DNA Isolation Method Is a Source of Global DNA Methylation Variability Measured with LUMA. Experimental Analysis and a Systematic Review

<div><p>In DNA methylation, methyl groups are covalently bound to CpG dinucleotides. However, the assumption that methyl groups are not lost during routine DNA extraction has not been empirically tested. To avoid nonbiological associations in DNA methylation studies, it is essential to account for potential batch effect bias in the assessment of this epigenetic mechanism. Our purpose was to determine if the DNA isolation method is an independent source of variability in methylation status. We quantified Global DNA Methylation (GDM) by luminometric methylation assay (LUMA), comparing the results from 3 different DNA isolation methods. In the controlled analysis (n = 9), GDM differed slightly for the same individual depending on extraction method. In the population analysis (n = 580) there were significant differences in GDM between the 3 DNA isolation methods (medians, 78.1%, 76.5% and 75.1%; p<0.001). A systematic review of published data from LUMA GDM studies that specify DNA extraction methods is concordant with our findings. DNA isolation method is a source of GDM variability measured with LUMA. To avoid possible bias, the method used should be reported and taken into account in future DNA methylation studies.</p></div

Demographic characteristics of the Population assay.

<p>Individuals recruited from the 3 cohorts. DNA was extracted with a different isolation method for each cohort: method 1 (Autopure) and method 2 (Gentra), method 3 (Chemagic).</p>*<p>Adjusted p value for all variables in the table comparing method 1 <i>vs</i> 2 cohorts.</p>+<p>Adjusted p value for all variables in the table comparing method 1 <i>vs</i> 3 cohorts.</p>†<p>Adjusted p value for all variables in the table comparing method 2 <i>vs</i> 3 cohorts.</p><p>ns, not significant.</p

Box plot of global methylation of the 9 healthy controls using LUMA.

<p>DNA extracted by three different methods: Autopure LS (method 1), Puregen Manual kit (method 2) and Chemagic (method 3).</p

Description and characteristics of DNA isolation methods employed in the Population Analysis (n = 580).

<p>Description and characteristics of DNA isolation methods employed in the Population Analysis (n = 580).</p

Box plot of global methylation of 580 healthy subjects using LUMA.

<p>DNA extracted by three different methods. Significant methylation differences were found between the three DNA isolation methods (medians: 78.1%, method 1; 76.5%, method 2; 75.1%, method 3; ***p<0.001).</p

Comparison of global DNA methylation between groups of DNA isolation method.

<p>Table shows the differences of weighted means (with 95% confidence interval). All the comparisons were statistically significant (p<0.001).</p

Suggestive SNP-SNP interactions—primary analysis.

<p>SNP: single nucleotide polymorphism, Chr: chromosome; MAF: minor allele frequency; BHF-FHS: British Heart Foundation Family Heart Study; MIGen: Myocardial Infarction Genetics Consortium; SNPA Pvalue: level of nominal statistical significance for single marker association with coronary artery disease for SNP A; SNPB Pvalue: level of nominal statistical significance for single marker association with coronary artery disease for SNP B; Int. Pvalue BHF-FHS: interaction P value in BHF-FHS; Int. Pvalue MIGen: interaction P value in MIGen; N/A: replication not available. PDE11A: phosphodiesterase 11A; SEC1P: secretory blood group 1, pseudogene; SERPINA12: serpin peptidase inhibitor, clade A; SRI: sorcin; ZHX2: zinc fingers and homeoboxes 2; NR5A2: nuclear receptor subfamily 5, group A, member 2; ANGPTL4: angiopoietin-like 4; NRG3: neuregulin 3; RPSAP15: ribosomal protein SA pseudogene 15; CSRP3: cysteine and glycine-rich protein 3; GSTM3: glutathione S-transferase mu 3; RYR2: ryanodine receptor 2; TBXAS1: thromboxane A synthase 1; TAC1: tachykinin, precursor 1; P2RX4: purinergic receptor P2X, ligand-gated ion channel, 4; SCARB2: scavenger receptor class B, member 2; NOD1: nucleotide-binding oligomerization domain containing 1; PDGFD: platelet derived growth factor D.</p><p>Suggestive SNP-SNP interactions—primary analysis.</p

Baseline Characteristics of cases in the British Heart Foundation-Family Heart Study and the Myocardial Infarction Genetics studies.

<p>Data are means and standard deviations or counts and percentages, BHF-FHS: British Heart Foundation Family Heart Study; MIGen: Myocardial Infarction Genetics Consortium; MI: myocardial infarction; BMI: body mass index.</p><p>Baseline Characteristics of cases in the British Heart Foundation-Family Heart Study and the Myocardial Infarction Genetics studies.</p

A schematic illustration of SNP selection process for the primary analysis.

<p>A schematic illustration of SNP selection process for the primary analysis.</p

SNP selection process for the secondary analysis.

<p>SNP selection process for the secondary analysis.</p