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
References
Mill J, Heijmans BT (2013) From promises to practical strategies in epigenetic epidemiology. Nat Rev Genet 14(8):585–594
Rao JS, Keleshian VL, Klein S, Rapoport SI (2012) Epigenetic modifications in frontal cortex from Alzheimer’s disease and bipolar disorder patients. Transl Psychiatry 2:e132
Xiao Y, Camarillo C, Ping Y, Arana TB, Zhao H, Thompson PM, Xu C, Su BB et al (2014) The DNA methylome and transcriptome of different brain regions in schizophrenia and bipolar disorder. PLoS One 9(4):e95875
Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, Jia P, Assadzadeh A et al (2008) Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. Am J Hum Genet 82(3):696–711
Ladd-Acosta C, Hansen KD, Briem E, Fallin MD, Kaufmann WE, Feinberg AP (2014) Common DNA methylation alterations in multiple brain regions in autism. Mol Psychiatry 19(8):862–871
Dempster EL, Pidsley R, Schalkwyk LC, Owens S, Georgiades A, Kane F, Kalidindi S, Picchioni M et al (2011) Disease-associated epigenetic changes in monozygotic twins discordant for schizophrenia and bipolar disorder. Hum Mol Genet 20(24):4786–4796
Kato T, Iwamoto K (2014) Comprehensive DNA methylation and hydroxymethylation analysis in the human brain and its implication in mental disorders. Neuropharmacology 80:133–139
Pidsley R, Viana J, Hannon E, Spiers H, Troakes C, Al-Saraj S, Mechawar N, Turecki G et al (2014) Methylomic profiling of human brain tissue supports a neurodevelopmental origin for schizophrenia. Genome Biol 15(10):483
Wockner LF, Noble EP, Lawford BR, Young RM, Morris CP, Whitehall VL, Voisey J (2014) Genome-wide DNA methylation analysis of human brain tissue from schizophrenia patients. Transl Psychiatry 4:e339
Koshiba M, Ogawa K, Hamazaki S, Sugiyama T, Ogawa O, Kitajima T (1993) The effect of formalin fixation on DNA and the extraction of high-molecular-weight DNA from fixed and embedded tissues. Pathol Res Pract 189(1):66–72
Grafstrom RC, Fornace A Jr, Harris CC (1984) Repair of DNA damage caused by formaldehyde in human cells. Cancer Res 44(10):4323–4327
Ludyga N, Grunwald B, Azimzadeh O, Englert S, Hofler H, Tapio S, Aubele M (2012) Nucleic acids from long-term preserved FFPE tissues are suitable for downstream analyses. Virchows Arch 460(2):131–140
Do H, Dobrovic A (2015) Sequence artifacts in DNA from formalin-fixed tissues: causes and strategies for minimization. Clin Chem 61(1):64–71
Gilbert MT, Haselkorn T, Bunce M, Sanchez JJ, Lucas SB, Jewell LD, Van Marck E, Worobey M (2007) The isolation of nucleic acids from fixed, paraffin-embedded tissues-which methods are useful when? PLoS One 2(6):e537
Williams C, Ponten F, Moberg C, Soderkvist P, Uhlen M, Ponten J, Sitbon G, Lundeberg J (1999) A high frequency of sequence alterations is due to formalin fixation of archival specimens. Am J Pathol 155(5):1467–1471
Quach N, Goodman MF, Shibata D (2004) In vitro mutation artifacts after formalin fixation and error prone translesion synthesis during PCR. BMC Clin Pathol 4(1):1
Gu H, Bock C, Mikkelsen TS, Jager N, Smith ZD, Tomazou E, Gnirke A, Lander ES et al (2010) Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution. Nat Methods 7(2):133–136
Weng L, Wu X, Gao H, Mu B, Li X, Wang JH, Guo C, Jin JM et al (2010) MicroRNA profiling of clear cell renal cell carcinoma by whole-genome small RNA deep sequencing of paired frozen and formalin-fixed, paraffin-embedded tissue specimens. J Pathol 222(1):41–51
Wood HM, Belvedere O, Conway C, Daly C, Chalkley R, Bickerdike M, McKinley C, Egan P et al (2010) Using next-generation sequencing for high resolution multiplex analysis of copy number variation from nanogram quantities of DNA from formalin-fixed paraffin-embedded specimens. Nucleic Acids Res 38(14):e151
Li Q, Ma Y, Li W, Xu W, Ma L, Fu G, Tian X, Wang Y et al (2014) A promoter that drives gene expression preferentially in male transgenic rats. Transgenic Res 23(2):341–349
Senguven B, Baris E, Oygur T, Berktas M (2014) Comparison of methods for the extraction of DNA from formalin-fixed, paraffin-embedded archival tissues. Int J Med Sci 11(5):494–499
Leong KJ, James J, Wen K, Taniere P, Morton DG, Bach SP, Matthews GM (2013) Impact of tissue processing, archiving and enrichment techniques on DNA methylation yield in rectal carcinoma. Exp Mol Pathol 95(3):343–349
Savioz A, Blouin JL, Guidi S, Antonarakis SE, Bouras C (1997) A method for the extraction of genomic DNA from human brain tissue fixed and stored in formalin for many years. Acta Neuropathol 93(4):408–413
Thirlwell C, Eymard M, Feber A, Teschendorff A, Pearce K, Lechner M, Widschwendter M, Beck S (2010) Genome-wide DNA methylation analysis of archival formalin-fixed paraffin-embedded tissue using the Illumina Infinium HumanMethylation27 BeadChip. Methods 52(3):248–254
Tournier B, Chapusot C, Courcet E, Martin L, Lepage C, Faivre J, Piard F (2012) Why do results conflict regarding the prognostic value of the methylation status in colon cancers? The role of the preservation method. BMC Cancer 12:12
Balic M, Pichler M, Strutz J, Heitzer E, Ausch C, Samonigg H, Cote RJ, Dandachi N (2009) High quality assessment of DNA methylation in archival tissues from colorectal cancer patients using quantitative high-resolution melting analysis. The Journal of molecular diagnostics : JMD 11(2):102–108
de Ruijter TC, de Hoon JP, Slaats J, de Vries B, Janssen MJ, van Wezel T, Aarts MJ, van Engeland M et al (2015) Formalin-fixed, paraffin-embedded (FFPE) tissue epigenomics using Infinium HumanMethylation450 BeadChip assays. Lab Investig 95(7):833–842
Sadi AM, Wang DY, Youngson BJ, Miller N, Boerner S, Done SJ, Leong WL (2011) Clinical relevance of DNA microarray analyses using archival formalin-fixed paraffin-embedded breast cancer specimens. BMC Cancer 11(253):251–213
Dorph-Petersen K-A, Rosenberg R (2006) The brain collection at Aarhus Psychiatric University Hospital in Risskov, Denmark. J Neural Transm 6(113):11–12
Thomsen R, Solvsten CA, Linnet TE, Blechingberg J, Nielsen AL (2010) Analysis of qPCR data by converting exponentially related Ct values into linearly related X0 values. J Bioinforma Comput Biol 8(5):885–900
Morris TJ, Butcher LM, Feber A, Teschendorff AE, Chakravarthy AR, Wojdacz TK, Beck S (2014) ChAMP: 450k Chip Analysis Methylation Pipeline. Bioinformatics 30(3):428–430
Aryee MJ, Jaffe AE, Corrada-Bravo H, Ladd-Acosta C, Feinberg AP, Hansen KD, Irizarry RA (2014) Minfi: a flexible and comprehensive Bioconductor package for the analysis of Infinium DNA methylation microarrays. Bioinformatics 30(10):1363–1369
Bonin S, Petrera F, Niccolini B, Stanta G (2003) PCR analysis in archival postmortem tissues. Molecular pathology : MP 56(3):184–186
Miething F, Hering S, Hanschke B, Dressler J (2006) Effect of fixation to the degradation of nuclear and mitochondrial DNA in different tissues. J Histochem Cytochem 54(3):371–374
Kitazawa S, Kitazawa R, Maeda S (2000) Identification of methylated cytosine from archival formalin-fixed paraffin-embedded specimens. Lab Investig 80(2):275–276
Srinivasan M, Sedmak D, Jewell S (2002) Effect of fixatives and tissue processing on the content and integrity of nucleic acids. Am J Pathol 161(6):1961–1971
Start RD, Cross SS, Smith JH (1992) Assessment of specimen fixation in a surgical pathology service. J Clin Pathol 45(6):546–547
Avila L, Yuen RK, Diego-Alvarez D, Penaherrera MS, Jiang R, Robinson WP (2010) Evaluating DNA methylation and gene expression variability in the human term placenta. Placenta 31(12):1070–1077
Vilahur N, Baccarelli AA, Bustamante M, Agramunt S, Byun HM, Fernandez MF, Sunyer J, Estivill X (2013) Storage conditions and stability of global DNA methylation in placental tissue. Epigenomics 5(3):341–348
Dietrich D, Uhl B, Sailer V, Holmes EE, Jung M, Meller S, Kristiansen G (2013) Improved PCR performance using template DNA from formalin-fixed and paraffin-embedded tissues by overcoming PCR inhibition. PLoS One 8(10):e77771
Golenberg EM, Bickel A, Weihs P (1996) Effect of highly fragmented DNA on PCR. Nucleic Acids Res 24(24):5026–5033
Hahn MA, Qiu R, Wu X, Li AX, Zhang H, Wang J, Jui J, Jin SG et al (2013) Dynamics of 5-hydroxymethylcytosine and chromatin marks in mammalian neurogenesis. Cell Rep 3(2):291–300
Globisch D, Munzel M, Muller M, Michalakis S, Wagner M, Koch S, Bruckl T, Biel M et al (2010) Tissue distribution of 5-hydroxymethylcytosine and search for active demethylation intermediates. PLoS One 5(12):e15367
Lister R, Mukamel EA, Nery JR, Urich M, Puddifoot CA, Johnson ND, Lucero J, Huang Y et al (2013) Global epigenomic reconfiguration during mammalian brain development. Science 341(6146):1237905
Bennett EA, Massilani D, Lizzo G, Daligault J, Geigl EM, Grange T (2014) Library construction for ancient genomics: single strand or double strand? BioTechniques 56(6):289-290–292-286, 298, passim
Gansauge MT, Meyer M (2013) Single-stranded DNA library preparation for the sequencing of ancient or damaged DNA. Nat Protoc 8(4):737–748
Gansauge MT, Meyer M (2014) Selective enrichment of damaged DNA molecules for ancient genome sequencing. Genome Res 24(9):1543–1549
Walker DL, Bhagwate AV, Baheti S, Smalley RL, Hilker CA, Sun Z, Cunningham JM (2015) DNA methylation profiling: comparison of genome-wide sequencing methods and the Infinium Human Methylation 450 Bead Chip. Epigenomics 7(8):1287–1302
Tost J, Gut IG (2007) DNA methylation analysis by pyrosequencing. Nat Protoc 2(9):2265–2275
Madi T, Balamurugan K, Bombardi R, Duncan G, McCord B (2012) The determination of tissue-specific DNA methylation patterns in forensic biofluids using bisulfite modification and pyrosequencing. Electrophoresis 33(12):1736–1745
Pruunsild P, Kazantseva A, Aid T, Palm K, Timmusk T (2007) Dissecting the human BDNF locus: bidirectional transcription, complex splicing, and multiple promoters. Genomics 90(3):397–406
Walsh PS, Erlich HA, Higuchi R (1992) Preferential PCR amplification of alleles: mechanisms and solutions. PCR Methods Appl 1(4):241–250
Shen L, Guo Y, Chen X, Ahmed S, Issa JP (2007) Optimizing annealing temperature overcomes bias in bisulfite PCR methylation analysis. BioTechniques 42(1):48–50, 52 passim
Rybakowski JK (2008) BDNF gene: functional Val66Met polymorphism in mood disorders and schizophrenia. Pharmacogenomics 9(11):1589–1593
Li S, Feng T, Fu L, Li Z, Lou C, Zhang X, Ma C, Cong B (2012) Pyrosequencing of a short fragment of the amelogenin gene for gender identification. Mol Biol Rep 39(6):6949–6957
Sun MA, Sun Z, Wu X, Rajaram V, Keimig D, Lim J, Zhu H, Xie H (2016) Mammalian brain development is accompanied by a dramatic increase in bipolar DNA methylation. Sci Rep 6:32298
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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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.
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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.)
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.)
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.)
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.)
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.)
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.)
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.)
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.)
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.)
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.)
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.)
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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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DOI: https://doi.org/10.1007/s12035-016-0345-x