influence on in the DNA sequence linked to a specific phenotype; disease risk and drug response, in Madrid in 2015 in the however, when the identified variants are not in gene VII SEFF conference. coding regions, these studies rarely provide insights into the regulatory mechanisms underlying the association. After the publication on 2003 of the human genome sequence, many branches of human biology have ben- Conclusions efited. However, despite this progress, scientists realized that DNA sequence cannot explain by itself cell function, Every individual is unique in his/her genetic and epi- development, aging, and many diseases. It became clear genetic profile, and this uniqueness will influence and that the epigenome would also be required to understand should be taken into account to perform personalized how genetic information is interpreted by the cell. medicine. Nowadays, with the development of new In 2012 the Encyclopedia of DNA Elements project high-throughput technologies such as NGS, national (ENCODE; https://www.encodeproject.org/), which aims and international efforts are being put into unveiling the to describe all the functional elements encoded in the genetic variability among individuals to get insights into human genome by mapping epigenetic modifications, the mechanisms underlying complex traits. These large- published its first results [18]. This pioneer project will scale resources such as the GTEx, EGA, ENCODE, or TCGA have a major impact on human genetics studies, but its projects compile diverse biologic information, including clinical application is limited because most of the results genomic, epigenetic, and phenotypic data. Integrative derive from a small number of laboratory cell lines. Now analyses using these resources will help to advance the the Roadmap Epigenomics Project (http://www.road- field of personalized medicine, facilitating the identi- mapepigenomics.org/) [19] has the goal of producing fication and prioritization of potential biomarkers and a public resource of epigenomic data from stem cells increasing our understanding on the mechanisms under- and mature cells from a variety of different tissues from lying the diversity in drug responses. However, there are healthy people, and from patients with different diseases, challenges ahead, such as dealing with large amounts of such as cancer, neurodegenerative diseases, and autoim- “omic” data, interpreting the genomic and epigenomic mune diseases. Recently, the Roadmap Epigenetics Con- data in relation with drug responses and predisposition to sortium published an integrative analysis of 111 reference diseases, or finding the optimal way to convey these data epigenomes [20], as well as the first human haplotype- to physicians and patients. On the whole, many difficul- resolved epigenomes, and showed these vary across ties lay ahead; however, advances in genomics and epi- tissues and individuals [21]. The new tools to study epigenomics can be exploited to personalize medicine and genetic modifications, in a systematic and genome-wide hold the promise of improving our health and well-being. 8 Apellaniz-Ruiz et al.: Human genetics: international projects and personalized medicine

Acknowledgments: We would like to thank to all of speak- 9. Fraga MF, Ballestar E, Paz MF, Ropero S, Setien F, Ballestar ML, ers and chairs of “Human genetics: international pro- et al. Epigenetic differences arise during the lifetime of monozygotic twins. Proc Natl Acad Sci USA 2005;102:10604–9. jects & new technologies” Symposium in the VII SEFF 10. Wong CC, Meaburn EL, Ronald A, Price TS, Jeffries AR, Conference. Schalkwyk LC, et al. Methylomic analysis of monozygotic twins Author contributions: All the authors have accepted discordant for autism spectrum disorder and related behav- responsibility for the entire content of this submitted ioural traits. Mol Psychiatry 2014;19:495–503. manuscript and approved submission. 11. The GTEx Consortium. The Genotype-Tissue Expression (GTEx) Research funding: María Apellániz-Ruiz is a predoctoral pilot analysis: Multitissue gene regulation in humans. Science 2015;348:648–60. fellow of “la Caixa”/ CNIO international PhD programme, 12. Melé M, Ferreira PG, Reverter F, Deluca DS, Monlong J, Sara Ruiz-Pinto is a predoctoral fellow supported by the Sammeth M, et al. The human transcriptome across tissues and Severo Ochoa Excellence Programme (Project SEV-2011- individuals. Science 2015;348:660–5. 0191). The Human Genotyping lab is a member of CeGen, 13. Rivas MA, Pirinen M, Conrad DF, Lek M, Tsang EK, Karczewski KJ, PRB2-ISCIII and is supported by grant PT13/0001, of the et al. Effect of predicted protein-truncating genetic variants on the human transcriptome. Science 2015;348:666–9. PE I+D+i 2013-2016, funded by ISCIII and FEDER (Fondo 14. Baran Y, Subramaniam M, Biton A, Tukiainen T, Tsang EK, Europeo de Desarrollo Regional). Rivas MA, et al. The landscape of genomic imprinting across Employment or leadership: None declared. diverse adult human tissues. Genome Res 2015. Honorarium: None declared. 15. Pierson E, Koller D, Battle A, Mostafavi S. Sharing and specific- Competing interests: The funding organization(s) played ity of co-expression networks across 35 human tissues. PLoS no role in the study design; in the collection, analysis, and Comput Biol 2015;11:1–19. 16. Pirinen M, Lappalainen T, Zaitlen NA, and GTEx Consortium, interpretation of data; in the writing of the report; or in the Dermitzakis ET, Donnelly P, McCarthy MI, et al. Assessing decision to submit the report for publication. allele specific expression across multiple tissues from RNA- seq read data. Bioinformatics 2015, Epub ahead of print 27 Mar 2015. 17. Berger SL, Kouzarides T, Shiekhattar R, Shilatifard A. An opera- References tional definition of epigenetics. Genes Dev 2009;23:781–3. 18. Ecker JR, Bickmore WA, Barroso I, Pritchard JK, Gilad Y, Segal E. 1. Jorde LB, Wooding SP. Genetic variation, classification and Genomics: ENCODE explained. Nature 2012;489:52–5. “race.” Nat Genet 2004;36 S28–33. 19. Skipper M, Eccleston A, Gray N, Heemels T, Le Bot N, 2. Ma Q, Lu AY. Pharmacogenetics, pharmacogenomics and indi- Marte B, et al. Presenting the epigenome roadmap. Nature vidualized medicine. Pharmacol Rev 2011;63:437–59. 2015;518:313. 3. William EE, McLeod HL. Pharmacogenomics – drug disposition, 20. Consortium RE, Kundaje A, Meuleman W, Ernst J, Bilenky M, drug targets, and side effects. New Engl J Med 2003;348:538–49. Yen A, et al. Integrative analysis of 111 reference human epig- 4. Lander ES, Linton LM, Birren B, Nusbaum C, Zody MC, Baldwin J, enomes. Nature 2015;518:317–30. et al. Initial sequencing and analysis of the human genome. 21. Leung D, Jung I, Rajagopal N, Schmitt A, Selvaraj S, Lee AY, et al. Nature 2001;409:860–921. Integrative analysis of haplotype-resolved epigenomes across 5. Venter JC, Adams MD, Myers EW, Li PW, Mural RJ, Sutton GG, et al. human tissues. Nature 2015;518:350–4. The sequence of the human genome. Science 2001;291:1304–51. 22. Calvanese V, Lara E, Kahn A, Fraga MF. The role of epigenetics 6. Watson J, Crick FH. Molecular structure of nucleic acids. Nature in aging and age-related diseases. Ageing Res Rev 2009;8: 1953;171:737–8. 268–76. 7. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain- 23. Johnson AA, Akman K, Calimport SR, Wuttke D, Stolzing A, terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463–7. de Magalhães JP. The role of DNA methylation in aging, 8. Feinberg AP, Tycko B. The history of cancer epigenetics. Nat Rev rejuvenation, and age-related Disease. Rejuvenation Res Cancer 2004;4:143–53. 2012;15:483–94.