Comparison of Genomic and Epigenomic Expression in Monozygotic Twins Discordant for Rett Syndrome.

Monozygotic (identical) twins have been widely used in genetic studies to determine the relative contributions of heredity and the environment in human diseases. Discordance in disease manifestation between affected monozygotic twins has been attributed to either environmental factors or different patterns of X chromosome inactivation (XCI). However, recent studies have identified genetic and epigenetic differences between monozygotic twins, thereby challenging the accepted experimental model for distinguishing the effects of nature and nurture. Here, we report the genomic and epigenomic sequences in skin fibroblasts of a discordant monozygotic twin pair with Rett syndrome, an X-linked neurodevelopmental disorder characterized by autistic features, epileptic seizures, gait ataxia and stereotypical hand movements. The twins shared the same de novo mutation in exon 4 of the MECP2 gene (G269AfsX288), which was paternal in origin and occurred during spermatogenesis. The XCI patterns in the twins did not differ in lymphocytes, skin fibroblasts, and hair cells (which originate from ectoderm as does neuronal tissue). No reproducible differences were detected between the twins in single nucleotide polymorphisms (SNPs), insertion-deletion polymorphisms (indels), or copy number variations. Differences in DNA methylation between the twins were detected in fibroblasts in the upstream regions of genes involved in brain function and skeletal tissues such as Mohawk Homeobox (MKX), Brain-type Creatine Kinase (CKB), and FYN Tyrosine Kinase Protooncogene (FYN). The level of methylation in these upstream regions was inversely correlated with the level of gene expression. Thus, differences in DNA methylation patterns likely underlie the discordance in Rett phenotypes between the twins

Mutation analyses of the <i>MECP2</i> gene in the RTT twins.

<p>(A) DNA sequencing demonstrated that both twin 1 (RS-1M) and twin 2 (RS-2T) had the same mutation (1 bp “G” deletion in exon 4). (B) Determination of the parental origin of the mutation. (Upper) Sequencing of <i>MECP2</i> in a somatic hybrid cell clone carrying an RS-1M derived maternal X chromosome. (Lower) Sequencing of <i>MECP2</i> in a somatic hybrid cell clone carrying an RS-1M derived paternal X chromosome. The parent of origin of the X chromosome was determined by the HUMARA X chromosome inactivation assay.</p

X chromosome inactivation analyses.

<p>Xi: X inactivation pattern based on the inactive X chromosome, Xa: X-inactivation pattern based on the active X chromosome. Mat: X chromosome inherited from the mother, Pat: X chromosome inherited from the father. Xi and Xa are differentiated by their methylation status at the androgen receptor gene locus. The maternal X and the paternal X are also differentiated by CAG repeat polymorphism at this locus. The X chromosome inactivation patterns showed no differences between the twins in lymphoblasts (A), skin fibroblasts (B), or hair cells (C).</p

DNA methylation and expression analyses for the <i>MECP2</i> gene.

<p>(A) DNA methylation profile in the upstream region of <i>MECP2</i> by bisulfite sequencing. The upper number indicates the position of the CpG from the transcription start site of <i>MECP2</i>. (B) Results of a qRT-PCR assay for MECP2 gene.</p

Validation of DNA methylation status and expression difference between the twins.

<p>(A) DNA methylation difference between the twins by bisulfite sequencing in the upstream regions of <i>Mohawk Homeobox (MKX), Brain-type Creatine Kinase (CKB)</i>, and <i>FYN Tyrosine Kinase Protooncogene (FYN)</i> genes. The proportion (%) of methylated CpG sites is shown. (B) Expression difference between the twins. *p<0.05.</p

Results of genome-wide analysis of DNA methylation in the twins.

<p>(A) Correlation of DNA methylation at the CpG sites analyzed between the twins based on an average (AVG) beta value. (B) Differentially methylated CpG sites between the twins. Red indicates hypermethylation and green indicates hypomethylation.</p