Epigenetic differences arise in MZ twins. (A) Two representative examples of the determination of monozygosity using microsatellite markers. (B) Quantification of X chromosome inactivation by PCR amplification of the androgen receptor locus after digestion with the DNA methylation-sensitive and -insensitive restriction enzymes HpaII and MSp I, respectively. Two examples of a different pattern of X inactivation between MZ twin pairs are shown. (C Upper) Quantification of global 5mC DNA content (Left), histone H4 acetylation (Center), and histone H3 acetylation (Right) by HPLC and high-performance capillary electrophoresis. (C Lower) Comparison of epigenetic values between the siblings of each 3- and 50-year-old twin pair. Results are expressed as mean ± SD.

Mapping sequences with differential DNA methylation between MZ twins using AIMS. (A Left) Example of an AIMS analysis in 3- and 50-year-old twin pairs. Differential anonymous bands corresponding to sibling-specific changes of DNA methylation are indicated with arrows. (A Right) Number of differential bands obtained by AIMS in 3- and 50-year-old twin pairs. (B) Bisulfite genomic sequencing of 12 clones of the repetitive DNA sequence Alu-SP obtained by AIMS in 3- and 50-year-old twin pairs. Schematic representations of the methylation status of each CpG dinucleotide. Black and white dots indicate methylated and unmethylated CpGs, respectively.

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 hybridization 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 hypomethylation events, whereas the 3-year-old twins have a very similar distribution of DNA methylation indicated by the presence of the yellow color obtained by equal amounts of the green and red dyes. Significant DNA methylation changes are indicated as thick red and green blocks in the ideograms.

Differences in promoter CpG island DNA methylation and expression profiles in MZ twins are associated with aging. (A) Example of an RLGS profile from a 50-year-old twin pair showing the presence of an RLGS fragment C14orf162 in Twin A, indicated by the arrow, in whose sister (Twin B) it is significantly reduced. (B) Southern blot analysis confirms the CpG island methylation difference in C14orf162 between twins. All lines contain DNA digested with EcoRV. The DNA was also digested with NotI, as indicated by +. (C) Bisulfite genomic sequencing of 12 clones of the C14orf162 promoter CpG island in the 50-year-old twin pair. Black and white dots indicate methylated and unmethylated CpGs, respectively. Below the sequences are two representative electropherograms showing sequence-specific DNA methylation changes at the C14orf162 promoter in the 50-year-old twin pair. (D) The older MZ twins display the greatest differences in gene expression profiles between siblings. (Left) Scatter plots showing expression profiles of 3- and 50-year-old twin pairs. (Right) Number of overexpressed and repressed genes obtained by DNA array analyses in the 3-year-old compared with the 50-year-old twin pair.