High-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.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Duplication of 7p11.2-p13, Including GRB10, in Silver-Russell Syndrome

Silver-Russell syndrome (SRS) is characterized by pre- and postnatal growth failure and other dysmorphic features. The syndrome is genetically heterogenous, but maternal uniparental disomy of chromosome 7 has been demonstrated in ∼7% of cases. This suggests that at least one gene on chromosome 7 is imprinted and involved in the pathogenesis of SRS. We have identified a de novo duplication of 7p11.2-p13 in a proband with features characteristic of SRS. FISH confirmed the presence of a tandem duplication encompassing the genes for growth factor receptor–binding protein 10 (GRB10) and insulin-like growth factor–binding proteins 1 and 3 (IGFBP1 and -3) but not that for epidermal growth factor–receptor (EGFR). Microsatellite markers showed that the duplication was of maternal origin. These findings provide the first evidence that SRS may result from overexpression of a maternally expressed imprinted gene, rather than from absent expression of a paternally expressed gene. GRB10 lies within the duplicated region and is a strong candidate, since it is a known growth supressor. Futhermore, the mouse homologue (Grb10/Meg1) is reported to be maternally expressed and maps to the imprinted region of proximal mouse chromosome 11 that demonstrates prenatal growth failure when it is maternally disomic. We have demonstrated that the GRB10 genomic interval replicates asynchronously in human lymphocytes, suggestive of imprinting. An additional 36 SRS probands were investigated for duplication of GRB10, but none were found. However, it remains possible that GRB10 and/or other genes within 7p11.2-p13 are responsible for some cases of SRS

Maternal inheritance of a promoter variant in the imprinted PHLDA2 gene significantly increases birth weight.

Birth weight is an important indicator of both perinatal and adult health, but little is known about the genetic factors contributing to its variability. Intrauterine growth restriction is a leading cause of perinatal morbidity and mortality and is also associated with adult disease. A significant correlation has been reported between lower birth weight and increased expression of the maternal PHLDA2 allele in term placenta (the normal imprinting pattern was maintained). However, a mechanism that explains the transcriptional regulation of PHLDA2 on in utero growth has yet to be described. In this study, we sequenced the PHLDA2 promoter region in 263 fetal DNA samples to identify polymorphic variants. We used a luciferase reporter assay to identify in the PHLDA2 promoter a 15 bp repeat sequence (RS1) variant that significantly reduces PHLDA2-promoter efficiency. RS1 genotyping was then performed in three independent white European normal birth cohorts. Meta-analysis of all three (total n = 9,433) showed that maternal inheritance of RS1 resulted in a significant 93 g increase in birth weight (p = 0.01; 95% confidence interval [CI] = 22-163). Moreover, when the mother was homozygous for RS1, the influence on birth weight was 155 g (p = 0.04; 95% CI = 9-300), which is a similar magnitude to the reduction in birth weight caused by maternal smoking

The role and interaction of imprinted genes in human fetal growth.

Identifying the genetic input for fetal growth will help to understand common, serious complications of pregnancy such as fetal growth restriction. Genomic imprinting is an epigenetic process that silences one parental allele, resulting in monoallelic expression. Imprinted genes are important in mammalian fetal growth and development. Evidence has emerged showing that genes that are paternally expressed promote fetal growth, whereas maternally expressed genes suppress growth. We have assessed whether the expression levels of key imprinted genes correlate with fetal growth parameters during pregnancy, either early in gestation, using chorionic villus samples (CVS), or in term placenta. We have found that the expression of paternally expressing insulin-like growth factor 2 (IGF2), its receptor IGF2R, and the IGF2/IGF1R ratio in CVS tissues significantly correlate with crown-rump length and birthweight, whereas term placenta expression shows no correlation. For the maternally expressing pleckstrin homology-like domain family A, member 2 (PHLDA2), there is no correlation early in pregnancy in CVS but a highly significant negative relationship in term placenta. Analysis of the control of imprinted expression of PHLDA2 gave rise to a maternally and compounded grand-maternally controlled genetic effect with a birthweight increase of 93/155 g, respectively, when one copy of the PHLDA2 promoter variant is inherited. Expression of the growth factor receptor-bound protein 10 (GRB10) in term placenta is significantly negatively correlated with head circumference. Analysis of the paternally expressing delta-like 1 homologue (DLK1) shows that the paternal transmission of type 1 diabetes protective G allele of rs941576 single nucleotide polymorphism (SNP) results in significantly reduced birth weight (-132 g). In conclusion, we have found that the expression of key imprinted genes show a strong correlation with fetal growth and that for both genetic and genomics data analyses, it is important not to overlook parent-of-origin effects