The transcriptome profile of human trisomy 21 blood cells.

BACKGROUND: Trisomy 21 (T21) is a genetic alteration characterised by the presence of an extra full or partial human chromosome 21 (Hsa21) leading to Down syndrome (DS), the most common form of intellectual disability (ID). It is broadly agreed that the presence of extra genetic material in T21 gives origin to an altered expression of genes located on Hsa21 leading to DS phenotype. The aim of this study was to analyse T21 and normal control blood cell gene expression profiles obtained by total RNA sequencing (RNA-Seq). RESULTS: The results were elaborated by the TRAM (Transcriptome Mapper) software which generated a differential transcriptome map between human T21 and normal control blood cells providing the gene expression ratios for 17,867 loci. The obtained gene expression profiles were validated through real-time reverse transcription polymerase chain reaction (RT-PCR) assay and compared with previously published data. A post-analysis through transcriptome mapping allowed the identification of the segmental (regional) variation of the expression level across the whole genome (segment-based analysis of expression). Interestingly, the most over-expressed genes encode for interferon-induced proteins, two of them (MX1 and MX2 genes) mapping on Hsa21 (21q22.3). The altered expression of genes involved in mitochondrial translation and energy production also emerged, followed by the altered expression of genes encoding for the folate cycle enzyme, GART, and the folate transporter, SLC19A1. CONCLUSIONS: The alteration of these pathways might be linked and involved in the manifestation of ID in DS.

Human trisomy 21 fibroblasts rescue methotrexate toxic effect after treatment with 5-methyl-tetrahydrofolate and 5-formyl-tetrahydrofolate.

Trisomy 21 causes Down syndrome (DS), the most common human genetic disorder and the leading genetic cause of intellectual disability. The alteration of one-carbon metabolism was described as the possible metabolic cause of the intellectual disability development in subjects with DS. One of the biochemical pathways involved in the one-carbon group transfer is the folate cycle. The cytotoxic drug methotrexate (MTX) is a folic acid (FA) analogue which inhibits the activity of dihydrofolate reductase enzyme involved in the one-carbon metabolic cycle. Trisomy 21 cells are more sensitive to the MTX effect than euploid cells, and in 1986 Jérôme Lejeune and Coll. demonstrated that MTX was twice as toxic in trisomy 21 lymphocytes than in control cells. In the present work, the rescue effect on MTX toxicity mediated by FA and some of its derivatives, tetrahydrofolate (THF), 5-formyl-THF, and 5-methyl-THF, in both normal and trisomy 21 skin fibroblast cells, was evaluated. A statistically significant rescue effect was obtained by 5-formyl-THF, 5-methyl-THF, and their combination, administered together with MTX. In conclusion, trisomy 21 fibroblast cell lines showed a good response to the rescue effects of 5-formyl-THF and 5-methyl-THF on the MTX toxicity almost as normal cell lines.

Systematic reanalysis of partial trisomy 21 cases with or without Down syndrome suggests a small region on 21q22.13 as critical to the phenotype.

A 'Down Syndrome critical region' (DSCR) sufficient to induce the most constant phenotypes of Down syndrome (DS) had been identified by studying partial (segmental) trisomy 21 (PT21) as an interval of 0.6-8.3 Mb within human chromosome 21 (Hsa21), although its existence was later questioned. We propose an innovative, systematic reanalysis of all described PT21 cases (from 1973 to 2015). In particular, we built an integrated, comparative map from 125 cases with or without DS fulfilling stringent cytogenetic and clinical criteria. The map allowed to define or exclude as candidates for DS fine Hsa21 sequence intervals, also integrating duplication copy number variants (CNVs) data. A highly restricted DSCR (HR-DSCR) of only 34 kb on distal 21q22.13 has been identified as the minimal region whose duplication is shared by all DS subjects and is absent in all non-DS subjects. Also being spared by any duplication CNV in healthy subjects, HR-DSCR is proposed as a candidate for the typical DS features, the intellectual disability and some facial phenotypes. HR-DSCR contains no known gene and has relevant homology only to the chimpanzee genome. Searching for HR-DSCR functional loci might become a priority for understanding the fundamental genotype-phenotype relationships in DS.

Extensive microRNA-mediated crosstalk between lncRNAs and mRNAs in mouse embryonic stem cells.

Recently, a handful of intergenic long noncoding RNAs (lncRNAs) have been shown to compete with mRNAs for binding to miRNAs and to contribute to development and disease. Beyond these reports, little is yet known of the extent and functional consequences of miRNA-mediated regulation of mRNA levels by lncRNAs. To gain further insight into lncRNA-mRNA miRNA-mediated crosstalk, we reanalyzed transcriptome-wide changes induced by the targeted knockdown of over 100 lncRNA transcripts in mouse embryonic stem cells (mESCs). We predicted that, on average, almost one-fifth of the transcript level changes induced by lncRNAs are dependent on miRNAs that are highly abundant in mESCs. We validated these findings experimentally by temporally profiling transcriptome-wide changes in gene expression following the loss of miRNA biogenesis in mESCs. Following the depletion of miRNAs, we found that >50% of lncRNAs and their miRNA-dependent mRNA targets were up-regulated coordinately, consistent with their interaction being miRNA-mediated. These lncRNAs are preferentially located in the cytoplasm, and the response elements for miRNAs they share with their targets have been preserved in mammals by purifying selection. Lastly, miRNA-dependent mRNA targets of each lncRNA tended to share common biological functions. Post-transcriptional miRNA-mediated crosstalk between lncRNAs and mRNA, in mESCs, is thus surprisingly prevalent, conserved in mammals, and likely to contribute to critical developmental processes.

Genotype-phenotype correlation for congenital heart disease in Down syndrome through analysis of partial trisomy 21 cases.

Among Down syndrome (DS) children, 40-50% have congenital heart disease (CHD). Although trisomy 21 is not sufficient to cause CHD, three copies of at least part of chromosome 21 (Hsa21) increases the risk for CHD. In order to establish a genotype-phenotype correlation for CHD in DS, we built an integrated Hsa21 map of all described partial trisomy 21 (PT21) cases with sufficient indications regarding presence or absence of CHD (n=107), focusing on DS PT21 cases. We suggest a DS CHD candidate region on 21q22.2 (0.96Mb), being shared by most PT21 cases with CHD and containing three known protein-coding genes (DSCAM, BACE2, PLAC4) and four known non-coding RNAs (DSCAM-AS1, DSCAM-IT1, LINC00323, MIR3197). The characterization of a DS CHD candidate region provides a useful approach to identify specific genes contributing to the pathology and to orient further investigations and possibly more effective therapy in relation to the multifactorial pathogenesis of CHD.

Integrated Transcriptome Map Highlights Structural and Functional Aspects of the Normal Human Heart.

A systematic meta-analysis of the available gene expression profiling datasets for the whole normal human heart generated a quantitative transcriptome reference map of this organ. Transcriptome Mapper (TRAM) software integrated 32 gene expression profile datasets from different sources returning a reference value of expression for each of the 43,360 known, mapped transcripts assayed by any of the experimental platforms used in this regard. Main findings include the visualization at the gene and chromosomal levels of the classical description of the basic histology and physiology of the heart, the identification of suitable housekeeping reference genes, the analysis of stoichiometry of gene products, and the focusing on chromosome 21 genes, which are present in one excess copy in Down syndrome subjects, presenting cardiovascular defects in 30-40% of cases. Independent in vitro validation showed an excellent correlation coefficient (r = 0.98) with the in silico data. Remarkably, heart/non-cardiac tissue expression ratio may also be used to anticipate that effects of mutations will most probably affect or not the heart. The quantitative reference global portrait of gene expression in the whole normal human heart illustrates the structural and functional aspects of the whole organ and is a general model to understand the mechanisms underlying heart pathophysiology. J. Cell. Physiol. 232: 759-770, 2017. © 2016 Wiley Periodicals, Inc.

A molecular view of the normal human thyroid structure and function reconstructed from its reference transcriptome map.

BACKGROUND: The thyroid is the earliest endocrine structure to appear during human development, and thyroid hormones are necessary for proper organism development, in particular for the nervous system and heart, normal growth and skeletal maturation. To date a quantitative, validated transcriptional atlas of the whole normal human thyroid does not exist and the availability of a detailed expression map might be an excellent occasion to investigate the many features of the thyroid transcriptome. RESULTS: We present a view at the molecular level of the normal human thyroid histology and physiology obtained by a systematic meta-analysis of all the available gene expression profiles for the whole organ. A quantitative transcriptome reference map was generated by using the TRAM (Transcriptome Mapper) software able to combine, normalize and integrate a total of 35 suitable datasets from different sources thus providing a typical reference expression value for each of the 27,275 known, mapped transcripts obtained. The experimental in vitro validation of data was performed by "Real-Time" reverse transcription polymerase chain reaction showing an excellent correlation coefficient (r = 0.93) with data obtained in silico. CONCLUSIONS: Our study provides a quantitative global reference portrait of gene expression in the normal human thyroid and highlights differential expression between normal human thyroid and a pool of non-thyroid tissues useful for modeling correlations between thyroidal gene expression and specific thyroid functions and diseases. The experimental in vitro validation supports the possible usefulness of the human thyroid transcriptome map as a reference for molecular studies of the physiology and pathology of this organ.

A quantitative transcriptome reference map of the normal human hippocampus.

We performed an innovative systematic meta-analysis of 41 gene expression profiles of normal human hippocampus to provide a quantitative transcriptome reference map of it, i.e. a reference typical value of expression for each of the 30,739 known mapped and the 16,258 uncharacterized (unmapped) transcripts. For this aim, we used the software called TRAM (Transcriptome Mapper), which is able to generate transcriptome maps based on gene expression data from multiple sources. We also analyzed differential expression by comparing the hippocampus with the whole brain transcriptome map to identify a typical expression pattern of this subregion compared with the whole organ. Finally, due to the fact that the hippocampus is one of the main brain region to be severely affected in trisomy 21 (the best known genetic cause of intellectual disability), a particular attention was paid to the expression of chromosome 21 (chr21) genes. Data were downloaded from microarray databases, processed, and analyzed using TRAM software. Among the main findings, the most over-expressed loci in the hippocampus are the expressed sequence tag cluster Hs.732685 and the member of the calmodulin gene family CALM2. The tubulin folding cofactor B (TBCB) gene is the best gene at behaving like a housekeeping gene. The hippocampus vs. the whole brain differential transcriptome map shows the over-expression of LINC00114, a long non-coding RNA mapped on chr21. The hippocampus transcriptome map was validated in vitro by assaying gene expression through several magnitude orders by "Real-Time" reverse transcription polymerase chain reaction (RT-PCR). The highly significant agreement between in silico and experimental data suggested that our transcriptome map may be a useful quantitative reference benchmark for gene expression studies related to human hippocampus. Furthermore, our analysis yielded biological insights about those genes that have an intrinsic over-/under-expression in the hippocampus.

A quantitative transcriptome reference map of the normal human brain.

We performed an innovative systematic meta-analysis of 60 gene expression profiles of whole normal human brain, to provide a quantitative transcriptome reference map of it, i.e. a reference typical value of expression for each of the 39,250 known, mapped and 26,026 uncharacterized (unmapped) transcripts. To this aim, we used the software named Transcriptome Mapper (TRAM), which is able to generate transcriptome maps based on gene expression data from multiple sources. We also analyzed differential expression by comparing the brain transcriptome with those derived from human foetal brain gene expression, from a pool of human tissues (except the brain) and from the two normal human brain regions cerebellum and cerebral cortex, which are two of the main regions severely affected when cognitive impairment occurs, as happens in the case of trisomy 21. Data were downloaded from microarray databases, processed and analyzed using TRAM software and validated in vitro by assaying gene expression through several magnitude orders by 'real-time' reverse transcription polymerase chain reaction (RT-PCR). The excellent agreement between in silico and experimental data suggested that our transcriptome maps may be a useful quantitative reference benchmark for gene expression studies related to the human brain. Furthermore, our analysis yielded biological insights about those genes which have an intrinsic over-/under-expression in the brain, in addition offering a basis for the regional analysis of gene expression. This could be useful for the study of chromosomal alterations associated to cognitive impairment, such as trisomy 21, the most common genetic cause of intellectual disability.

Improving mRNA 5' coding sequence determination in the mouse genome.

The incomplete determination of the mRNA 5' end sequence may lead to the incorrect assignment of the first AUG codon and to errors in the prediction of the encoded protein product. Due to the significance of the mouse as a model organism in biomedical research, we performed a systematic identification of coding regions at the 5' end of all known mouse mRNAs, using an automated expressed sequence tag (EST)-based approach which we have previously described. By parsing almost 4 million BLAT alignments we found 351 mouse loci, out of 20,221 analyzed, in which an extension of the mRNA 5' coding region was identified. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for Apc2 and Mknk2 cDNAs. We also generated a list of 16,330 mouse mRNAs where the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5' end in the current form. Systematic searches in the main mouse genome databases and genome browsers showed that 82% of our results are original and have not been identified by their annotation pipelines. Moreover, the same information is not easily derivable from RNA-Seq data, due to short sequence length and laboriousness in building full-length transcript structures. In conclusion, our results improve the determination of full-length 5' coding sequences and might be useful in order to reduce errors when studying mouse gene structure and function in biomedical research.

Characterization of human gene locus CYYR1: a complex multi-transcript system.

Cysteine/tyrosine-rich 1 (CYYR1) is a gene we previously identified on human chromosome 21 starting from an in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.3), where no coding region had previously been predicted. CYYR1 was initially characterized as a four-exon gene, whose brain-derived cDNA sequencing predicts a 154-amino acid product. In this study we provide, with in silico and in vitro analyses, the first detailed description of the human CYYR1 locus. The analysis of this locus revealed that it is composed of a multi-transcript system, which includes at least seven CYYR1 alternative spliced isoforms and a new CYYR1 antisense gene (named CYYR1-AS1). In particular, we cloned, for the first time, the following isoforms: CYYR1-1,2,3,4b and CYYR1-1,2,3b, which present a different 3' transcribed region, with a consequent different carboxy-terminus of the predicted proteins; CYYR1-1,2,4 lacks exon 3; CYYR1-1,2,2bis,3,4 presents an additional exon between exon 2 and exon 3; CYYR1-1b,2,3,4 presents a different 5' untranslated region when compared to CYYR1. The complexity of the locus is enriched by the presence of an antisense transcript. We have cloned a long transcript overlapping with CYYR1 as an antisense RNA, probably a non-coding RNA. Expression analysis performed in different normal tissues, tumour cell lines as well as in trisomy 21 and euploid fibroblasts has confirmed a quantitative and qualitative variability in the expression pattern of the multi-transcript locus, suggesting a possible role in complex diseases that should be further investigated.

Universal tight correlation of codon bias and pool of RNA codons (codonome): The genome is optimized to allow any distribution of gene expression values in the transcriptome from bacteria to humans.

Codon bias is the phenomenon in which distinct synonymous codons are used with different frequencies. We define here the "codonome value" as the total number of codons present across all the expressed mRNAs in a given biological condition. We have developed the "CODONOME" software, which calculates the codon bias and, following integration with a gene expression profile, estimates the actual frequency of each codon at the transcriptome level (codonome bias) of a given tissue. Systematic analysis across different human tissues and multiple species shows a surprisingly tight correlation between the codon bias and the codonome bias. An aneuploidy and cancer condition such as that of Down Syndrome-related acute megakaryoblastic leukemia (DS-AMKL), does not appear to alter this relationship. The law of correlation between codon bias and codonome emerges as a property of the distribution and range of the number, sequence and expression level of the genes in a genome.

Corrigendum: One-carbon pathway metabolites are altered in the plasma of subjects with Down syndrome: relation to chromosomal dosage.

[This corrects the article DOI: 10.3389/fmed.2022.1006891.].

The functional roles of S-adenosyl-methionine and S-adenosyl-homocysteine and their involvement in trisomy 21.

The one-carbon metabolism pathway is involved in critical human cellular functions such as cell proliferation, mitochondrial respiration, and epigenetic regulation. In the homocysteine-methionine cycle S-adenosyl-methionine (SAM) and S-adenosyl-homocysteine (SAH) are synthetized, and their levels are finely regulated to ensure proper functioning of key enzymes which control cellular growth and differentiation. Here we review the main biological mechanisms involving SAM and SAH and the known related human diseases. It was recently demonstrated that SAM and SAH levels are altered in plasma of subjects with trisomy 21 (T21) but how this metabolic dysregulation influences the clinical manifestation of T21 phenotype has not been previously described. This review aims at providing an overview of the biological mechanisms which are altered in response to changes in the levels of SAM and SAH observed in DS.

Zinc metabolism and its role in immunity status in subjects with trisomy 21: chromosomal dosage effect.

Introduction: Trisomy 21 (T21), which causes Down syndrome (DS), is the most common chromosomal aneuploidy in humankind and includes different clinical comorbidities, among which the alteration of the immune system has a heavy impact on patient's lives. A molecule with an important role in immune response is zinc and it is known that its concentration is significantly lower in children with T21. Different hypotheses were made about this metabolic alteration and one of the reasons might be the overexpression of superoxide dismutase 1 (SOD1) gene, as zinc is part of the SOD1 active enzymatic center. Methods: The aim of our work is to explore if there is a linear correlation between zinc level and immune cell levels measured in a total of 217 blood samples from subjects with T21. Furthermore, transcriptome map analyses were performed using Transcriptome Mapper (TRAM) software to investigate whether a difference in gene expression is detectable between subjects with T21 and euploid control group in tissues and cells involved in the immune response such as lymphoblastoid cells, thymus and white blood cells. Results: Our results have confirmed the literature data stating that the blood zinc level in subjects with T21 is lower compared to the general population; in addition, we report that the T21/control zinc concentration ratio is 2:3, consistent with a chromosomal dosage effect due to the presence of three copies of chromosome 21. The transcriptome map analyses showed an alteration of some gene's expression which might explain low levels of zinc in the blood. Discussion: Our data suggest that zinc level is not associated with the levels of immunity cells or proteins analyzed themselves and rather the main role of this ion might be played in altering immune cell function.

Genome-scale analysis of human mRNA 5' coding sequences based on expressed sequence tag (EST) database.

The "5' end mRNA artifact" issue refers to the incorrect assignment of the first AUG codon in an mRNA, due to the incomplete determination of its 5' end sequence. We performed a systematic identification of coding regions at the 5' end of all human known mRNAs, using an automated expressed sequence tag (EST)-based approach. Following parsing of more than 7 million BLAT alignments, we found 477 human loci, out of 18,665 analyzed, in which an extension of the mRNA 5' coding region was identified. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1, QARS and TDP2 cDNAs, and the consequences for the functional studies of these loci are discussed. We also generated a list of 20,775 human mRNAs where the presence of an in-frame stop codon upstream of the known start codon indicates completeness of the coding sequence at 5' in the current form.

An estimation of the number of cells in the human body.

BACKGROUND: All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 10(12) and 10(16) and it is widely mentioned without a proper reference. AIM: To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. SUBJECTS AND METHODS: A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. RESULTS: A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72 × 10(13). CONCLUSIONS: Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

Machine learning based analysis for intellectual disability in Down syndrome.

Down syndrome (DS) or trisomy 21 is the most common genetic cause of intellectual disability (ID), but a pathogenic mechanism has not been identified yet. Studying a complex and not monogenic condition such as DS, a clear correlation between cause and effect might be difficult to find through classical analysis methods, thus different approaches need to be used. The increased availability of big data has made the use of artificial intelligence (AI) and in particular machine learning (ML) in the medical field possible. The purpose of this work is the application of ML techniques to provide an analysis of clinical records obtained from subjects with DS and study their association with ID. We have applied two tree-based ML models (random forest and gradient boosting machine) to the research question: how to identify key features likely associated with ID in DS. We analyzed 109 features (or variables) in 106 DS subjects. The outcome of the analysis was the age equivalent (AE) score as indicator of intellectual functioning, impaired in ID. We applied several methods to configure the models: feature selection through Boruta framework to minimize random correlation; data augmentation to overcome the issue of a small dataset; age effect mitigation to take into account the chronological age of the subjects. The results show that ML algorithms can be applied with good accuracy to identify variables likely involved in cognitive impairment in DS. In particular, we show how random forest and gradient boosting machine produce results with low error (MSE <0.12) and an acceptable R2 (0.70 and 0.93). Interestingly, the ranking of the variables point to several features of interest related to hearing, gastrointestinal alterations, thyroid state, immune system and vitamin B12 that can be considered with particular attention for improving care pathways for people with DS. In conclusion, ML-based model may assist researchers in identifying key features likely correlated with ID in DS, and ultimately, may improve research efforts focused on the identification of possible therapeutic targets and new care pathways. We believe this study can be the basis for further testing/validating of our algorithms with multiple and larger datasets.

One-carbon pathway metabolites are altered in the plasma of subjects with Down syndrome: Relation to chromosomal dosage.

Introduction: Down syndrome (DS) is the most common chromosomal disorder and it is caused by trisomy of chromosome 21 (Hsa21). Subjects with DS show a large heterogeneity of phenotypes and the most constant clinical features present are typical facies and intellectual disability (ID). Several studies demonstrated that trisomy 21 causes an alteration in the metabolic profile, involving among all the one-carbon cycle. Methods: We performed enzyme-linked immunosorbent assays (ELISAs) to identify the concentration of 5 different intermediates of the one-carbon cycle in plasma samples obtained from a total of 164 subjects with DS compared to 54 euploid subjects. We investigated: tetrahydrofolate (THF; DS n = 108, control n = 41), 5-methyltetrahydrofolate (5-methyl-THF; DS n = 140, control n = 34), 5-formyltetrahydrofolate (5-formyl-THF; DS n = 80, control n = 21), S-adenosyl-homocysteine (SAH; DS n = 94, control n = 20) and S-adenosyl-methionine (SAM; DS n = 24, control n = 15). Results: Results highlight specific alterations of THF with a median concentration ratio DS/control of 2:3, a decrease of a necessary molecule perfectly consistent with a chromosomal dosage effect. Moreover, SAM and SAH show a ratio DS/control of 1.82:1 and 3.6:1, respectively. Discussion: The relevance of these results for the biology of intelligence and its impairment in trisomy 21 is discussed, leading to the final proposal of 5-methyl-THF as the best candidate for a clinical trial aimed at restoring the dysregulation of one-carbon cycle in trisomy 21, possibly improving cognitive skills of subjects with DS.