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Epigenetic Differences Arise During the Lifetime of Monozygotic Twins

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Epigenetic Differences Arise During the Lifetime of Monozygotic Twins Epigenetic differences arise during the lifetime of monozygotic twins Mario F. Fraga*, Esteban Ballestar*, Maria F. Paz*, Santiago Ropero*, Fernando Setien*, Maria L. Ballestar†, Damia Heine-Sun˜ er‡, Juan C. Cigudosa§, Miguel Urioste¶, Javier Benitez¶, Manuel Boix-Chornet†, Abel Sanchez-Aguilera†, Charlotte Lingʈ, Emma Carlssonʈ, Pernille Poulsen**, Allan Vaag**, Zarko Stephan††, Tim D. Spector††, Yue-Zhong Wu‡‡, Christoph Plass‡‡, and Manel Esteller*§§ *Epigenetics, §Cytogenetics, and ¶Genetic Laboratories, Spanish National Cancer Centre (CNIO), Melchor Fernandez Almagro 3, 28029 Madrid, Spain; †Department of Behavioral Science, University of Valencia, 46010 Valencia, Spain; ‡Molecular Genetics Laboratory, Genetics Department, Son Dureta Hospital, 07014 Palma de Mallorca, Spain; ʈDepartment of Clinical Sciences, University Hospital Malmo¨, Lund University, S-205 02 Malmo¨, Sweden; **Steno Diabetes Center, 2820 Gentofte, Denmark; ††Twin Research and Genetic Epidemiology Unit, St. Thomas’ Hospital, London SE1 7EH, United Kingdom; and ‡‡Human Cancer Genetics Program, Department of Molecular Virology, Immunology, and Medical Genetics, Ohio State University, Columbus, OH 43210 Edited by Stanley M. Gartler, University of Washington, Seattle, WA, and approved May 23, 2005 (received for review January 17, 2005) Monozygous twins share a common genotype. However, most Materials and Methods monozygotic twin pairs are not identical; several types of pheno- Subjects. Eighty volunteer Caucasian twins from Spain were re- typic discordance may be observed, such as differences in suscep- cruited in the study, including 30 male and 50 female subjects. Their tibilities to disease and a wide range of anthropomorphic features. mean (ϮSD) age was 30.6 (Ϯ14.2) years (range, 3–74 years). Twins There are several possible explanations for these observations, but studied included monochorionic and dichorionic. All subjects, or in one is the existence of epigenetic differences. To address this issue, the case of children, the parents, gave their informed written we examined the global and locus-specific differences in DNA consent to be included in the study. Lymphocyte cells were purified methylation and histone acetylation of a large cohort of monozy- by standard procedures and stored at Ϫ80°C. In eight cases, gotic twins. We found that, although twins are epigenetically epithelial skin cells were obtained from buccal smears. Muscle indistinguishable during the early years of life, older monozygous biopsy tissues (n ϭ 14) from the vastus lateralis muscle and s.c. twins exhibited remarkable differences in their overall content and abdominal tissue (n ϭ 4) were obtained by needle suction under genomic distribution of 5-methylcytosine DNA and histone acety- local anesthesia from volunteer MZ twins from Denmark and the lation, affecting their gene-expression portrait. These findings United Kingdom, respectively. Homozygosity was determined by indicate how an appreciation of epigenetics is missing from our using highly polymorphic short tandem-repeat loci. With five understanding of how different phenotypes can be originated markers, the probability that any twin pair was MZ if all markers from the same genotype. were concordant was 99% (5). DNA methylation ͉ epigenetics ͉ histones X-Inactivation Analysis. Androgen receptor locus methylation anal- ysis was performed based on PCR on genomic DNA digested with uman monozygotic (MZ) twins account for 1 in 250 live births the methylation-sensitive restriction enzyme HpaII where only the H(1). The origin of MZ twins is attributed to two or more androgen receptor gene residing on the inactivated X chromosome daughter cells of a single zygote undergoing independent mitotic is amplified (6, 7). divisions, leading to independent development and births. They are considered genetically identical, but significant phenotypic discor- High-Performance Capillary Electrophoresis Quantification of Global dance between them may exist. This quality is particularly notice- Histone H3 and H4 Acetylation. Global histone H4 acetylation able for psychiatric diseases, such as schizophrenia and bipolar (AcH4) and histone H3 acetylation (AcH3) were quantified as disorder (2). MZ twins have been used to demonstrate the role of described in ref. 8. In brief, individual histone H3 and histone H4 environmental factors in determining complex diseases and phe- fractions were prepared from cell nuclei and further purified by notypes, but the true nature of the phenotypic discordance never- reversed-phase high-performance liquid chromatography (HPLC) theless remains extremely poorly understood. In this context, on a Jupiter C18 column (Phenomenex, Torrance, CA). Histones differences in the placenta, amniotic sac, and vascularization of the were eluted with an acetonitrile gradient (20–60%) in 0.3% trif- separate cell masses or even mosaicism in genetic and cytogenetic luoroacetic acid using an HPLC gradient system (Beckman markers in MZ may exist (3), although the published studies are Coulter). Nonacetylated, monoacetylated, and diacetylated histone H3 and H4 derivatives were resolved by high-performance capillary very few in number. Thus, the real causes for MZ twin discordance electrophoresis. All samples were analyzed in duplicate, and three for common diseases and traits remain to be established. Epigenetic measurements were made per replicate. differences may be an important part of the solution to this puzzle. Indeed, epigenetic profiles may represent the link between an Quantification of Total DNA Methylation. 5-Methylcytosine (5mC) environmental factor and phenotypic differences in MZ twins. content was determined by high-performance capillary electro- Cloned animals provide another example of how epigenetics may phoresis, as described in ref. 8. Briefly, genomic DNA samples were explain phenotypic differences in beings that have identical genetic sequences. In this case, inefficient epigenetic reprogramming of the transplanted nucleus is associated with aberrations in imprinting, This paper was submitted directly (Track II) to the PNAS office. aberrant growth, and lethality beyond a threshold of faulty epige- Freely available online through the PNAS open access option. netic control (4). MZ twins are another phenomenon in which Abbreviations: AIMS, amplification of intermethylated sites; ESD, Euclidean squared dis- epigenetics can ‘‘make the difference.’’ To address this possibility, tance; 5mC, 5-methylcytosine; MZ, monozygotic; RLGS, restriction landmark genomic we have profiled the epigenetic patterns related to global and scanning. locus-specific DNA methylation and histone H3 and H4 acetylation See Commentary on page 10413. in the largest series of MZ twins for which molecular studies have §§To whom correspondence should be addressed. E-mail: [email protected]. been reported. © 2005 by The National Academy of Sciences of the USA 10604–10609 ͉ PNAS ͉ July 26, 2005 ͉ vol. 102 ͉ no. 30 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0500398102 Downloaded by guest on September 25, 2021 boiled, treated with nuclease P1 for 16 h at 37°C, and treated with Affymetrix GeneChip. Biotinylated target RNA was prepared from alkaline phosphatase (Sigma) for an additional2hat37°C.After 5 ␮g of total RNA by following the Affymetrix protocol and was hydrolysis, total cytosine and 5mC content was measured by hybridized on the Human U133 Plus 2.0 array GeneChips (Af- SEE COMMENTARY capillary electrophoresis using a P͞ACE MDQ system (Beckman fymetrix, Santa Clara, CA). The hybridization reactions were Coulter). Relative 5mC content was expressed as a percentage with processed and scanned according to the standard Affymetrix respect to the total cytosine content. protocols. To select genes that were differently expressed in twin siblings, ANOVA P values were adjusted by using multiple com- Amplification of Intermethylated Sites (AIMS). AIMS was developed parison procedures. The expression profiles of twins were com- as described in ref. 9. The nonmethylated sites were cut in an initial pared in scatter plots. digestion using the methylation-sensitive endonuclease SmaI (Am- ersham Pharmacia Biotech, Buckinghamshire, U.K.), which leaves Questionnaires. All subjects were interviewed by properly trained blunt ends. A second digestion was performed using the isoschi- personnel to complete the questionnaire about their health, nutri- zomer PspAI (Stratagene), which leaves a CCGG overhang. DNA tional habits, physical activities, pharmacological treatments, and fragments flanked by two ligated adaptors were amplified by PCR tobacco, alcohol, and drug consumption. Weight and height were using specific primers that hybridize to the adaptor sequence and measured, and a family tree of genetic history was drawn up by the interviewer. The data collected in the questionnaires were entered the restriction site and one or more additional, arbitrarily chosen into a database as nominal (natural health history), ordinal (per- nucleotides. The PCR products were run on polyacrylamide urea centage of lifetime shared, nutritional habits, physical activity, and sequencing gels. Bands appearing differentially methylated were consumption of folates, alcohol, tobacco, and drugs), and numerical excised from dried polyacrylamide gels and cloned into plasmid (age, weight, and height) variables, which were subsequently used vectors. Automatic sequencing of multiple colonies was performed to estimate the phenotypic͞environmental distance between twin to ascertain the unique identity of the
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