Archived Blood Spots Research Articles

Variable DNA methylation in neonates mediates the association between prenatal smoking and birth weight.

There is great interest in the role epigenetic variation induced by non-genetic exposures may play in the context of health and disease. In particular, DNA methylation has previously been shown to be highly dynamic during the earliest stages of development and is influenced by in utero exposures such as maternal smoking and medication. In this study we sought to identify the specific DNA methylation differences in blood associated with prenatal and birth factors, including birth weight, gestational age and maternal smoking. We quantified neonatal methylomic variation in 1263 infants using DNA isolated from a unique collection of archived blood spots taken shortly after birth (mean = 6.08 days; s.d. = 3.24 days). An epigenome-wide association study (EWAS) of gestational age and birth weight identified 4299 and 18 differentially methylated positions (DMPs) respectively, at an experiment-wide significance threshold of p < 1 × 10-7. Our EWAS of maternal smoking during pregnancy identified 110 DMPs in neonatal blood, replicating previously reported genomic loci, including AHRR. Finally, we tested the hypothesis that DNA methylation mediates the relationship between maternal smoking and lower birth weight, finding evidence that methylomic variation at three DMPs may link exposure to outcome. These findings complement an expanding literature on the epigenomic consequences of prenatal exposures and obstetric factors, confirming a link between the maternal environment and gene regulation in neonates. This article is part of the theme issue 'Developing differences: early-life effects and evolutionary medicine'.

Elevated polygenic burden for autism is associated with differential DNA methylation at birth

BackgroundAutism spectrum disorder (ASD) is a severe neurodevelopmental disorder characterized by deficits in social communication and restricted, repetitive behaviors, interests, or activities. The etiology of ASD involves both inherited and environmental risk factors, with epigenetic processes hypothesized as one mechanism by which both genetic and non-genetic variation influence gene regulation and pathogenesis. The aim of this study was to identify DNA methylation biomarkers of ASD detectable at birth.MethodsWe quantified neonatal methylomic variation in 1263 infants—of whom ~ 50% went on to subsequently develop ASD—using DNA isolated from archived blood spots taken shortly after birth. We used matched genotype data from the same individuals to examine the molecular consequences of ASD-associated genetic risk variants, identifying methylomic variation associated with elevated polygenic burden for ASD. In addition, we performed DNA methylation quantitative trait loci (mQTL) mapping to prioritize target genes from ASD GWAS findings.ResultsWe identified robust epigenetic signatures of gestational age and prenatal tobacco exposure, confirming the utility of DNA methylation data generated from neonatal blood spots. Although we did not identify specific loci showing robust differences in neonatal DNA methylation associated with later ASD, there was a significant association between increased polygenic burden for autism and methylomic variation at specific loci. Each unit of elevated ASD polygenic risk score was associated with a mean increase in DNA methylation of − 0.14% at two CpG sites located proximal to a robust GWAS signal for ASD on chromosome 8.ConclusionsThis study is the largest analysis of DNA methylation in ASD undertaken and the first to integrate genetic and epigenetic variation at birth. We demonstrate the utility of using a polygenic risk score to identify molecular variation associated with disease, and of using mQTL to refine the functional and regulatory variation associated with ASD risk variants.

Long-term storage limits PCR-based analyses of malaria parasites in archival dried blood spots

BackgroundBlood samples collected in epidemiological and clinical investigations and then stored, often at room temperature, as blood spots dried on a filter paper have become one of the most popular source of material for further molecular analyses of malaria parasites. The dried blood spots are often archived so that they can be used for further retrospective investigations of parasite prevalence, or as new genetic markers come to the fore. However, the suitability of the template obtained from dried blood spots that have been stored for long periods for DNA amplification is not known.MethodsDNA from 267 archived blood spots collected over a period of 12 years from persons with microscopically confirmed Plasmodium falciparum infection was purified by one of two methods, Chelex and Qiagen columns. These templates were subjected to highly sensitive nested PCR amplification targeting three parasite loci that differ in length and/or copy number.ResultsWhen a 1.6 kb fragment of the parasites’ small subunit ribosomal RNA was targeted (primary amplification), the efficiency of P. falciparum detection decreased in samples archived for more than six years, reaching very low levels for those stored for more than 10 years. Positive amplification was generally obtained more often with Qiagen-extracted templates. P. falciparum could be detected in 32 of the 40 negative Qiagen-extracted templates when a microsatellite of about 180 bp was targeted. The remaining eight samples gave a positive amplification when a small region of 238 bp of the higher copy number (20 to 200) mitochondrial genome was targeted.ConclusionsThe average length of DNA fragments that can be recovered from dried blood spots decreases with storage time. Recovery of the DNA is somewhat improved, especially in older samples, by the use of a commercial DNA purification column, but targets larger than 1.5 kb are unlikely to be present 10 years after the initial blood collection, when the average length of the DNA fragments present is likely to be around a few hundred bp. In conclusion, the utility of archived dried blood spots for molecular analyses decreases with storage time.

Novel approach for deriving genome wide SNP analysis data from archived blood spots

BackgroundThe ability to transport and store DNA at room temperature in low volumes has the advantage of optimising cost, time and storage space. Blood spots on adapted filter papers are popular for this, with FTA (Flinders Technology Associates) Whatman™TM technology being one of the most recent. Plant material, plasmids, viral particles, bacteria and animal blood have been stored and transported successfully using this technology, however the method of porcine DNA extraction from FTA Whatman™TM cards is a relatively new approach, allowing nucleic acids to be ready for downstream applications such as PCR, whole genome amplification, sequencing and subsequent application to single nucleotide polymorphism microarrays has hitherto been under-explored.FindingsDNA was extracted from FTA Whatman™TM cards (following adaptations of the manufacturer’s instructions), whole genome amplified and subsequently analysed to validate the integrity of the DNA for downstream SNP analysis. DNA was successfully extracted from 288/288 samples and amplified by WGA. Allele dropout post WGA, was observed in less than 2% of samples and there was no clear evidence of amplification bias nor contamination. Acceptable call rates on porcine SNP chips were also achieved using DNA extracted and amplified in this way.ConclusionsDNA extracted from FTA Whatman cards is of a high enough quality and quantity following whole genomic amplification to perform meaningful SNP chip studies.

Archived blood spots could be epigenetic jackpot

Archived blood spots could be epigenetic jackpot

Genetic Information from Archived Blood Spots

Genetic Information from Archived Blood Spots

Microarray-based Detection of Mannose-binding Lectin 2 (MBL2) Polymorphisms in a Routine Clinical Setting

The assessment of allelic variants in the human mannose-binding lectin 2 (MBL2) gene is of great clinical importance in newborns or immune-suppressed patients at high risk for a variety of infections. Here, we present a study on the genotyping accuracy of a DNA microarray-based on-chip PCR method suited for the detection of five different polymorphisms in the MBL2 gene. We tested 153 genomic DNA samples, prepared from archival blood spots on Guthrie cards, for the presence of allelic variants in the human MBL2 gene by the on-chip PCR method and compared the obtained results of three variants to standard DNA capillary sequencing. The genotyping power of the described assay was readily comparable to DNA sequencing (453/459 correct genotype calls in 153 DNA samples; 98.7% accuracy), mainly due to intrinsic technical benefits of microarrays such as high number of test replicates and automated data analysis. This study demonstrates, for the first time, the accuracy and reliability of a microarray-based on-chip PCR genotyping assay for measuring allelic variants in a routine clinical setting.