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. 2010 Apr 19:11:25.
doi: 10.1186/1471-2156-11-25.

High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

Affiliations

High-throughput analysis of candidate imprinted genes and allele-specific gene expression in the human term placenta

Caroline Daelemans et al. BMC Genet. .

Abstract

Background: Imprinted genes show expression from one parental allele only and are important for development and behaviour. This extreme mode of allelic imbalance has been described for approximately 56 human genes. Imprinting status is often disrupted in cancer and dysmorphic syndromes. More subtle variation of gene expression, that is not parent-of-origin specific, termed 'allele-specific gene expression' (ASE) is more common and may give rise to milder phenotypic differences. Using two allele-specific high-throughput technologies alongside bioinformatics predictions, normal term human placenta was screened to find new imprinted genes and to ascertain the extent of ASE in this tissue.

Results: Twenty-three family trios of placental cDNA, placental genomic DNA (gDNA) and gDNA from both parents were tested for 130 candidate genes with the Sequenom MassArray system. Six genes were found differentially expressed but none imprinted. The Illumina ASE BeadArray platform was then used to test 1536 SNPs in 932 genes. The array was enriched for the human orthologues of 124 mouse candidate genes from bioinformatics predictions and 10 human candidate imprinted genes from EST database mining. After quality control pruning, a total of 261 informative SNPs (214 genes) remained for analysis. Imprinting with maternal expression was demonstrated for the lymphocyte imprinted gene ZNF331 in human placenta. Two potential differentially methylated regions (DMRs) were found in the vicinity of ZNF331. None of the bioinformatically predicted candidates tested showed imprinting except for a skewed allelic expression in a parent-specific manner observed for PHACTR2, a neighbour of the imprinted PLAGL1 gene. ASE was detected for two or more individuals in 39 candidate genes (18%).

Conclusions: Both Sequenom and Illumina assays were sensitive enough to study imprinting and strong allelic bias. Previous bioinformatics approaches were not predictive of new imprinted genes in the human term placenta. ZNF331 is imprinted in human term placenta and might be a new ubiquitously imprinted gene, part of a primate-specific locus. Demonstration of partial imprinting of PHACTR2 calls for re-evaluation of the allelic pattern of expression for the PHACTR2-PLAGL1 locus. ASE was common in human term placenta.

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Figures

Figure 1
Figure 1
Correlation of Sequenom and Illumina allele quantification. Pearson's correlation coefficient (r) is calculated for the allele-specific quantification on the two platforms and plotted against cDNA intensity (average log2 fluorescence for the 23 placentas) on Illumina. The correlation drops when cDNA average intensity is lower than the 11.25 cut-off (dashed red line).
Figure 2
Figure 2
ROC plots for the mixture control data set.
Figure 3
Figure 3
Log-ratios for three SNPs tested in different DNA proportions of two control HapMap samples (NA12892:NA19092, from 0:100 to 100:0) on the Illumina ASE Beadarray. From top to bottom, we see SNPs that exhibit extreme allelic imbalance, intermediate allelic imbalance and no allelic imbalance, respectively. The first two examples are true positives while the last one is a true negative for our sensitivity and specificity analysis.
Figure 4
Figure 4
Informative samples for SNP rs2839702 (Fig. 2A) and rs2075745 (Fig. 2B) in the human H19 imprinted gene. For each informative (heterozygous placental genomic DNA) SNP, allelic log-ratios were plotted to compare gDNA and cDNA results. Each family trio is represented by a number on the X-axis and consists of placental gDNA (green), placental cDNA (purple), paternal gDNA (orange), and maternal gDNA (yellow). In the placental gDNA, both alleles occur almost equally and the log-ratio is close to zero. H19 is an imprinted gene. Hence in the placental cDNA, only one allele is expressed. The sign of the log-ratio for the cDNA sample changes depending upon the allele expressed. Imprinted genes cDNA log-ratio will show a typical oscillation of signal across the y-axis because it is not the allele that is important but its parent-of-origin [21]. When at least one parent is homozygous for the SNP under study, the parent-of-origin of the expressed allele can be ascertained. In the case of H19, the maternal allele is the one expressed as expected (i.e. for homozygous parents gDNA, the maternal gDNA allelic log-ratio has the same sign as the placental cDNA log-ratio and the paternal gDNA allelic log-ratio has the opposite sign to the placental cDNA).
Figure 5
Figure 5
Monoallelic maternal expression of ZNF331 as detected by two SNPs rs12982082 and rs8100247. Bar chart designed as in Figure 4.
Figure 6
Figure 6
Methylation levels of the CpG islands within ZNF331. The figure shows the position of ZNF331 and the CpG islands in the UCSC genome browser. In the bottom part of the figure, the methylation pattern of the three CpG islands studied is represented. Bisulphite sequencing data was compiled with the BDPC webtool [53]. The blue colour indicates metylated CpGs and the yellow colour unmethylated CpGs. Each column represents a single CpG site and each row represents one clone. The sequences for two CpGs (45 and 83) were sorted according to their genotype.
Figure 7
Figure 7
Methylation levels of CpG86 in ZNF331 locus. The figure, generated with UCSC genome browser, indicates the position of the CpG island in relation to the location of ZNF331, its neighbouring genes and miRNAs. Bisulphite sequencing data was compiled with the BDPC webtool as in Figure 6. No SNP was present in the amplified sequence.
Figure 8
Figure 8
rs1082 PHACTR2. The ASE pattern of rs1082-PHACTR2 is consistent with partial imprinting (maternal expression bias). Bar chart designed as in Figure 4.
Figure 9
Figure 9
PHACTR2 cDNA allelic expression ratio is biased towards the maternal allele. Sequences for rs1082 (3'UTR) of all available informative term placenta samples in cDNA and gDNA are shown with corresponding maternal and paternal genotyping data.
Figure 10
Figure 10
Lack of complete repression of the silenced allele for imprinted genes. Average quantification of the expressed allele (dark blue) and of the silenced allele (light blue) in all informative samples for all expressed control imprinted genes (PEG3, H19, MEST, PEG10, PHLDA2, PLAGL1, DLK1 and IGF2AS), for ZNF331 and for PHACTR2.

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