Table 1. Summary of DNA sequences with methylation changes identified by AIMS in MZ twin pairs CpG methylation, 出 Accession Function Chr. loc. CpGs Twin a Twin B Change AC006271 19p132 16.8 F3 NG_002795 Polycystic kidney disease 83 7 8p11+1 GUKT BC006249 Guanylate kinase 1 28 6.9 Chr. loc. chromosome location; -, unknown *Twelve clones per lo we performed ANOVA(see Table 4, which is published as sup- single-copy genes To confirm that these sequences featured DNA porting information on the PNAS web site). ANOVA was used to methylation differences in the twins, we carried out bisulfite sequencing of multiple clones. The analysis of four dif- pigenetic variability in the entire population, and ESD between ferent Alu sequences and two single-copy genes demonstrated that twins, as a measure of variability between twin pairs. First, we in those MZ twin pairs from which they were isolated, one sibling compared it with the resr igenetic values for each individual and had dense CpG hypermethylation, whereas the other was predom- yzed the descriptive the population, organized into young inantly hypomethylated at that particular sequence(Fig. 2B). Most (28 years old)vs. old(28 years old)age-groups. Statistical importantly, for both Alus and single-copy genes, differential analysis of individual descriptive parameters provides information methylation was associatedwith a different expression of at MZ twins are significantly more different within their pairs. We Table 1 summarizes the results n silencing or reduced express found that the epigenetic variability among individuals is high and on the PNAS web site), regardless of the age group to which individuals belong(Pearson test, P>0.05). In contrast, the variance corresponding to the ESD in the older MZ twin group is signifi- A tly higher than that obtained for the test,P< 0.05). These results suggest that older twins are epige- E difference is not associated with an increased variance in the etically more different between pairs than younger twins, and this 圈围 descriptive epigenetic parameters of the older population Finally, we also found that those twin pairs who, according to the questionnaire, had spent less of their lifetime together and/or had a more different natural health-medical history were those whe also showed the greatest differences in levels of 5mC DNA and acetylation of histones H3 and H4 levels(Pearson test, P<0.05) Genomic Screening and Loci Identification of DNA Methylation Dif- ferences in MZ Twin Pairs. We next examined where in the mz twi genomes these epigenetic differences arose by using a global methylation DNA fingerprinting technique, AIMS (9). The AIMS approach provides a methylation fingerprint constituted by multiple anonymous bands or tags, representing DNA sequences flanked by two methylated sites, which can be isolated. An illustrative AIMS result in a mz twin pair is shown in Fig. 24. Approximately 600 B AIMS bands were resolved in the gels, and we found that between 5 图圉 0.5% and 35% of the bands were different(on the basis of their resence or absence)within MZ twin pairs. Those MZ twins with the most differential aiMs bands were those with the greatest differences in 5mCDNAlevels and acetylation levels of histones H3 S 国图 and H4(Pearson test, P<0.05). Most importantly, the twin pairs with the most differential AIMs bands corresponded to MZ twins who were older, had spent less of their lifetimes together, or had Fig 3. Mapping chromosomal regions with differential DNA met different natural health-medical histories(Pearson test, P<0.05). MZ twins by using comparative genomic hybridization for methylated DNA. We then selected a subset of 53 AIMs bands differentially Competitive hybridization onto normal metaphase chromosomes of the AIMS were cloned, sequenced, and blasted against multiple sequence ization of chromosomes 1, 3, 12, and 17 are displayed. The 50-year-old twin pair databases. We found that 43%(23 of 53)of the clones matched Alu shows abundant changes in the pattem of DNa methylation observed by the lences(mainly from the Alu 6, Alu 7, and Alu 8 subfamilies), presence of green and red signals that indicate hypermethylation and hypom- 9%(5of53)matched other repetitive sequences(2 LINES, 2 MER, DNAmethylation indicated by the presence ofthe yellow color obtained byequa and 1 MIr), 34%(18 of 53)matched ESTs deposited in databases, amounts of the green and red dyes. Significant DNA methylation changes are and 13%(7 of 53)of the clones corresponded to identified indicated as thick red and green blocks in the ideograms Fraga et al. PNAs|y262005|vol.102|no.30|10607we performed ANOVA (see Table 4, which is published as sup￾porting information on the PNAS web site). ANOVA was used to compare two parameters: individual epigenetic values, to compare epigenetic variability in the entire population, and ESD between twins, as a measure of variability between twin pairs. First, we analyzed the descriptive epigenetic values for each individual and compared it with the rest of the population, organized into young (28 years old) vs. old (28 years old) age-groups. Statistical analysis of individual descriptive parameters provides information about variability within the whole population and in the two age groups. Second, we used the ESD to test whether older vs. younger MZ twins are significantly more different within their pairs. We found that the epigenetic variability among individuals is high and similar (see Table 5, which is published as supporting information on the PNAS web site), regardless of the age group to which individuals belong (Pearson test, P  0.05). In contrast, the variance corresponding to the ESD in the older MZ twin group is signifi￾cantly higher than that obtained for the younger group (Pearson test, P  0.05). These results suggest that older twins are epige￾netically more different between pairs than younger twins, and this difference is not associated with an increased variance in the descriptive epigenetic parameters of the older population. Finally, we also found that those twin pairs who, according to the questionnaire, had spent less of their lifetime together andor had a more different natural health–medical history were those who also showed the greatest differences in levels of 5mC DNA and acetylation of histones H3 and H4 levels (Pearson test, P  0.05). Genomic Screening and Loci Identification of DNA Methylation Dif￾ferences in MZ Twin Pairs. We next examined where in the MZ twin genomes these epigenetic differences arose by using a global methylation DNA fingerprinting technique, AIMS (9). The AIMS approach provides a methylation fingerprint constituted by multiple anonymous bands or tags, representing DNA sequences flanked by two methylated sites, which can be isolated. An illustrative AIMS result in a MZ twin pair is shown in Fig. 2A. Approximately 600 AIMS bands were resolved in the gels, and we found that between 0.5% and 35% of the bands were different (on the basis of their presence or absence) within MZ twin pairs. Those MZ twins with the most differential AIMS bands were those with the greatest differences in 5mC DNA levels and acetylation levels of histones H3 and H4 (Pearson test, P  0.05). Most importantly, the twin pairs with the most differential AIMS bands corresponded to MZ twins who were older, had spent less of their lifetimes together, or had different natural health–medical histories (Pearson test, P  0.05). We then selected a subset of 53 AIMS bands differentially present in MZ twin pairs for further characterization. These bands were cloned, sequenced, and blasted against multiple sequence databases. We found that 43% (23 of 53) of the clones matched Alu sequences (mainly from the Alu 6, Alu 7, and Alu 8 subfamilies), 9% (5 of 53) matched other repetitive sequences (2 LINEs, 2 MER, and 1 MIR), 34% (18 of 53) matched ESTs deposited in databases, and 13% (7 of 53) of the clones corresponded to identified single-copy genes. To confirm that these sequences featured DNA methylation differences in the twins, we carried out bisulfite genomic sequencing of multiple clones. The analysis of four dif￾ferent Alu sequences and two single-copy genes demonstrated that in those MZ twin pairs from which they were isolated, one sibling had dense CpG hypermethylation, whereas the other was predom￾inantly hypomethylated at that particular sequence (Fig. 2B). Most importantly, for both Alus and single-copy genes, differential methylation was associated with a different expression of that particular sequence in the MZ twin pair, the presence of DNA methylation being associated with silencing or reduced expression. Table 1 summarizes the results. Fig. 3. Mapping chromosomal regions with differential DNA methylation in MZ twins by using comparative genomic hybridization for methylated DNA. Competitive hybridization onto normal metaphase chromosomes of the AIMS products generated from 3- and 50-year-old twin pairs. Examples of the hybrid￾ization of chromosomes 1, 3, 12, and 17 are displayed. The 50-year-old twin pair shows abundant changes in the pattern of DNA methylation observed by the presence of green and red signals that indicate hypermethylation and hypom￾ethylation events, whereas the 3-year-old twins have a very similar distribution of DNAmethylationindicatedbythepresenceoftheyellowcolorobtainedbyequal amounts of the green and red dyes. Significant DNA methylation changes are indicated as thick red and green blocks in the ideograms. Table 1. Summary of DNA sequences with methylation changes identified by AIMS in MZ twin pairs Locus Accession Function Chr. loc. No. CpGs CpG methylation,* % Twin A Twin B Change Alu-Sx AC006271 — 19p13.2 30 72.1 55.3 16.8 Alu-Sp U14572 — 19p133 17 68.1 29.9 38.2 Alu-E2F3 AF547386 — 6p22 2 81.8 100 18.2 PKD1P2 NG
002795 Polycystic kidney disease 1 16p13 7 90.9 83.5 7.4 Alu-Sc U14571 — 8p111 4 66.6 63.3 3.3 GUK1 BC006249 Guanylate kinase 1 1q42 18 2.8 9.7 6.9 Chr. loc., chromosome location; —, unknown. *Twelve clones per locus. Fraga et al. PNAS July 26, 2005 vol. 102 no. 30 10607 MEDICAL SCIENCES SEE COMMENTARY