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Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples

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Abstract

We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.

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Abbreviations

5mC:

DNA methylation

5hmC:

DNA hydroxymethylation

Alz:

Alzheimer’s disease

B:

Brain

Bd:

Bipolar disorder

C:

Cortex

FFPE:

Formalin-fixed paraffin-embedded

FF:

Fresh frozen

H:

Hippocampus

hMeDIP:

Hydroxymethylated DNA immunoprecipitation

MeDIP:

Methylated DNA immunoprecipitation

NC:

Negative control

NGS:

Next-generation sequencing

SD:

Standard deviation

Sz:

Schizophrenia

AUBC:

The Brain Collection at Aarhus University Hospital, Risskov, Denmark

QC:

Quality control

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Acknowledgements

We thank Ida E. Holm, Department of Pathology, Randers Hospital, Denmark, for support of fresh frozen brain material. This study was supported by the The Lundbeck Foundation Initiative for Integrative Psychiatric Research (iPSYCH), Villum Foundation (for the Centre for Stochastic Geometry and Advanced Bioimaging), Fonden til Lægevidenskabens Fremme, and The Toyota Foundation.

Authors’ Contributions

STB, NHS, MN, AB, JRN, OM, KADP, and ALN conceived and designed the study. STB, NHS, AS, and TFD performed biological experiments. All authors contributed to the writing of the manuscript.

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Authors and Affiliations

Authors

Corresponding author

Correspondence to Anders L. Nielsen.

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Conflict of Interest

The authors declare that they have no conflict of interest.

Ethics Statement

Usage of samples from The Brain Collection, Psychiatric Hospital, Aarhus University Hospital, Risskov, Denmark, as well as the experimental work was approved by the Danish National Committee for Health Research Ethics (license no. 1400077). All patient identification data are anonymized.

Additional information

Stine T. Bak and Nicklas H. Staunstrup contributed equally to the work.

Electronic supplementary material

Suppl. Table S1

Primers utilized for qPCR. *, amplicon size information not provided by the manufacturer. (PDF 317 kb.)

Suppl. Table S2

Primers for pyrosequencing experiments and sequence information for the applied assays. (PDF 317 kb.)

Suppl. Table S3

Overview of DNA concentration levels from hippocampus samples B40H and B39H estimated by Picogreen, Oligreen, and Nanodrop. (PDF 284 kb.)

Suppl. Table S4

Infinium HD FFPE QC assay. Quality DNA score (delta Ct) obtained with the Infinium HD FFPE QC assay performed on DNA extracted from cortex samples B39C and B40C, as well as hippocampus samples B39H and B40H. (PDF 279 kb.)

Suppl. Table S5

Summary of two-step QC assay. (PDF 284 kb.)

Suppl. Fig S1

Overview of the brain samples and controls used for the various methods employed in the current work. (GIF 115 kb.)

High resolution image (EPS 4703 kb.)

Suppl. Fig S2

(A) Experimental setup for characterization of the extracted DNA. RNase treated DNA samples were either digested with ssDNA specific nucleases also targeting dsDNA with gabs or nicks, dsDNA specific nucleases or left untreated before quantification. (B) Amplification efficiency (Eamp) using genomic control DNA and Sat2 primers spanning amplicon lengths of 67 bp, 105 bp, and 160 bp, measured at an undiluted and at an ×10, ×30, and ×100 diluted concentration. The Ct-values were plotted versus corresponding concentrations with the start concentration set to 100 ng. Eamp was measured to be 76%, 79%, and 73% for Sat2–67, Sat2–105, and Sat2–160, respectively. (GIF 25 kb.)

High resolution image (EPS 1354 kb.)

Suppl. Fig S3

DNA methylation and hydroxymethylation in archived FFPE brain samples. (A) Relative levels of whole genome DNA methylation (5mC) measured by ELISA in the four FFPE brain samples B40H, B40C, B39H and B39C and two fresh frozen brain samples, BFF1H and BFF1C. Values are presented relative to BFF1C. NC, negative control. (B) Global levels of DNA hydroxymethylation measured by ELISA in the four FFPE brain samples B40H, B40C, B39H and B39C and two fresh frozen brain samples (BFF1H and BFF1C). (C) Recovery of the methylated long interspersed nuclear element 1 (LINE-1) from FFPE B40 samples measured after MeDIP with LINE-1 targeting primers. (D) Enrichment of DNA hydroxymethylation after immunoprecipitation with an hmeDNA specific antibody or an isotype (IgG) control. Recovery was measured by qPCR employing methylated DNA (meDNA), unmethylated DNA (unDNA), hydroxymethylated DNA (hmeDNA), Chr.1–1 (positive control), and GAPDH (negative control) specific primers. (E) Enrichment of hydroxymethylated DNA relative to unmethylated DNA for the B40C sample. Values are presented relative to BFF1C. NC, negative control. (GIF 45 kb.)

High resolution image (EPS 953 kb.)

Suppl. Fig S4

Methylation microarray analysis based on the brain FFPE samples. (A) Amount of failed probes for each brain and blood (A1-A4) sample in the methylation microarray analysis. (B) Density plot of DNA methylation signals present in each sample. (GIF 102 kb.)

High resolution image (EPS 1439 kb.)

Suppl. Fig S5

DNA methylation analysis of CpG- sites in ST6GALNAC1, BDNF, and HIST3H3. (A) Methylation profiling of CG13015534 in ST6GALNAC1. Clonal methylation analysis of bisulfite converted B39H and B40H DNA with methylated sites marked as black and un-methylated sites marked as white circles. Methylation percentages are indicated. (B) Bisulfite pyrosequencing of BDNF promoter 4 showing representative results for B39H, B40H, and BFF1H. (C) Bisulfite pyrosequencing of HIST3H3 promoter showing representative results for B39H, B40H, and BFF1H. For the bisulfite pyrosequencing results methylation percentages are indicated in each panel. Orange bars; intrinsic control for bisulfite conversion efficiency. (GIF 117 kb.)

High resolution image (EPS 1877 kb.)

Suppl. Fig S6

Bisulfite pyrosequencing of CpG site CG13015534 in ST6GALNAC1. (A) Experimental setup for bisulfite pyrosequencing in triplicates. (B) Representative triplicates for hippocampus samples B39H, B40H, and the positive control BFF1H. (C) Representative triplicates for cortex B39C, B40C, and the positive control BFF1C. Methylation percentages are displayed in each panel. Note that the second position analyzed is spiked in and not a bona fide methylation site. Orange bars; intrinsic control for bisulfite conversion efficiency. (GIF 321 kb.)

High resolution image (EPS 3859 kb.)

Suppl. Fig S7

CpG-site and SNP used for the genotype-specific methylation assay (A) Schematic illustration of the G/A SNP rs6265 and the potential change in DNA methylation due to the underlying CG to CA sequence alteration. Position of the SNP rs6265 is marked by a red square revealing the expected G/A-heterozygosity. (B) Pyrosequencing based genotyping of BDNF SNP rs6265. (GIF 83 kb.)

High resolution image (EPS 1129 kb.)

Suppl. Fig S8

Pyrosequencing of BDNF SNP rs6265 and the corresponding DNA methylation. (A) Representative bisulfite pyrosequencing results on B39H, B40H, and BFF1H DNA. Methylation percentages for CG2 and CG1 are presented in each panel. (B) Bisulfite pyrosequencing analysis of CG2 and CG1 in B39C with methylation percentages displayed. The presented experiment was selected on the basis of equal G/A heights. For a B39H pyrosequencing result with equal G/A heights see upper left pyrogram in panel A. Orange bars; intrinsic control for bisulfite conversion efficiency. (GIF 216 kb.)

High resolution image (EPS 2360 kb.)

Suppl. Fig S9

Sex prediction using an AMLXY pyrosequencing assay. Representative pyrosequencing results depicted for a female control, a male control, B39H and B40H. (GIF 139 kb.)

High resolution image (EPS 1317 kb.)

Suppl. Fig S10

Sex prediction by bisulfite pyrosequencing analyses of a SLC9A7 promoter region. (A) Screenshot of the methylation status of CG18799866 in the SLC9A7 promoter as a function of age from the BrainCloudMethyl database. (B) Braincloud screenshot showing the relative methylation status of CG18799866 stratified by sex with females on the left (red) and males on the right (blue). (C) Representative bisulfite pyrosequencing results for a female control, a male control, B39H, and B40H. CG18799866 corresponds to the second of the two CpGs encountered in the pyrosequencing assay. Methylation percentage for each individual was calculated as the mean methylation for these CpGs. Orange bars; intrinsic control for bisulfite conversion efficiency. (GIF 190 kb.)

High resolution image (EPS 1755 kb.)

Suppl. Fig S11

DNA integrity in FFPE brain samples display storage time dependency. QC evaluated brain samples were divided into three equally-width bins as a function of sample storage time (years), age of individual at death (years) or post mortem interval (hours). Chi-square statistics indicated an association between storage time and QC outcome (Chi-square statistic =17.7828, p-value =0.000138). (GIF 31 kb.)

High resolution image (EPS 1133 kb.)

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Bak, S.T., Staunstrup, N.H., Starnawska, A. et al. Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples. Mol Neurobiol 55, 668–681 (2018). https://doi.org/10.1007/s12035-016-0345-x

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