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

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. Background Although diploid organisms have two copies of each gene, they are not always equally expressed. For some genes, only one allele is active while the other is almost completely silenced. Two different groups of genes fall into this category: genes that exhibit random monoallelic expression, e.g. the odorant receptor genes and genes coding for immunoglobulins [1,2]; and imprinted genes that exhibit monoallelic expression in a parent-of-origin specific manner [3]. Imprinted genes have been shown to be important in fetal and placental development, postnatal growth, behaviour and metabolism [4]. Their regula* Correspondence: G.Moore@ich.ucl.ac.uk, dunham@ebi.ac.uk 1 Wellcome Trust Sanger Institute, Hinxton, Cambridge, CB10 1 SA, UK 2 Molecular and Clinical Genetics Unit, Institute of Child Health, London, WC1 1EH, UK † Contributed equally Full list of author information is available at the end of the article Daelemans et al. BMC Genetics 2010, 11:25 http://www.biomedcentral.com/1471-2156/11/25 Page 2 of 20 tion has been found to be disturbed in numerous cancers and dysmorphic syndromes [5]. To date, 56 genes have been identified as imprinted in humans and 98 in mice [6]. A catalogue of human imprinted genes is kept and regularly updated at http:// igc.otago.ac.nz/home.html[7]. However, since most imprinted have been discovered by direct approaches, the total number of imprinted genes is not yet known. Recently, a bioinformatics approach based on DNA sequence characteristics of known imprinted genes predicted 600 imprinted genes in mice [8]. In the human, statistical models have been developed to identify genes with unequal representation of alternative alleles in the public EST libraries, suggesting a further 55 candidate imprinted genes [9]. Many imprinted genes are expressed in a parent-of-origin specific manner in the placenta, making it a "first choice" tissue in which to screen for new imprinted genes [10]. Imprinted expression is at the extreme end of the autosomal allelic imbalance spectrum. However, more subtle allelic variations around the expected 50:50 ratio of expression have been documented. Yan et al. were the first to report such ASE in human [11]. They studied 13 genes and detected 1.3 to 4.3-fold expression differences between alleles for six of them. Lo et al. studied 1063 genes (using Affymetrix HuSNP array) in seven fetuses, where of the 602 genes that were heterozygous, 326 showed preferential expression of one allele in at least one individual (54%), while 170 (28%) showed more than a four-fold difference between the two alleles [12]. Several oligonucleotide microarrays have been used to study ASE in lymphoblastoid cell lines (LCLs). Pant et al. used a custom made microarray (Perlegen, USA) and found allelic expression differences in at least one individual in 53% of the 1389 genes targeted by heterozygous single nucleotide polymorphisms (SNPs) [13]. More recently, Gimelbrant et al. found monoallelic expression for 7.3% of the genes they tested in clonal lymphoblastoid cells [14]. Strong ASE differences (ASE ratio >4 or <1/4) have been found by Bjornsson et al. in 10% of SNPs in LCLs [15]. Hence, it seems that ASE is frequent, possibly underlying much of human variability [11-15]. We have screened human term placenta for novel imprinted genes and ASE using two technologies that have been shown to be able to quantify allelic expression in a medium and high-throughput manner: the MassArray system (Sequenom, Inc.) [16] and the Illumina ASE Bead ArrayTM[17], respectively. Results Sequenom The MassArray system (Sequenom, Inc.) was used to test 143 genes for ASE in at least 23 family-trios. Each trio consisted of placental genomic DNA (gDNA), placental cDNA and both parental gDNAs. We analysed six imprinted control genes, seven biallelically expressed genes, seven orthologues of mouse imprinted genes, 99 orthologues of mouse imprinted candidate genes [8], and 26 human imprinted candidate genes [9] (Additional file 1: Supplemental Table S1). For 123 genes (86%), the cDNA amplification was successful and at least two placentas were heterozygous. A t-test (followed by FDRmoderation) was used to verify the null hypothesis that there was no allelic imbalance between the ratios of alleles in gDNA and in cDNA (Table 1 and Methods). Five imprinted control genes exhibited imprinting (no informative sample for rs2066707-ATP10A). In the subset of genes with acceptable cDNA genotyping success (arbitrarily set at a ratio between cDNA and gDNA genotyping higher than 75%, see Methods), six candidate genes were significant for allelic imbalance in cDNA (p < 0.05) (Table 1). None of these genes had an allelic expression pattern that was compatible with imprinting. Of these, RASGRF1 had the most allelic difference (76%) and it is notable that the mouse orthologue Rasgrf1 is imprinted in the brain [18]. Its mode of allelic expression in human term placenta was compatible with random monoallelic expression (no allelic preference; four paternal, one maternal and three biallelic mode of expression; data not shown). We checked the mode of expression of RASGFR1 in the human term placenta by Sanger sequencing. Biallelic expression (with sometimes a very slight random bias between alleles) was found in seven informative term placenta samples (data not shown). The average fluorescence level of RASGFR1 on the Illumina array was below our cut-off suggesting low expression level (see below). We thus considered RASGRF1 random monoallelic ASE to be a false positive. Using rs4911163 as a readout, ACSS2 showed a statistically significant (two-tailed t-test, p = 0.0075) preferential mode of ASE (Additional file 2). Using the Genevar database (T-P. Yang and E. Dermitzakis, manuscript in preparation), variable level of expression for ACSS2 in relation to rs4911163 genotype was also found in lymphoblastoid cells of HapMap3 individuals (B. Stranger and E. Dermitzakis, manuscript in preparation; [19,20]). ACSS2 is a cytosolic enzyme that catalyzes the activation of acetate for use in lipid synthesis and energy generation. It has no known function in relation to placenta. The four other genes presented a much less convincing ASE pattern and were probably false positives. Three of them (DISC1, C9orf93, TF) were present on the Illumina array (see below) and had low expression levels (average log2 fluorescence lower than 11.25). In conclusion, the Sequenom platform can detect ASE and imprinting, but no new imprinted gene was found in this study. Daelemans et al. BMC Genetics 2010, 11:25 http://www.biomedcentral.com/1471-2156/11/25 Page 3 of 20 ASE Illumina Array To test more candidate genes, we increased our screening throughput by using the ASE BeadArrayTM (Illumina, Inc., USA). With this technique a total of 1536 SNPs, located in 932 genes (214 expected to be expressed in placenta, see Methods) (Additional file 1: Supplemental Table S2), were tested for ASE and imprinting across 23 of the family-trios. The candidate imprinted genes included ten orthologues of known murine imprinted genes whose status was unknown in human, 124 orthologues of 600 mouse candidate imprinted genes [8], ten human candidate imprinted genes [9], and 18 known control imprinted genes [6,13,21] (Additional file 1: Supplemental Table S2). Genes specifically expressed in the placenta compared to other tissues and genes differentially expressed according to the birth weight may influence fetal growth and so may also be imprinted. We therefore tested 46 such genes [22]. The remaining 1179 SNPs (718 genes) on the array were chosen for unrelated research purposes and were thus randomly selected in ter