Allele-specific Gene Expression Research Articles

Transcriptome profiling and comparison of maize ear heterosis during the spikelet and floret differentiation stages.

BackgroundHybridization is a prominent process in the evolution of crop plants that can give rise to gene expression variation, phenotypic novelty and heterosis. Maize is the most successful crop in utilizing heterosis. The development of hybrid maize ears exhibits strong heterotic vigor and greatly affects maize yield. However, a comprehensive perspective on transcriptional variation and its correlation with heterosis during maize ear development is not available.ResultsUsing RNA sequencing technology, we investigated the transcriptome profiles of maize ears in the spikelet and floret differentiation stages of hybrid ZD808 and its parents CL11 and NG5. Our results revealed that 53.9% (21,258) of maize protein-coding genes were transcribed in at least one genotype. In both development stages, significant numbers of genes were differentially expressed between the hybrid and its parents. Gene expression inheritance analysis revealed approximately 80% of genes were expressed additively, which suggested that the complementary effect may play a foundation role in maize ear heterosis. Among non-additively expressed genes, NG5-dominant genes were predominant. Analyses of the allele-specific gene expression in hybrid identified pervasive allelic imbalance and significant preferential expression of NG5 alleles in both developmental stages. The results implied that NG5 may provide beneficial alleles that contribute greatly to heterosis. Further comparison of parental and hybrid allele-specific expression suggested that gene expression variation is largely attributable to cis-regulatory variation in maize. The cis-regulatory variations tend to preserve the allelic expression levels in hybrid and result in additive expression. Comparison between the two development stages revealed that allele-specific expression and cis-/trans-regulatory variations responded differently to developmental cues, which may lead to stage-specific vigor phenotype during maize ear development.ConclusionOur research suggests that cis-regulated additive expression may fine-tune gene expression level into an optimal status and play a foundation role in maize ear heterosis. Our work provides a comprehensive insight into transcriptional variation and its correlation with heterosis during maize ear development. The knowledge gained from this study presents novel opportunity to improve our maize varieties.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-3296-8) contains supplementary material, which is available to authorized users.

Allele-specific gene expression in carcinogenesis

Recent large-scale genomic studies established the occurrence of multiple DNA sequence variants in genomes of healthy individuals that differ from the reference sequence. Among these variants mostly represented by germline single nucleotide polymorphisms disease-related alleles are detected including alleles which are associated with monogenic disorders, and putative deleterious genetic variants. Apart from functional significance of a particular variant and of a gene harboring it, the penetrance of these allelic variants depends on their expression level and can be determined by preferential expression of a particular allele, or allele-specific expression. It is estimated that 20–30 % of genes present in the human genome display allelic bias in a tissue-specific manner. Allele-specific expression is defined by a range of genetic and epigenetic mechanisms including cis-regulatory polymorphisms, allele-specific binding of transcription factors, allele-specific DNA methylation and regulation through non-coding RNA. Although the data on the issue are scarce, allele-specific expression has been reported to be implicated in several hereditary disorders including benign and malignant tumors of the large intestine. Recent studies that estimate allele-specific expression incidence in tumors and identify wide range of genes displaying allelic imbalance indicate that allele-specific expression might play a significant role in carcinogenesis. Eventually, estimation of transcriptional rate of allelic variants which cause dysfunction of oncogenes and tumor suppressors may prove to be essential for rational choice of antitumor therapeutic strategy. In this review, we outline the main concepts and mechanisms of allele-specific expression and the data on allelic imbalance in tumors.

Ribosomal protein genes are highly enriched among genes with allele-specific expression in the interspecific F1 hybrid catfish.

Interspecific hybrids provide a rich source for the analysis of allele-specific expression (ASE). In this work, we analyzed ASE in F1 hybrid catfish using RNA-Seq datasets. While the vast majority of genes were expressed with both alleles, 7-8% SNPs exhibited significant differences in allele ratios of expression. Of the 66,251 and 177,841 SNPs identified from the datasets of the liver and gill, 5420 (8.2%) and 13,390 (7.5%) SNPs were identified as significant ASE-SNPs, respectively. With these SNPs, a total of 1519 and 3075 ASE-genes were identified. Gene Ontology analysis revealed that genes encoding cytoplasmic ribosomal proteins (RP) were highly enriched among ASE genes. Parent-of-origin was determined for 27 and 30 ASE RP genes in the liver and gill, respectively. The results indicated that genes from both channel catfish and blue catfish were involved in ASE. However, each RP gene appeared to be almost exclusively expressed from only one parent, indicating that ribosomes in the hybrid catfish were in the "hybrid" form. Overall representation of RP transcripts among the transcriptome appeared lower in the F1 hybrid catfish than in channel catfish or blue catfish, suggesting that the "hybrid" ribosomes may work more efficiently for translation in the F1 hybrid catfish.

A Bayesian approach for estimating allele-specific expression from RNA-Seq data with diploid genomes.

BackgroundRNA-sequencing (RNA-Seq) has become a popular tool for transcriptome profiling in mammals. However, accurate estimation of allele-specific expression (ASE) based on alignments of reads to the reference genome is challenging, because it contains only one allele on a mosaic haploid genome. Even with the information of diploid genome sequences, precise alignment of reads to the correct allele is difficult because of the high-similarity between the corresponding allele sequences.ResultsWe propose a Bayesian approach to estimate ASE from RNA-Seq data with diploid genome sequences. In the statistical framework, the haploid choice is modeled as a hidden variable and estimated simultaneously with isoform expression levels by variational Bayesian inference. Through the simulation data analysis, we demonstrate the effectiveness of the proposed approach in terms of identifying ASE compared to the existing approach. We also show that our approach enables better quantification of isoform expression levels compared to the existing methods, TIGAR2, RSEM and Cufflinks. In the real data analysis of the human reference lymphoblastoid cell line GM12878, some autosomal genes were identified as ASE genes, and skewed paternal X-chromosome inactivation in GM12878 was identified.ConclusionsThe proposed method, called ASE-TIGAR, enables accurate estimation of gene expression from RNA-Seq data in an allele-specific manner. Our results show the effectiveness of utilizing personal genomic information for accurate estimation of ASE. An implementation of our method is available at http://nagasakilab.csml.org/ase-tigar.

Extensive variation between tissues in allele specific expression in an outbred mammal.

BackgroundAllele specific gene expression (ASE), with the paternal allele more expressed than the maternal allele or vice versa, appears to be a common phenomenon in humans and mice. In other species the extent of ASE is unknown, and even in humans and mice there are several outstanding questions. These include; to what extent is ASE tissue specific? how often does the direction of allele expression imbalance reverse between tissues? how often is only one of the two alleles expressed? is there a genome wide bias towards expression of the paternal or maternal allele; and finally do genes that are nearby on a chromosome share the same direction of ASE? Here we use gene expression data (RNASeq) from 18 tissues from a single cow to investigate each of these questions in turn, and then validate some of these findings in two tissues from 20 cows.ResultsBetween 40 and 100 million sequence reads were generated per tissue across three replicate samples for each of the eighteen tissues from the single cow (the discovery dataset). A bovine gene expression atlas was created (the first from RNASeq data), and differentially expressed genes in each tissue were identified. To analyse ASE, we had access to unambiguously phased genotypes for all heterozygous variants in the cow’s whole genome sequence, where these variants were homozygous in the whole genome sequence of her sire, and as a result we were able to map reads to parental genomes, to determine SNP and genes showing ASE in each tissue. In total 25,251 heterozygous SNP within 7985 genes were tested for ASE in at least one tissue. ASE was pervasive, 89 % of genes tested had significant ASE in at least one tissue. This large proportion of genes displaying ASE was confirmed in the two tissues in a validation dataset.For individual tissues the proportion of genes showing significant ASE varied from as low as 8–16 % of those tested in thymus to as high as 71–82 % of those tested in lung. There were a number of cases where the direction of allele expression imbalance reversed between tissues. For example the gene SPTY2D1 showed almost complete paternal allele expression in kidney and thymus, and almost complete maternal allele expression in the brain caudal lobe and brain cerebellum. Mono allelic expression (MAE) was common, with 1349 of 4856 genes (28 %) tested with more than one heterozygous SNP showing MAE. Across all tissues, 54.17 % of all genes with ASE favoured the paternal allele. Genes that are closely linked on the chromosome were more likely to show higher expression of the same allele (paternal or maternal) than expected by chance. We identified several long runs of neighbouring genes that showed either paternal or maternal ASE, one example was five adjacent genes (GIMAP8, GIMAP7 copy1, GIMAP4, GIMAP7 copy 2 and GIMAP5) that showed almost exclusive paternal expression in brain caudal lobe.ConclusionsInvestigating the extent of ASE across 18 bovine tissues in one cow and two tissues in 20 cows demonstrated 1) ASE is pervasive in cattle, 2) the ASE is often MAE but ranges from MAE to slight overexpression of the major allele, 3) the ASE is most often tissue specific and that more than half the time displays divergent allele specific expression patterns across tissues, 4) across all genes there is a slight bias towards expression of the paternal allele and 5) genes expressing the same parental allele are clustered together more than expected by chance, and there are several runs of large numbers of genes expressing the same parental allele.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2174-0) contains supplementary material, which is available to authorized users.

Identification of genes escaping X inactivation by allelic expression analysis in a novel hybrid mouse model.

X chromosome inactivation (XCI) is a female-specific mechanism that serves to balance gene dosage between the sexes whereby one X chromosome in females is inactivated during early development. Despite this silencing, a small portion of genes escape inactivation and remain expressed from the inactive X (Xi). Little is known about the distribution of escape from XCI in different tissues in vivo and about the mechanisms that control tissue-specific differences. Using a new binomial model in conjunction with a mouse model with identifiable alleles and skewed X inactivation we are able to survey genes that escape XCI in vivo. We show that escape from X inactivation can be a common feature of some genes, whereas others escape in a tissue specific manner. Furthermore, we characterize the chromatin environment of escape genes and show that expression from the Xi correlates with factors associated with open chromatin and that CTCF co-localizes with escape genes. Here, we provide a detailed description of the experimental design and data analysis pipeline we used to assay allele-specific expression and epigenetic characteristics of genes escaping X inactivation. The data is publicly available through the GEO database under ascension numbers GSM1014171, GSE44255, and GSE59779. Interpretation and discussion of these data are included in a previously published study (Berletch et al., 2015) [1].

Altered Gene Expression Associated with microRNA Binding Site Polymorphisms.

Allele-specific gene expression associated with genetic variation in regulatory regions can play an important role in the development of complex traits. We hypothesized that polymorphisms in microRNA (miRNA) response elements (MRE-SNPs) that either disrupt a miRNA binding site or create a new miRNA binding site can affect the allele-specific expression of target genes. By integrating public expression quantitative trait locus (eQTL) data, miRNA binding site predictions, small RNA sequencing, and Argonaute crosslinking immunoprecipitation (AGO-CLIP) datasets, we identified genetic variants that can affect gene expression by modulating miRNA binding efficiency. We also identified MRE-SNPs located in regions associated with complex traits, indicating possible causative mechanisms associated with these loci. The results of this study expand the current understanding of gene expression regulation and help to interpret the mechanisms underlying eQTL effects.

Recommendations for Accurate Resolution of Gene and Isoform Allele-Specific Expression in RNA-Seq Data.

Genetic variation modulates gene expression transcriptionally or post-transcriptionally, and can profoundly alter an individual’s phenotype. Measuring allelic differential expression at heterozygous loci within an individual, a phenomenon called allele-specific expression (ASE), can assist in identifying such factors. Massively parallel DNA and RNA sequencing and advances in bioinformatic methodologies provide an outstanding opportunity to measure ASE genome-wide. In this study, matched DNA and RNA sequencing, genotyping arrays and computationally phased haplotypes were integrated to comprehensively and conservatively quantify ASE in a single human brain and liver tissue sample. We describe a methodological evaluation and assessment of common bioinformatic steps for ASE quantification, and recommend a robust approach to accurately measure SNP, gene and isoform ASE through the use of personalized haplotype genome alignment, strict alignment quality control and intragenic SNP aggregation. Our results indicate that accurate ASE quantification requires careful bioinformatic analyses and is adversely affected by sample specific alignment confounders and random sampling even at moderate sequence depths. We identified multiple known and several novel ASE genes in liver, including WDR72, DSP and UBD, as well as genes that contained ASE SNPs with imbalance direction discordant with haplotype phase, explainable by annotated transcript structure, suggesting isoform derived ASE. The methods evaluated in this study will be of use to researchers performing highly conservative quantification of ASE, and the genes and isoforms identified as ASE of interest to researchers studying those loci.

Genomic imprinting effects on complex traits in domesticated animal species.

Monoallelically expressed genes that exert their phenotypic effect in a parent-of-origin specific manner are considered to be subject to genomic imprinting, the most well understood form of epigenetic regulation of gene expression in mammals. The observed differences in allele specific gene expression for imprinted genes are not attributable to differences in DNA sequence information, but to specific chemical modifications of DNA and chromatin proteins. Since the discovery of genomic imprinting some three decades ago, over 100 imprinted mammalian genes have been identified and considerable advances have been made in uncovering the molecular mechanisms regulating imprinted gene expression. While most genomic imprinting studies have focused on mouse models and human biomedical disorders, recent work has highlighted the contributions of imprinted genes to complex trait variation in domestic livestock species. Consequently, greater understanding of genomic imprinting and its effect on agriculturally important traits is predicted to have major implications for the future of animal breeding and husbandry. In this review, we discuss genomic imprinting in mammals with particular emphasis on domestic livestock species and consider how this information can be used in animal breeding research and genetic improvement programs.

The effect of metallothionein 2A core promoter region single-nucleotide polymorphism on accumulation of toxic metals in sinonasal inverted papilloma tissues

Metallothioneins (MTs) are intracellular thiol-rich heavy metal-binding proteins which join trace metal ions protecting cells against heavy metal toxicity and regulate metal distribution and donation to various enzymes and transcription factors. The goal of this study was to identify the −5 A/G (rs28366003) single-nucleotide polymorphism (SNP) in the core promoter region of the MT2A gene, and to investigate its effect on allele-specific gene expression and Cd, Zn, Cu and Ni content in sinonasal inverted papilloma tissue (IP), with non-cancerous sinonasal mucosa (NCM) as a control. The MT2A promoter region −5 A/G SNP was identified by restriction fragment length polymorphism using 117 IP and 132 NCM. MT2A gene analysis was performed by quantitative real-time PCR. Metal levels were analyzed by flame atomic absorption spectrometry. The frequency of A allele carriage was 99.2% and 100% in IP and NCM, respectively. The G allele carriage was detected in 23.9% of IP and in 12.1% of the NCM samples. As a result, a significant association of −5 A/G SNP in MT2A gene with mRNA expression in both groups was determined. A significant association was identified between the −5 A/G SNP in the MT2A gene with mRNA expression in both groups. A highly significant association was detected between the rs28366003 genotype and Cd and Zn content in IP. Furthermore, significant differences were identified between A/A and A/G genotype with regard to the type of metal contaminant. The Spearman rank correlation results showed the MT2A gene expression and both Cd and Cu levels were negatively correlated. The results obtained in this study suggest that the −5 A/G SNP in the MT2A gene may have an effect on allele-specific gene expression and toxic metal accumulation in sinonasal inverted papilloma.

Editorial: Genetics and epigenetics of fetal alcohol spectrum disorders.

Editorial: Genetics and epigenetics of fetal alcohol spectrum disorders.

Germline and somatic imprinting in the nonhuman primate highlights species differences in oocyte methylation.

Genomic imprinting is an epigenetic mechanism resulting in parental allele-specific gene expression. Defects in normal imprinting are found in cancer, assisted reproductive technologies, and several human syndromes. In mouse models, germline-derived DNA methylation is shown to regulate imprinting. Though imprinting is largely conserved between mammals, species- and tissue-specific domains of imprinted expression exist. Using the cynomolgus macaque (Macaca fascicularis) to assess primate-specific imprinting, we present a comprehensive view of tissue-specific imprinted expression and DNA methylation at established imprinted gene clusters. For example, like mouse and unlike human, macaque IGF2R is consistently imprinted, and the PLAGL1, INPP5F transcript variant 2, and PEG3 imprinting control regions are not methylated in the macaque germline but acquire this post-fertilization. Methylome data from human early embryos appear to support this finding. These suggest fundamental differences in imprinting control mechanisms between primate species and rodents at some imprinted domains, with implications for our understanding of the epigenetic programming process in humans and its influence on disease.

Global epigenomic analysis indicates that Epialleles contribute to Allele-specific expression via Allele-specific histone modifications in hybrid rice

BackgroundFor heterozygous genes, alleles on the chromatin from two different parents exhibit histone modification variations known as allele-specific histone modifications (ASHMs). The regulation of allele-specific gene expression (ASE) by ASHMs has been reported in animals. However, to date, the regulation of ASE by ASHM genes remains poorly understood in higher plants.ResultsWe used chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) to investigate the global ASHM profiles of trimethylation on histone H3 lysine 27 (H3K27me3) and histone H3 lysine 36 (H3K36me3) in two rice F1 hybrids. A total of 522 to 550 allele-specific H3K27me3 genes and 428 to 494 allele-specific H3K36me3 genes were detected in GL × 93-11 and GL × TQ, accounting for 11.09% and 26.13% of the total analyzed genes, respectively. The epialleles between parents were highly related to ASHMs. Further analysis indicated that 52.48% to 70.40% of the epialleles were faithfully inherited by the F1 hybrid and contributed to 33.18% to 46.55% of the ASHM genes. Importantly, 66.67% to 82.69% of monoallelic expression genes contained the H3K36me3 modification. Further studies demonstrated a significant positive correlation of ASE with allele-specific H3K36me3 but not with H3K27me3, indicating that ASHM-H3K36me3 primarily regulates ASE in this study.ConclusionsOur results demonstrate that epialleles from parents can be inherited by the F1 to produce ASHMs in the F1 hybrid. Our findings indicate that ASHM-H3K36me3, rather than H3K27me3, mainly regulates ASE in hybrid rice.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1454-z) contains supplementary material, which is available to authorized users.

Escape from X inactivation varies in mouse tissues.

X chromosome inactivation (XCI) silences most genes on one X chromosome in female mammals, but some genes escape XCI. To identify escape genes in vivo and to explore molecular mechanisms that regulate this process we analyzed the allele-specific expression and chromatin structure of X-linked genes in mouse tissues and cells with skewed XCI and distinguishable alleles based on single nucleotide polymorphisms. Using a binomial model to assess allelic expression, we demonstrate a continuum between complete silencing and expression from the inactive X (Xi). The validity of the RNA-seq approach was verified using RT-PCR with species-specific primers or Sanger sequencing. Both common escape genes and genes with significant differences in XCI status between tissues were identified. Such genes may be candidates for tissue-specific sex differences. Overall, few genes (3–7%) escape XCI in any of the mouse tissues examined, suggesting stringent silencing and escape controls. In contrast, an in vitro system represented by the embryonic-kidney-derived Patski cell line showed a higher density of escape genes (21%), representing both kidney-specific escape genes and cell-line specific escape genes. Allele-specific RNA polymerase II occupancy and DNase I hypersensitivity at the promoter of genes on the Xi correlated well with levels of escape, consistent with an open chromatin structure at escape genes. Allele-specific CTCF binding on the Xi clustered at escape genes and was denser in brain compared to the Patski cell line, possibly contributing to a more compartmentalized structure of the Xi and fewer escape genes in brain compared to the cell line where larger domains of escape were observed.

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance.

Complex human traits are influenced by variation in regulatory DNA through mechanisms that are not fully understood. Since regulatory elements are conserved between humans and mice, a thorough annotation of cis regulatory variants in mice could aid in this process. Here we provide a detailed portrait of mouse gene expression across multiple tissues in a three-way diallel. Greater than 80% of mouse genes have cis regulatory variation. These effects influence complex traits and usually extend to the human ortholog. Further, we estimate that at least one in every thousand SNPs creates a cis regulatory effect. We also observe two types of parent-of-origin effects, including classical imprinting and a novel, global allelic imbalance in favor of the paternal allele. We conclude that, as with humans, pervasive regulatory variation influences complex genetic traits in mice and provide a new resource toward understanding the genetic control of transcription in mammals.

ASEQ: fast allele-specific studies from next-generation sequencing data.

BackgroundSingle base level information from next-generation sequencing (NGS) allows for the quantitative assessment of biological phenomena such as mosaicism or allele-specific features in healthy and diseased cells. Such studies often present with computationally challenging burdens that hinder genome-wide investigations across large datasets that are now becoming available through the 1,000 Genomes Project and The Cancer Genome Atlas (TCGA) initiatives.ResultsWe present ASEQ, a tool to perform gene-level allele-specific expression (ASE) analysis from paired genomic and transcriptomic NGS data without requiring paternal and maternal genome data. ASEQ offers an easy-to-use set of modes that transparently to the user takes full advantage of a built-in fast computational engine. We report its performances on a set of 20 individuals from the 1,000 Genomes Project and show its detection power on imprinted genes. Next we demonstrate high level of ASE calls concordance when comparing it to AlleleSeq and MBASED tools. Finally, using a prostate cancer dataset we report on a higher fraction of ASE genes with respect to healthy individuals and show allele-specific events nominated by ASEQ in genes that are implicated in the disease.ConclusionsASEQ can be used to rapidly and reliably screen large NGS datasets for the identification of allele specific features. It can be integrated in any NGS pipeline and runs on computer systems with multiple CPUs, CPUs with multiple cores or across clusters of machines.Electronic supplementary materialThe online version of this article (doi:10.1186/s12920-015-0084-2) contains supplementary material, which is available to authorized users.

Analysis of the expression and polymorphism of APOE, HSP, BDNF, and GRIN2B genes associated with the neurodegeneration process in the pathogenesis of primary open angle glaucoma.

Glaucoma is characterized by optic neuropathy of the RGC or retinal nerve fiber. The aim of this study was to evaluate a relationship between the neurodegenerative genes' polymorphisms of the APOE (rs449647), BDNF (rs2030324), GRIN2B (rs3764028), and HSP70-1 (rs1043618) and the occurrence risk of POAG and to investigate its effect on allele-specific gene expression. Genomic DNA was extracted from peripheral blood. Analysis of the genes' polymorphisms was performed using PCR-RFLP. The level of mRNA expression was determined by QRT-PCR. We showed a statistically significant association of BDNF and APOE genes' polymorphisms with a risk of POAG occurrence. There was a statistically significant association of the rs2030324 polymorphism with progression of POAG based on cup disc ratio value and rs1043618 polymorphism based on nerve fiber index and rim area. Furthermore, we found that mean HSP70-1 mRNA expression was significantly lower in the case of individuals with the G/G genotype than in the case of minor allele carriers, that is, G/C and C/C. We also found that BDNF and HSP70-1 expression level are associated with the progression of POAG based on rim area value. In conclusion, our results suggest that BDNF, APOE, and HSP70-1 genes might be associated with a risk of POAG occurrence in the Polish population.

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance

Analyses of allele-specific gene expression in highly divergent mouse crosses identifies pervasive allelic imbalance

Thrombomodulin Has a Significant Impact on Transplant Outcomes after HLA-Fully-Matched Unrelated Bone Marrow Transplantation for Standard Risk Hematologic Malignancies

Background:Endothelial injury is involved in graft-versus-host-disease (GVHD), thrombotic microangiopathy, or sinusoidal obstruction syndrome. Thrombomodulin (THBD) plays a role of anti-coagulation or anti-inflammatory responses, and there are increasing evidences suggesting recombinant human soluble thrombomodulin (rTHBD) is effective on clinical outcomes of patients with coagulopathy after hematopoietic stem cell transplantation. THBD gene has several single nucleotide polymorphisms (SNP) which are associated with the risk of venous thrombotic events or coronary heart disease. In this study, we analyzed the impact of THBD polymorphism A2729C on transplant outcomes in recipients undergoing unrelated HLA-fully-matched T-cell-replete bone marrow transplantation through the Japan Donor Marrow Program and examined allele-specific gene expression of A2729C. Moreover, we investigated the effect of treatment with rTHBD prior to total body irradiation (TBI) on endothelial cells in vitro models.Methods and Results: The SNP A2729C was analyzed using the TaqMan system (Applied Biosystems), and the results were analyzed using the Allelic Discrimination software program (Applied Biosystems). The THBD polymorphisms were retrospectively analyzed in a cohort of 399 pairs of patients with hematologic malignancies and their unrelated donors. The genotype frequencies of A/A, A/C and C/C were 10%, 35% and 55% in recipients and 51%, 39% and 10% in donors (p=0.41). In standard risk disease group, the recipient C/C genotype was associated with a better overall survival (OS) than the recipient A/A and A/C genotype (p<0.10), while the recipient C/C genotype was not significantly associated with a better transplant-related mortality (TRM) than the recipient A/A and A/C genotype (p=0.11). On the multivariate analysis, the beneficial effects of the recipient C/C genotype remained significant for OS (hazard ratio [HR], 0.13; 95% confidence interval [CI], 0.02 to 0.99; p<0.05) and TRM (HR, 4.2x10-5; 95% CI, 1.3x10-5 to 1.4x10-4; p<0.0001). In standard risk disease group, moreover, the donor C/C genotype was associated with a better TRM than the donor A/A and A/C genotype (p<0.10), but was not associated with OS (p=0.80). On the multivariate analysis for TRM, there was the beneficial effect of the donor C/C genotype (HR, 3.6x10-5; 95% CI, 0.30 to 1.6; p<0.0001). The donor/recipient THBD genotype showed no significant effects on the relapse, aGVHD and cGVHD. To further investigate the effect of rs3176123 on transcription of mRNA, we performed Allele-specific transcript quantification on human primary monocytes from 6 healthy donors with heterozygous genotypes of rs3176123. In these cells the mean ratio (C allele vs A allele) was 0.53, which is significantly higher than that of DNA amplicons (ratio 0.50 ; p<0.01). In vitro, human umbilical vein endothelial cells (HUVECs) were pretreated with 1 μg/ml rTHBD for 1 hour, and irradiated with X-rays with single doses of 10 Gy. Total RNA was extracted 3 hours after radiation, and mRNA levels were determined by RT-PCR. Pretreatment with rTHBD significantly inhibited irradiation-induced tissue factor (p<0.03), IL-6 (p<0.02), E-selectin (p<0.03) and ICAM-1 (p<0.05) up-regulation in HUVECs.Conclusions: These results suggest an association of the recipient THBD genotype with overall survival and TRM after unrelated BMT in the standard risk disease group, and may substantiate that the higher THBD activity by donor with the THBD C allele likely accounts for a reduced risk for TRM in their recipients.These could therefore be useful in selecting the donor and creating therapeutic strategies for improving the final outcome of allogeneic BMT. DisclosuresNo relevant conflicts of interest to declare.

Angelman syndrome imprinting center encodes a transcriptional promoter

Clusters of imprinted genes are often controlled by an imprinting center that is necessary for allele-specific gene expression and to reprogram parent-of-origin information between generations. An imprinted domain at 15q11–q13 is responsible for both Angelman syndrome (AS) and Prader–Willi syndrome (PWS), two clinically distinct neurodevelopmental disorders. Angelman syndrome arises from the lack of maternal contribution from the locus, whereas Prader–Willi syndrome results from the absence of paternally expressed genes. In some rare cases of PWS and AS, small deletions may lead to incorrect parent-of-origin allele identity. DNA sequences common to these deletions define a bipartite imprinting center for the AS–PWS locus. The PWS–smallest region of deletion overlap (SRO) element of the imprinting center activates expression of genes from the paternal allele. The AS–SRO element generates maternal allele identity by epigenetically inactivating the PWS–SRO in oocytes so that paternal genes are silenced on the future maternal allele. Here we have investigated functional activities of the AS–SRO, the element necessary for maternal allele identity. We find that, in humans, the AS–SRO is an oocyte-specific promoter that generates transcripts that transit the PWS–SRO. Similar upstream promoters were detected in bovine oocytes. This result is consistent with a model in which imprinting centers become DNA methylated and acquire maternal allele identity in oocytes in response to transiting transcription.