Around the world, newborn babies are routinely screened for inborn errors of metabolism and other congenital disorders through testing of neonatal blood spot cards, a technique pioneered in the early 1960s by Robert Guthrie, after whom the cards are named. Collected within a few days of birth from heel pricks, Guthrie cards usually contain four blood spots 6 to 10 mm in diameter. The duration of Guthrie card archiving varies between and within countries, ranging from a few months to indefinitely. Thus, in many countries, Guthries represent a near-perfect national biorepository. In addition to serum analytes, DNA has been extracted from Guthries and has been used for such purposes as carrier screening for cystic fibrosis, detection of HIV and, more recently, genome-wide association studies. Modest degradation of DNA occurs during storage and extraction that could affect data quality [
5], although the quality appears sufficient for genomic assays. Remarkably, gene expression has also been analyzed in RNA from 20-year-old Guthries [
6]. As for the epigenetic regulators of gene expression, it has been shown that DNA methylation can be analyzed at individual genes using the widely used technique of bisulfite conversion, which converts methylation differences to sequence differences [
7]; this has been applied to Guthrie-based methylation screening of the
FMR1 (fragile × mental retardation 1) gene to predict cognitive impairment in individuals with fragile × syndrome [
5].
A recent paper by Beyan and colleagues [
8] has now taken this one stage further, conducting a proof-of-principle epigenome-wide pilot study using Guthrie card methylomics. On average, 200 ng of DNA from each 6 mm-diameter Guthrie spot was extracted and used in two methods of genome-scale methylation profiling: one array-based, the other based on immunoprecipitation of methylated DNA followed by high-throughput sequencing. For the array-based method, DNA was extracted from 10-year-old Guthries and compared with fresh blood and sperm from unrelated individuals. This approach identified tissue-specific differentially methylated regions between sperm and blood. There was an excellent genome-wide correlation between archived Guthrie DNA and fresh blood, but a weaker correlation for the subset of regions showing small (<20%) differences in methylation between the two tissues. No comparisons were reported between fresh and aged DNA from the same individual, which would have been an ideal control for the effect of storage of Guthries on measurement of DNA methylation. This represents an important caveat of the present study. For the immunoprecipitation-based method, which usually requires 2 µg of DNA, the method was adapted to work with 200 ng. The team then attempted to define the regions of the genome that differed between individuals but remained constant from birth to 3 years of age. This was an important comparison because it has been proposed that such 'metastable epialleles' are influenced by environmental and stochastic factors
in utero, remain constant thereafter, and can act as stable biomarkers for disease risk [
9]. For this, the team was careful to exclude genomic regions for which genetic heterogeneity could influence epigenetic variation and focused on clusters of variable, stable regions. Unfortunately, due to the low DNA yields, the longitudinal comparisons were limited to the array-based technique at birth and the immunoprecipitation-based technique at 3 years of age. Nevertheless, up to a dozen metastable epialleles were identified, two of which had previously been associated with human disease.
3
This article has been 