Additional file 2: of The importance of distinguishing between the odds ratio and the incidence rate ratio in GWAS

Supplementary Figures S1–S10 with captions. (PDF 3.29 mb

Additional file 1: of The importance of distinguishing between the odds ratio and the incidence rate ratio in GWAS

The R code for simulations. (ZIP 2.06 kb

Triploidy—Observations in 154 Diandric Cases

<div><p>Hydatidiform moles (HMs) are abnormal human pregnancies with vesicular chorionic villi, imposing two clinical challenges; miscarriage and a risk of gestational trophoblastic neoplasia (GTN). The parental type of most HMs are either diandric diploid (PP) or diandric triploid (PPM). We consecutively collected 154 triploid or near-triploid samples from conceptuses with vesicular chorionic villi. We used analysis of DNA markers and/or methylation sensitive-MLPA and collected data from registries and patients records. We performed whole genome SNP analysis of one case of twinning (PP+PM).In all 154 triploids or near-triploids we found two different paternal contributions to the genome (P1P2M). The ratios between the sex chromosomal constitutions XXX, XXY, and XYY were 5.7: 6.9: 1.0. No cases of GTN were observed. Our results corroborate that all triploid human conceptuses with vesicular chorionic villi have the parental type P1P2M. The sex chromosomal ratios suggest approximately equal frequencies of meiosis I and meiosis II errors with selection against the XYY conceptuses or a combination of dispermy, non-disjunction in meiosis I and meiosis II and selection against XYY conceptuses. Although single cases of GTN after a triploid HM have been reported, the results of this study combined with data from previous prospective studies estimate the risk of GTN after a triploid mole to 0% (95% CI: 0–1,4%).</p></div

Parental types in triploid conceptuses with molar phenotype.

<p>Parental types in triploid conceptuses with molar phenotype.</p

reproduced [7]. Possible fertilizations, endoreduplications, and abnormal cell divisions in mosaic hydatidiform moles (HMs), and twin gestations including an HM.

<p>(a) Fertilisation by two sperms; one giving rise to the paternal genome set in the diploid biparental cell population, the other giving rise to both genome sets in the diploid androgenetic cell population via endoreduplication. (b) Fertilisation by two sperms; one giving rise to one of the paternal genome sets in the diploid androgenetic cell population, the other contributing one genome set to both cell populations via endoreduplication. (c) Fertilisation by one sperm that via two endoreduplications gives rise to three identical paternal genome sets, of which two constitute the genome of the diploid androgenetic cell population and one is the paternal genome set in the diploid biparental cell population.</p

Number of fully informative loci in 152 (near) triploid cases with vesicular chorionic villi, analyzed along with a maternal sample.

<p>Number of fully informative loci in 152 (near) triploid cases with vesicular chorionic villi, analyzed along with a maternal sample.</p

SNP6 array analysis, C0301.

<p>SNP6 array analysis of individual C0301q and her twin pregnancy (C0301B (mole, PP) and C0301V (normal conceptus, PM)) demonstrating that the molar pregnancy was homozygous for all markers across the autosomal chromosomes and the X chromosome. Upper panel: Regions with stretches of markers showing homozygousity are displayed in purple. Lower panel: Genotypes (AA, AB, and BB) on chromosomes 1 for the three samples, illustrating the general absence of markers with genotype AB for the mole pregnancy. Each dot represents a genotyped SNP.</p

Expression of <i>BRD1</i> transcripts and methylation proportions in region 2 and 3 in SH-SY5Y cells following drug treatment.

<p>(a) Expression of <i>BRD1</i> transcripts in SH-SY5Y cells following Lithium, Valproate, and Carbamazepine treatment. The total <i>BRD1</i> expression and expression of transcript variants containing exon 1C, 1B, and 1A were measured in RNA extracted from control cells or following exposure of the cells to the lowest and highest therapeutic dosage of the drugs for 72 hours in SH-SY5Y cells. Data are presented as mean percentages of the mean value of the control group (no drug treatment) +SEM (n = 3/group). ***<i>p</i><0.001, one-way ANOVA with Dunnett's post-hoc tests. (b) Methylation proportions as determined by pyrosequencing of region 2 and 3 following Carbamazepine treatment in SH-SY5Y cells. Methylation proportions were measured by pyrosequencing of region 2 and 3 in DNA extracted from SH-SY5Y cells following exposure to 0 or 0.1 mM Carbamazepine for 72 hours. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 3/group). Group means were compared by unpaired t-test.</p

Structure of the <i>BRD1</i> promoter regions and methylation proportions in region 1–4 in cell lines.

<p>(a) Structure of the <i>BRD1</i> promoter regions. <i>BRD1</i> comprises 12 coding exons with the first coding exon 1 illustrated in the figure. <i>BRD1</i> was initially described to feature two splice variants, a long and a short resulting from alternative splicing of exon 7 and with transcription initiated from respectively, exon 1 with a 487 bp 5’ UTR (exon 1C) or from a non-coding exon located more than 3 kb upstream of the common exon 1 (exon 1A). We confirm the presence of a third non-coding exon located 1.4 kb upstream exon 1 (exon 1B). Additionaly, the alternative versions of exon 1 (exon 1C, 1B, and 1A) are supported by sequence evidence from different cell types including respectively, GenBank mRNA variant AF005067, CR456408, and AK292428 as well as EST sequences. On the figure the genomic localizations of SNP rs138880 and 6 CpG probes from the Illumina Infinium Human Methylation 450K Bead Array are illustrated: cg15144773, cg21032013, cg02550151, cg15145965, cg06057569, and cg16001335. For DNA methylation analysis, four regions (region 1–4) were analyzed by bisulfite sequencing. The number of CpG sites in each region is: region 1: 33, region 2: 4, region 3: 12, and region 4: 66. Region 2 and 3 was also examined by pyrosequencing. Pyrosequencing of region 2 included CpG sites 3 and 4 from the region 2 bisulfite sequencing assay, including cg15145965 (region 2, CpG site 3) and SNP rs138880. Pyrosequencing of region 3 covered the same region as examined by bisulfite sequencing but because of sequencing limitations it only included CpG sites 1–6 and 9–12. These CpG sites include cg06057569 (region 3, CpG site 6) and cg16001335 (region 3, CpG site 12). The genomic localizations of three CpG islands in the region are shown as well. (b) Methylation proportions as determined by bisulfite sequencing of region 1–4 in HeLa cells. Cells were cultured under standard conditions and DNA was isolated for bisulfite sequencing with primers listed in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170121#pone.0170121.s007" target="_blank">S3 Table</a>. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 9–10 clones for each region). For Region 1 and 4 CpG positions were merged when the methylation proportions were equal for adjacent sites. (c) Methylation proportions as determined by bisulfite sequencing of region 1–4 in SH-SY5Y cells using the same conditions as described for HeLa cells. Data are presented as mean cytosine methylation proportions (C-methylation) at each CpG site +SEM (n = 9–10 clones for each region).</p

Methylation proportion changes in the <i>BRD1</i> locus during fetal neocortex development.

<p>Methylation proportions in the <i>BRD1</i> locus during fetal neocortex development. A public available dataset was utilized to correlate DNA methylation proportions with fetal age (n = 179, range = 23–184 days post-conception). DNA methylation data was available for probes from the Illumina Infinium Human Methylation 450K Bead Array. Probes located near the promoter region of <i>BRD1</i> were assessed for correlation between DNA methylation proportions and fetal age. All probes that revealed age related changes in methylation proportions and some that did not are illustrated. Statistical analysis was performed with Pearson’s correlation test.</p