Global DNA hypermethylation in down syndrome placenta.

Down syndrome (DS), commonly caused by an extra copy of chromosome 21 (chr21), occurs in approximately one out of 700 live births. Precisely how an extra chr21 causes over 80 clinically defined phenotypes is not yet clear. Reduced representation bisulfite sequencing (RRBS) analysis at single base resolution revealed DNA hypermethylation in all autosomes in DS samples. We hypothesize that such global hypermethylation may be mediated by down-regulation of TET family genes involved in DNA demethylation, and down-regulation of REST/NRSF involved in transcriptional and epigenetic regulation. Genes located on chr21 were up-regulated by an average of 53% in DS compared to normal villi, while genes with promoter hypermethylation were modestly down-regulated. DNA methylation perturbation was conserved in DS placenta villi and in adult DS peripheral blood leukocytes, and enriched for genes known to be causally associated with DS phenotypes. Our data suggest that global epigenetic changes may occur early in development and contribute to DS phenotypes.

Improved reduced representation bisulfite sequencing for epigenomic profiling of clinical samples.

BACKGROUND: DNA methylation plays crucial roles in epigenetic gene regulation in normal development and disease pathogenesis. Efficient and accurate quantification of DNA methylation at single base resolution can greatly advance the knowledge of disease mechanisms and be used to identify potential biomarkers. We developed an improved pipeline based on reduced representation bisulfite sequencing (RRBS) for cost-effective genome-wide quantification of DNA methylation at single base resolution. A selection of two restriction enzymes (TaqαI and MspI) enables a more unbiased coverage of genomic regions of different CpG densities. We further developed a highly automated software package to analyze bisulfite sequencing results from the Solexa GAIIx system. RESULTS: With two sequencing lanes, we were able to quantify ~1.8 million individual CpG sites at a minimum sequencing depth of 10. Overall, about 76.7% of CpG islands, 54.9% of CpG island shores and 52.2% of core promoters in the human genome were covered with at least 3 CpG sites per region. CONCLUSIONS: With this new pipeline, it is now possible to perform whole-genome DNA methylation analysis at single base resolution for a large number of samples for understanding how DNA methylation and its changes are involved in development, differentiation, and disease pathogenesis.

DNMT1 and AIM1 Imprinting in human placenta revealed through a genome-wide screen for allele-specific DNA methylation.

BACKGROUND: Genomic imprinting is an epigenetically regulated process wherein genes are expressed in a parent-of-origin specific manner. Many imprinted genes were initially identified in mice; some of these were subsequently shown not to be imprinted in humans. Such discrepancy reflects developmental, morphological and physiological differences between mouse and human tissues. This is particularly relevant for the placenta. Study of genomic imprinting thus needs to be carried out in a species and developmental stage-specific manner. We describe here a new strategy to study allele-specific DNA methylation in the human placenta for the discovery of novel imprinted genes. RESULTS: Using this methodology, we confirmed 16 differentially methylated regions (DMRs) associated with known imprinted genes. We chose 28 genomic regions for further testing and identified two imprinted genes (DNMT1 and AIM1). Both genes showed maternal allele-specific methylation and paternal allele-specific transcription. Imprinted expression for AIM1 was conserved in the cynomolgus macaque placenta, but not in other macaque tissues or in the mouse. CONCLUSIONS: Our study indicates that while there are many genomic regions with allele-specific methylation in tissues like the placenta, only a small sub-set of them are associated with allele-specific transcription, suggesting alternative functions for such genomic regions. Nonetheless, novel tissue-specific imprinted genes remain to be discovered in humans. Their identification may help us better understand embryonic and fetal development.

Further improvement in quantifying male fetal DNA in maternal plasma.

BACKGROUND: Cell-free fetal DNA (cffDNA) in maternal plasma can be clinically useful for detecting prenatal disorders and pregnancy monitoring. More sensitive, specific, and quantitative detection of cffDNA in maternal plasma may expand the clinical utility of such measurements. METHODS: We developed a quantitative real-time PCR (qPCR) assay [Y chromosome repetitive sequence (YRS) assay] based on a highly repetitive short sequence specific for the Y chromosome. Both standard qPCR and digital qPCR were performed to compare the sensitivity and specificity of this new assay against already established male DNA-specific assays. RESULTS: The YRS assay was at least 10-fold more sensitive than the currently most sensitive DYS14 assay. The YRS assay was able to detect 0.5 genome equivalents (GE) per PCR reaction when fetal DNA was present at 0.2% of the total DNA. The background noise for the YRS assay was much lower than for the DYS14 assay in analyses of plasma samples from pregnancies with female fetuses. CONCLUSIONS: The YRS assay is a substantial improvement for quantifying rare male fetal DNA in maternal plasma. The higher sensitivity and specificity may expand the clinical and research utility of cffDNA.