Differential Methylation in the GSTT1 Regulatory Region in Sudden Unexplained Death and Sudden Unexpected Death in Epilepsy.

Sudden cardiac death (SCD) is a diagnostic challenge in forensic medicine. In a relatively large proportion of the SCDs, the deaths remain unexplained after autopsy. This challenge is likely caused by unknown disease mechanisms. Changes in DNA methylation have been associated with several heart diseases, but the role of DNA methylation in SCD is unknown. In this study, we investigated DNA methylation in two SCD subtypes, sudden unexplained death (SUD) and sudden unexpected death in epilepsy (SUDEP). We assessed DNA methylation of more than 850,000 positions in cardiac tissue from nine SUD and 14 SUDEP cases using the Illumina Infinium MethylationEPIC BeadChip. In total, six differently methylated regions (DMRs) between the SUD and SUDEP cases were identified. The DMRs were located in proximity to or overlapping genes encoding proteins that are a part of the glutathione S-transferase (GST) superfamily. Whole genome sequencing (WGS) showed that the DNA methylation alterations were not caused by genetic changes, while whole transcriptome sequencing (WTS) showed that DNA methylation was associated with expression levels of the GSTT1 gene. In conclusion, our results indicate that cardiac DNA methylation is similar in SUD and SUDEP, but with regional differential methylation in proximity to GST genes.

DNA methylation profile of human dura and leptomeninges.

Healthy meninges are used as control tissue in meningioma studies usually without specification of the exact meningeal layer or macroanatomical origin but the DNA methylation profile of human meninges has not been investigated on a macroanatomical level. We undertook a proof-of-principle analysis to determine whether (1) meningeal tissues show sufficiently homogenous DNA methylation profiles to function as normal control tissue without further specification and (2) if previously described location-specific molecular signatures of meningiomas correspond to region-specific DNA methylation patterns. Dura mater and arachnoid membrane specimens were dissected from 5 anatomical locations in 2 fresh human cadavers and analyzed with the Illumina Infinium MethylationEPIC array. Dura and leptomeninges showed marked differences in global DNA methylation patterns and between rostral and caudal anatomical locations. These differences did not reflect known anatomical predilection of meningioma molecular signatures. The highest numbers of differentially methylated probes were annotated to DIPC2 and FOXP1. Samples from foramen magnum showed hypomethylation of TFAP2B compared to those from remaining locations. Thus, the DNA methylation profiles of human meninges are heterogenous in terms of meningeal layer and anatomical location. The potential variability of DNA methylation data from meningiomas should be considered in studies using meningeal controls.

Fresh and frozen cardiac tissue are comparable in DNA methylation array ß-values, but formalin-fixed, paraffin-embedded tissue may overestimate DNA methylation levels.

Untreated fresh cardiac tissue is the optimal tissue material for investigating DNA methylation patterns of cardiac biology and diseases. However, fresh tissue is difficult to obtain. Therefore, tissue stored as frozen or formalin-fixed, paraffin-embedded (FFPE) is widely used for DNA methylation studies. It is unknown whether storage conditions alter the DNA methylation in cardiac tissue. In this study, we compared the DNA methylation patterns of fresh, frozen, and FFPE cardiac tissue to investigate if the storage method affected the DNA methylation results. We used the Infinium MethylationEPIC assay to obtain genome-wide methylation levels in fresh, frozen, and FFPE tissues from nine individuals. We found that the DNA methylation levels of 21.4% of the examined CpG sites were overestimated in the FFPE samples compared to that of fresh and frozen tissue, whereas 5.7% were underestimated. Duplicate analyses of the DNA methylation patterns showed high reproducibility (precision) for frozen and FFPE tissues. In conclusion, we found that frozen and FFPE tissues gave reproducible DNA methylation results and that frozen and fresh tissues gave similar results.

SNP allele calling of Illumina Infinium Omni5-4 data using the butterfly method.

We introduce a within-sample SNP calling method, called the "butterfly method", that improves the quality of SNP calling with the Illumina Infinium Omni5-4 SNP Kit. This was done by improving how no-calls are determined from allele signal intensities. High confidence of SNP allele calling is extremely important in forensic genetics and clinical diagnostics. This paper is accompanied by two open-source R packages, omni54manifest and snpbeadchip that make SNP calling easy by helping with bookkeeping and giving easy access to meta-information about the SNPs typed with the Illumina Infinium Omni5-4 Kit (including chromosome, probe type, and SNP bases). We compared the results from our method with those obtained with the Illumina GenomeStudio software (which does not provide sample and SNP specific genotype probabilities or other quality measures), and with whole-genome sequencing (WGS). Given the signal intensities, the SNP calling quality was optimised using a threshold for the a posteriori probability of a SNP belonging to a SNP cluster. By lowering the a posteriori probability threshold for no-calls, we obtained a higher call rate than GenomeStudio. Using a higher a posteriori probability threshold, we achieved a higher concordance with the WGS data than GenomeStudio. Our method had SNP call and concordance rates with WGS data of approximately 99%.

Reproducibility of the Infinium methylationEPIC BeadChip assay using low DNA amounts.

The Infinium MethylationEPIC BeadChip (EPIC) is a reliable method for measuring the DNA methylation of more than 850,000 CpG positions. In clinical and forensic settings, it is critical to be able to work with low DNA amounts without risking reduced reproducibility. We evaluated the EPIC for a range of DNA amounts using two-fold serial dilutions investigated on two different days. While the ß-value distributions were generally unaffected by decreasing DNA amounts, the median squared Pearson's correlation coefficient (R2) of between-days ß-value comparisons decreased from 0.994 (500 ng DNA) to 0.957 (16 ng DNA). The median standard deviation of the ß-values was 0.005 and up to 0.017 (median of medians: 0.014) for ß-values around 0.6-0.7. With decreasing amounts of DNA from 500 ng to 16 ng, the percentage of probes with standard deviations ≤ 0.1 decreased from 99.9% to 99.4%. This study showed that high reproducibility results are obtained with DNA amounts in the range 125-500 ng DNA, while DNA amounts equal to 63 ng or below gave less reproducible results.