boiled, treated with nuclease PI for 16 h at 37 C, and treated with Affymetrix Genechip Biotinylated target RNA was prepared from alkaline phosphatase(Sigma)for an additional 2 h at 37C. After 5 ug of total RNA by following the Affymetrix protocol and wasw hydrolysis, total cytosine and 5mC content was measured by hybridized on the Human U133 Plus 2.0 array G capillary electrophoresis using a P/ACE 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(AIM AIMS was dev eloped 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 plot gestion using the methylation-sensitive endonuclease Smal(Am- ersham Pharmacia Biotech, Buckinghamshire, U. K ) which leaves Questionnaires. All subjects were interviewed by properly trained zomer PspAI(Stratagene), which leaves a CCGG overhang. DNA tional habits, Physical activities, pharmacological treatments, and using specific primers that hybridize to the adaptor sequence and measured, and a family tree of genetic history was drawn up by the the restriction site and one or more additional, arbitrarily chosen interviewer. The data collected in the questionnaires were entered 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 excised from dried polyacrylamide gels and cloned into plasmid consumption of folates, alcohol, tobacco, and drugs), and numerical vectors. Automatic sequencing of multiple colones was per rmed (age, weight, and height)variables, which were subsequently used to ascertain the unique identity of the isolated band to estimate the phenotypic/environmental distance between twin palIs. Analysis of Sequence-Specific DNA Methylation. The methylation Statistics. First a descriptive value was obtained for each individual status of specific genomic DNA sequences was established by corresponding to each epigeneticvariable(5mC, AcH4, and AcH3 bisulfite genomic sequencing (9). Automatic sequencing of 12 The similarity between twin pairs for each variable was estimated for each sequence was performed to obtain data on the as the Euclidean squared distance(ESD) by subtracting their Ition status of every single CpG dinucleotide for statistical respective descriptive values. By using these distances, the relation- Primer sequences are available in Table 2, which is ships between the phenotypic-environmental data and the epige published as supporting information on the PNAS web site netic variables were examined by categorical principal component analysis, using SPSS software(Version 11.5; SPSS, Chicago). Cat Real-Time Quantitative RT-PCR Expression Analyses. Quantitative egorical principal component analysis reduced all of the origi RT-PCRs were performed in 96-well optical plates, in a volume of variables from the questionnaire to two new uncorrelated com 20 ul per well. Each reaction mixture contained 1 ul of the nents or variables ("aging"and"health"). The aging principal appropriate dilution of the experimental cDNA (1: 10 to 1: 100), 5 component included the variables of age, weight, and height and is mol of each primer, and 10 ul of 2X SYBRGreen PCR Master an indicator of the ontogenic development of the twins. The health triplicate Expression values were normalized against the expression macological treatments. To identifie ariables of diseases and phar- Incipal con ixed-effects models and to analyzed by using the Prism 7700 Sequence Detection System the dependent variable, the variance component procedure was (Applied Biosystems). uestionnaire variables and the new phenotypic-environmental Competitive Hybridization of AIMS Products to Metaphase Chromo- variables(aging and health)vs the epigenetic data was evaluated by within the chromosomes, we hybridized equimolecular quantities Resulf on test. To study the distribution of the AIMS-amplified DNA of the twins was labeled with Spectrum Red dUTP using a Inclusion of Cases and zygosity Typing. The starting biological comparative genomic hybridization nick-translation kit( Vysis), and material studied was DNA and rNa from peripheral lymphocytes the aIMS products of the other twin was labeled with Spectrum In all cases, the true Mz twin status was confirmed by microsatellite Green. The mixture was then hybridized onto normal metaphase analysis(Fig 1A)() chromosome spreads, following the comparative genomic hybrid- Comparison of X Chromosome Inactivation Patterns in MZ Twins.One ization protocol described in ref. 9. All experiments were carried of the most obvious epigenetic layers where MZ twins may differ is X chromosome inactivation in females. where discordant Restriction Landmark Genomic Scanning (RLGS). RLGS of genomic x-linked diseases(11). We found that among MZ female twins DNA was performed as described in ref 10. DNA samples were informative for our PCR approach, 81%(13 of 16) had the san analyzed by using the restriction enzyme combination of Notl- x chromosome methylation pattern, and of these, 85%(11 of 13) EcoRV-Hinfl. Briefly, high-molecular-weight DNA was digested had an unskewed random distribution of each methylated allele with the methylation-sensitive restriction enzyme Notl, and the Although the remaining 15%(2 of 13)displayed a skewed X digestion, thus preventing the site from becoming radioactively skewed X chromosome methylation pattern that differed between labeled.Next, the DNA was digested with EcoRV, electrophoresed corresponding siblings(Fig. 1B). The presence of a differential X vernight in a 0.8% agarose gel, digested in situ with Hinfl, and chromosome inactivation pattern in the latter cases was not asso- ide Gels were dried ciated with logical and clinicopathological feature but and exposed to x-ray film for 7 days. Southern blot analysis was raises the possibility that epigenetic discordance can arise early on carried out as described in ref. 10 in the development of some MZ twins. Fraga et al. NAs|Jy26.2005|vo.102|no.30|10605boiled, treated with nuclease P1 for 16 h at 37°C, and treated with alkaline phosphatase (Sigma) for an additional 2 h at 37°C. After hydrolysis, total cytosine and 5mC content was measured by capillary electrophoresis using a PACE MDQ system (Beckman Coulter). Relative 5mC content was expressed as a percentage with respect to the total cytosine content. Amplification of Intermethylated Sites (AIMS). AIMS was developed as described in ref. 9. The nonmethylated sites were cut in an initial digestion using the methylation-sensitive endonuclease SmaI (Am￾ersham Pharmacia Biotech, Buckinghamshire, U.K.), which leaves blunt ends. A second digestion was performed using the isoschi￾zomer PspAI (Stratagene), which leaves a CCGG overhang. DNA fragments flanked by two ligated adaptors were amplified by PCR using specific primers that hybridize to the adaptor sequence and the restriction site and one or more additional, arbitrarily chosen nucleotides. The PCR products were run on polyacrylamide urea sequencing gels. Bands appearing differentially methylated were excised from dried polyacrylamide gels and cloned into plasmid vectors. Automatic sequencing of multiple colonies was performed to ascertain the unique identity of the isolated band. Analysis of Sequence-Specific DNA Methylation. The methylation status of specific genomic DNA sequences was established by bisulfite genomic sequencing (9). Automatic sequencing of 12 colonies for each sequence was performed to obtain data on the methylation status of every single CpG dinucleotide for statistical analysis. Primer sequences are available in Table 2, which is published as supporting information on the PNAS web site. Real-Time Quantitative RT-PCR Expression Analyses. Quantitative RT-PCRs were performed in 96-well optical plates, in a volume of 20
l per well. Each reaction mixture contained 1
l of the appropriate dilution of the experimental cDNA (1:10 to 1:100), 5 pmol of each primer, and 10
l of 2 SYBRGreen PCR Master Mix (Applied Biosystems). All measurements were performed in triplicate. Expression values were normalized against the expression of GAPDH as an endogenous control. PCR reactions were run and analyzed by using the Prism 7700 Sequence Detection System (Applied Biosystems). Competitive Hybridization of AIMS Products to Metaphase Chromo￾somes. To study the distribution of the AIMS-amplified DNA within the chromosomes, we hybridized equimolecular quantities from both members of the twin pair. The AIMS product from one of the twins was labeled with Spectrum Red dUTP using a comparative genomic hybridization nick-translation kit (Vysis), and the AIMS products of the other twin was labeled with Spectrum Green. The mixture was then hybridized onto normal metaphase chromosome spreads, following the comparative genomic hybrid￾ization protocol described in ref. 9. All experiments were carried out twice. Restriction Landmark Genomic Scanning (RLGS). RLGS of genomic DNA was performed as described in ref. 10. DNA samples were analyzed by using the restriction enzyme combination of NotI– EcoRV–HinfI. Briefly, high-molecular-weight DNA was digested with the methylation-sensitive restriction enzyme NotI, and the restriction sites were filled in with [- 32P]dCTP and [- 32P]dGTP. Methylation of the NotI restriction site makes it resistant to digestion, thus preventing the site from becoming radioactively labeled. Next, the DNA was digested with EcoRV, electrophoresed overnight in a 0.8% agarose gel, digested in situ with HinfI, and electrophoresed overnight in a 5% acrylamide gel. Gels were dried and exposed to x-ray film for 7 days. Southern blot analysis was carried out as described in ref. 10. Affymetrix GeneChip. Biotinylated target RNA was prepared from 5
g of total RNA by following the Affymetrix protocol and was hybridized on the Human U133 Plus 2.0 array GeneChips (Af￾fymetrix, Santa Clara, CA). The hybridization reactions were processed and scanned according to the standard Affymetrix protocols. To select genes that were differently expressed in twin siblings, ANOVA P values were adjusted by using multiple com￾parison procedures. The expression profiles of twins were com￾pared in scatter plots. Questionnaires. All subjects were interviewed by properly trained personnel to complete the questionnaire about their health, nutri￾tional habits, physical activities, pharmacological treatments, and tobacco, alcohol, and drug consumption. Weight and height were measured, and a family tree of genetic history was drawn up by the interviewer. The data collected in the questionnaires were entered into a database as nominal (natural health history), ordinal (per￾centage of lifetime shared, nutritional habits, physical activity, and consumption of folates, alcohol, tobacco, and drugs), and numerical (age, weight, and height) variables, which were subsequently used to estimate the phenotypicenvironmental distance between twin pairs. Statistics. First, a descriptive value was obtained for each individual corresponding to each epigenetic variable (5mC, AcH4, and AcH3). The similarity between twin pairs for each variable was estimated as the Euclidean squared distance (ESD) by subtracting their respective descriptive values. By using these distances, the relation￾ships between the phenotypic-environmental data and the epige￾netic variables were examined by categorical principal component analysis, using SPSS software (Version 11.5; SPSS, Chicago). Cat￾egorical principal component analysis reduced all of the original variables from the questionnaire to two new uncorrelated compo￾nents or variables (‘‘aging’’ and ‘‘health’’). The aging principal component included the variables of age, weight, and height and is an indicator of the ontogenic development of the twins. The health principal component grouped the variables of diseases and phar￾macological treatments. To identify mixed-effects models and to estimate the contribution of each random effect to the variance of the dependent variable, the variance component procedure was assessed by ANOVA. The relationship between all of the single questionnaire variables and the new phenotypic-environmental variables (aging and health) vs. the epigenetic data was evaluated by the Pearson test. Results Inclusion of Cases and Zygosity Typing. The starting biological material studied was DNA and RNA from peripheral lymphocytes. In all cases, the true MZ twin status was confirmed by microsatellite analysis (Fig. 1A) (7). Comparison of X Chromosome Inactivation Patterns in MZ Twins. One of the most obvious epigenetic layers where MZ twins may differ is X chromosome inactivation in females, where discordant skewing of X chromosome inactivation has been observed for specific X-linked diseases (11). We found that among MZ female twins informative for our PCR approach, 81% (13 of 16) had the same X chromosome methylation pattern, and of these, 85% (11 of 13) had an unskewed random distribution of each methylated allele. Although the remaining 15% (2 of 13) displayed a skewed X chromosome pattern, this pattern was always concordant between siblings. Among all female MZ twins, only 19% (3 of 16) had a skewed X chromosome methylation pattern that differed between corresponding siblings (Fig. 1B). The presence of a differential X chromosome inactivation pattern in the latter cases was not asso￾ciated with any epidemiological and clinicopathological feature but raises the possibility that epigenetic discordance can arise early on in the development of some MZ twins. Fraga et al. PNAS July 26, 2005 vol. 102 no. 30 10605 MEDICAL SCIENCES SEE COMMENTARY