Dataset Information

Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21.

ABSTRACT: Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high-throughput RNA sequencing have revealed the complexity of transcriptomes, most of them rely on polyA enrichment protocols, able to detect only a small fraction of total RNA content. On the opposite end, massive-scale RNA sequencing on rRNA-depleted samples allows the survey of the complete set of coding and non-coding RNA species, now emerging as novel contributors to pathogenic mechanisms. Hence, in this work we analysed for the first time the complete transcriptome of human trisomic endothelial progenitor cells to an unprecedented level of resolution and sensitivity by RNA-sequencing. Our analysis allowed us to detect differential expression of even low expressed genes crucial for the pathogenesis, to disclose novel regions of active transcription outside yet annotated loci, and to investigate a plethora of non-polyadenylated long as well as short non coding RNAs. Novel splice isoforms for a large subset of crucial genes, and novel extended untranslated regions for known genes--possibly novel miRNA targets or regulatory sites for gene transcription--were also identified in this study. Coupling the rRNA depletion of samples, followed by high-throughput RNA-sequencing, to the easy availability of these cells renders this approach very feasible for transcriptome studies, offering the possibility of investigating in-depth blood-related pathological features of Down syndrome, as well as other genetic disorders.

SUBMITTER: Costa V

PROVIDER: S-EPMC3080369 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

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Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21.

Costa Valerio V Angelini Claudia C D'Apice Luciana L Mutarelli Margherita M Casamassimi Amelia A Sommese Linda L Gallo Maria Assunta MA Aprile Marianna M Esposito Roberta R Leone Luigi L Donizetti Aldo A Crispi Stefania S Rienzo Monica M Sarubbi Berardo B Calabrò Raffaele R Picardi Marco M Salvatore Paola P Infante Teresa T De Berardinis Piergiuseppe P Napoli Claudio C Ciccodicola Alfredo A

PloS one 20110420 4

Hybridization- and tag-based technologies have been successfully used in Down syndrome to identify genes involved in various aspects of the pathogenesis. However, these technologies suffer from several limits and drawbacks and, to date, information about rare, even though relevant, RNA species such as long and small non-coding RNAs, is completely missing. Indeed, none of published works has still described the whole transcriptional landscape of Down syndrome. Although the recent advances in high ...[more]

PMID: 21533138

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Project description:Down syndrome (DS) is due to the presence of an extra full or partial chromosome 21 (Hsa21). The identification of genes contributing to DS pathogenesis could be the key to any rational therapy of the associated intellectual disability. We aim at generating quantitative transcriptome maps in DS integrating all gene expression profile datasets available for any cell type or tissue, to obtain a complete model of the transcriptome in terms of both expression values for each gene and segmental trend of gene expression along each chromosome. We used the TRAM (Transcriptome Mapper) software for this meta-analysis, comparing transcript expression levels and profiles between DS and normal brain, lymphoblastoid cell lines, blood cells, fibroblasts, thymus and induced pluripotent stem cells, respectively. TRAM combined, normalized, and integrated datasets from different sources and across diverse experimental platforms. The main output was a linear expression value that may be used as a reference for each of up to 37,181 mapped transcripts analyzed, related to both known genes and expression sequence tag (EST) clusters. An independent example in vitro validation of fibroblast transcriptome map data was performed through "Real-Time" reverse transcription polymerase chain reaction showing an excellent correlation coefficient (r = 0.93, p < 0.0001) with data obtained in silico. The availability of linear expression values for each gene allowed the testing of the gene dosage hypothesis of the expected 3:2 DS/normal ratio for Hsa21 as well as other human genes in DS, in addition to listing genes differentially expressed with statistical significance. Although a fraction of Hsa21 genes escapes dosage effects, Hsa21 genes are selectively over-expressed in DS samples compared to genes from other chromosomes, reflecting a decisive role in the pathogenesis of the syndrome. Finally, the analysis of chromosomal segments reveals a high prevalence of Hsa21 over-expressed segments over the other genomic regions, suggesting, in particular, a specific region on Hsa21 that appears to be frequently over-expressed (21q22). Our complete datasets are released as a new framework to investigate transcription in DS for individual genes as well as chromosomal segments in different cell types and tissues.

Project description:ObjectivesTo validate the clinical efficacy and practical feasibility of massively parallel maternal plasma DNA sequencing to screen for fetal trisomy 21 among high risk pregnancies clinically indicated for amniocentesis or chorionic villus sampling.DesignDiagnostic accuracy validated against full karyotyping, using prospectively collected or archived maternal plasma samples.SettingPrenatal diagnostic units in Hong Kong, United Kingdom, and the Netherlands.Participants753 pregnant women at high risk for fetal trisomy 21 who underwent definitive diagnosis by full karyotyping, of whom 86 had a fetus with trisomy 21. Intervention Multiplexed massively parallel sequencing of DNA molecules in maternal plasma according to two protocols with different levels of sample throughput: 2-plex and 8-plex sequencing.Main outcome measuresProportion of DNA molecules that originated from chromosome 21. A trisomy 21 fetus was diagnosed when the z score for the proportion of chromosome 21 DNA molecules was >3. Diagnostic sensitivity, specificity, positive predictive value, and negative predictive value were calculated for trisomy 21 detection.ResultsResults were available from 753 pregnancies with the 8-plex sequencing protocol and from 314 pregnancies with the 2-plex protocol. The performance of the 2-plex protocol was superior to that of the 8-plex protocol. With the 2-plex protocol, trisomy 21 fetuses were detected at 100% sensitivity and 97.9% specificity, which resulted in a positive predictive value of 96.6% and negative predictive value of 100%. The 8-plex protocol detected 79.1% of the trisomy 21 fetuses and 98.9% specificity, giving a positive predictive value of 91.9% and negative predictive value of 96.9%.ConclusionMultiplexed maternal plasma DNA sequencing analysis could be used to rule out fetal trisomy 21 among high risk pregnancies. If referrals for amniocentesis or chorionic villus sampling were based on the sequencing test results, about 98% of the invasive diagnostic procedures could be avoided.

Dataset Information

Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21.

Publications

Massive-scale RNA-Seq analysis of non ribosomal transcriptome in human trisomy 21.

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