One of the biggest challenges in complex disease epigenomics is distinguishing cause from consequence. Simply comparing disease-affected vs. unaffected individuals will not reveal whether disease-associated epigenetic variation is causative for, or induced by, the disease. In this respect, comparing epigenetic profiles before and after disease onset would be invaluable, but prospective longitudinal birth-cohort studies are extremely expensive, time-consuming, and hence uncommon.

A Guthrie (or Dried Blood Spot, DBS) card is filter paper spotted with neonatal blood and used to screen for various diseases. For ~20 years, DBSs have been routinely generated in many countries (since 2000, greater than 90% of all neonates in the USA had DBSs made) and often stored indefinitely. This led us to consider whether DBSs can be used to generate whole-genome DNA methylation profiles (DNA methylomes). This would permit large-scale systematic analyses to determine whether disease-associated methylation variants are present at birth i.e. prior to overt disease. Importantly, this retrospective approach could be done at a fraction of the cost and time investment required for prospective studies. But, it would require the development of DNA methylomics methods that work on small amounts of relatively old DNA present in archived DBSs.

We have developed two different high throughput sequencing-based methods for generating DNA methylomes using nanogram quantities of DNA from greater than 10 year old DBSs. First, we developed the mini-MeDIPseq method that allows immunoprecipitation-based DNA methylomics from only 200ng of DNA. Then, we adapted BSseq (bisulfite conversion-based single bp-resolution DNA methylomes) for 100ng of DBS DNA. A range of bioinformatic analyses and comparisons with independent external data confirm the quality of the mini-MeDIPseq and BSseq data.

DBSs are already available for millions of people, of whom tens of thousands are disease-affected, and these numbers are increasing. DBS storage is also becoming more formalized. It is worth noting that spotting blood on filter paper is not limited to neonatal screening, but also used in home/field-based settings, and our methods can be applied to such samples. In summary,DBS-based DNA methylomics will help determine whether methylation variants in disease-affected individuals are present at birth, thus forming the basis of a novel retrospective longitudinal strategy for complex disease epigenomics.

Now, we would like to demonstrate, using the methods we have developed, that MZ twins are born epigenetically different, and that these differences are stable. To do this, we will need Guthrie cards from an MZ twin pair and then a follow up samples take any time between 1 - 13 years after birth. The sex, ethnicity, current age, disease-discordance etc. are of no relevance to this project. It would be good to have information on the chorionicity if available, and as many we would also be very grateful if you could provide samples for 2-3 different MZ pairs.