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IntroductionTrisomy 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.MethodsThe 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.ResultsOur 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.DiscussionOur 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.

Abstract Background Down syndrome (DS) is caused by the presence of an extra copy of full or partial human chromosome 21 (Hsa21). Partial (segmental) trisomy 21 (PT21) is the duplication of only a delimited region of Hsa21 and can be associated or not to DS: the study of PT21 cases is an invaluable model for addressing genotype–phenotype correlation in DS. Previous works reported systematic reanalyses of 132 subjects with PT21 and allowed the identification of a 34-kb highly restricted DS critical region (HR-DSCR) as the minimal region whose duplication is shared by all PT21 subjects diagnosed with DS. Methods We report clinical data and cytogenetic analysis of two children with PT21, one with DS and the other without DS. Moreover, we performed a systematic bibliographic search for any new PT21 report. Results Clinical and cytogenetic analyses of the two PT21 children have been reported: in Case 1 the duplication involves the whole long arm of Hsa21, except for the last 2.7 Mb, which are deleted as a consequence of an isodicentric 21: the HR-DSCR is within the duplicated regions and the child is diagnosed with DS. In Case 2 the duplication involves 7.1 Mb of distal 21q22, with a deletion of 2.1 Mb of proximal 20p, as a consequence of an unbalanced translocation: the HR-DSCR is not duplicated and the child presents with psychomotor development delay but no clinical signs of DS. Furthermore, two PT21 reports recently published (named Case 3 and 4) have been discussed: Case 3 has DS diagnosis, nearly full trisomy for Hsa21 and a monosomy for the 21q22.3 region. Case 4 is a baby without DS and a 0.56-Mb duplication of 21q22.3. Genotype–phenotype correlation confirmed the presence of three copies of the HR-DSCR in all DS subjects and two copies in all non-DS individuals. Conclusions The results presented here are fully consistent with the hypothesis that the HR-DSCR is critically associated with DS diagnosis. No exception to this pathogenetic model was found. Further studies are needed to detect genetic determinants likely located in the HR-DSCR and possibly responsible for core DS features, in particular intellectual disability.

Abstract Down syndrome (DS) is characterised by several clinical features including intellectual disability (ID) and craniofacial dysmorphisms. In 1976, Jackson and coll. identified a checklist of signs for clinical diagnosis of DS; the utility of these checklists in improving the accuracy of clinical diagnosis has been recently reaffirmed, but they have rarely been revised. The purpose of this work is to reassess the characteristic phenotypic signs and their frequencies in 233 DS subjects, following Jackson's checklist. 63.77% of the subjects showed more than 12 signs while none showed less than 5, confirming the effectiveness of Jackson's checklist for the clinical diagnosis of DS. An association between three phenotypic signs emerged, allowing us to distinguish two sub-phenotypes: Brachycephaly, short and broad Hands, short Neck (BHN), which is more frequent, and "non-BHN". The strong association of these signs might be interpreted in the context of the growth defects observed in DS children suggesting decreased cell proliferation. Lastly, cognitive assessments were investigated for 114 subjects. The lack of association between the presence of a physical sign or the number of signs present in a subject and cognitive skills disproves the stereotype that physical characteristics are predictive of degree of ID.

IntroductionDown 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.MethodsWe 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).ResultsResults 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.DiscussionThe 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.

Abstract 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.

Abstract This work investigates the role of metabolite levels in the intellectual impairment of subjects with Down syndrome (DS). Homocysteine, folate, vitamin B12, uric acid (UA), creatinine levels and MTHFR C677T genotype were analyzed in 147 subjects with DS. For 77 subjects, metabolite levels were correlated with cognitive tests. Griffiths-III test was administered to 28 subjects (3.08–6.16 years) and WPPSI-III test was administered to 49 subjects (7.08–16.08 years). Significant correlations were found among some metabolite levels and between homocysteine levels and MTHFR C677T genotype. Moreover, homocysteine, UA and creatinine levels resulted increased with age. We did not find any correlation between metabolites and cognitive test score in the younger group. Homocysteine showed statistically significant correlation with WPPSI-III subtest scores when its level is ≥ 7.35 µmol/L, remaining correlated in higher thresholds only for non-verbal area scores. Vitamin B12 showed correlations with all WPPSI-III subtest scores when its level is

Down syndrome (DS) is caused by trisomy of chromosome 21 and it is the most common genetic cause of intellectual disability (ID) in humans. Subjects with DS show a typical phenotype marked by facial dysmorphisms and ID. Partial trisomy 21 (PT21) is a rare genotype characterized by the duplication of a delimited chromosome 21 (Hsa21) portion and it may or may not be associated with DS diagnosis. The highly restricted Down syndrome critical region (HR-DSCR) is a region of Hsa21 present in three copies in all individuals with PT21 and a diagnosis of DS. This region, located on distal 21q22.13, is 34 kbp long and does not include characterized genes. The HR-DSCR is annotated as an intergenic region between KCNJ6-201 transcript encoding for potassium inwardly rectifying channel subfamily J member 6 and DSCR4-201 transcript encoding Down syndrome critical region 4. Two transcripts recently identified by massive RNA-sequencing (RNA-Seq) and automatically annotated on Ensembl database reveal that the HR-DSCR seems to be partially crossed by KCNJ6-202 and DSCR4-202 isoforms. KCNJ6-202 shares the coding sequence with KCNJ6-201 which is involved in many physiological processes, including heart rate in cardiac cells and circuit activity in neuronal cells. DSCR4-202 transcript has the first two exons in common with DSCR4-201, the only experimentally verified gene uniquely present in Hominidae. In this study, we performed in silico and in vitro analyses of the HR-DSCR. Bioinformatic data, obtained using Sequence Read Archive (SRA) and SRA-BLAST software, were confirmed by Reverse Transcription-Polymerase Chain Reaction (RT-PCR) and Sanger sequencing on a panel of human tissues. Our data demonstrate that the HR-DSCR cannot be defined as an intergenic region. Further studies are needed to investigate the functional role of the new transcripts, likely involved in DS phenotypes.

Abstract Objective A well-known limit of genome browsers is that the large amount of genome and gene data is not organized in the form of a searchable database, hampering full management of numerical data and free calculations. Due to the continuous increase of data deposited in genomic repositories, their content revision and analysis is recommended. Using GeneBase, a software with a graphical interface able to import and elaborate National Center for Biotechnology Information (NCBI) Gene database entries, we provide tabulated spreadsheets updated to 2019 about human nuclear protein-coding gene data set ready to be used for any type of analysis about genes, transcripts and gene organization. Results Comparison with previous reports reveals substantial change in the number of known nuclear protein-coding genes (now 19,116), the protein-coding non-redundant transcriptome space [now 59,281,518 base pair (bp), 10.1% increase], the number of exons (now 562,164, 36.2% increase) due to a relevant increase of the RNA isoforms recorded. Other parameters such as gene, exon or intron mean and extreme length appear to have reached a stability that is unlikely to be substantially modified by human genome data updates, at least regarding protein-coding genes. Finally, we confirm that there are no human introns shorter than 30 bp.

Abstract Objective Basic parameters commonly used to describe genomes including length, weight and relative guanine-cytosine (GC) content are widely cited in absence of a primary source. By using updated data and original software we determined these values to the best of our knowledge as standard reference for the whole human nuclear genome, for each chromosome and for mitochondrial DNA. We also devised a method to calculate the relative GC content in the whole messenger RNA sequence set and in transcriptomes by multiplying the GC content of each gene by its mean expression level. Results The male nuclear diploid genome extends for 6.27 Gigabase pairs (Gbp), is 205.00 cm (cm) long and weighs 6.41 picograms (pg). Female values are 6.37 Gbp, 208.23 cm, 6.51 pg. The individual variability and the implication for the DNA informational density in terms of bits/volume were discussed. The genomic GC content is 40.9%. Following analysis in different transcriptomes and species, we showed that the greatest deviation was observed in the pathological condition analysed (trisomy 21 leukaemic cells) and in Caenorhabditis elegans. Our results may represent a solid basis for further investigation on human structural and functional genomics while also providing a framework for other genome comparative analysis.

Abstract 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.

Caenorhabditis elegans is a nematode widely used in biology and genomics as a model organism. We provide an integrated, quantitative reference map for the transcriptome of whole, wild type Bristol N2 strain C. elegans worms. The map has been obtained by meta-analysis of 110 gene expression profiles available in Gene Expression Omnibus (GEO) repository and integrated using the computational biology tool Transcriptome Mapper (TRAM). Following probe assignment to the relative locus and intra- and inter-sample normalization (in particular using the scaled quantile method), a mean, consensus reference value is provided for 45,932 transcripts, along with standard deviation. Expression values are all mapped in the context of genomic coordinates. The map provides easy access to relationships among expression values of different genes in this standard condition, highlights genomic segments with relatively high over-/under-expression and may serve as a reference to test for gene expression variation for both individual genes and the whole transcriptome in specific biological conditions (e.g. mutated strains or differently grown worms). Keywords: C. elegans, Transcriptome map, Gene expression, Adult worms, Meta-analysis

Abstract Background Down syndrome (DS) is characterized by the presence of an extra full or partial human chromosome 21 (Hsa21). An invaluable model to define genotype‐phenotype correlations in DS is the study of the extremely rare cases of partial (segmental) trisomy 21 (PT21), the duplication of only a delimited region of Hsa21 associated or not to DS. A systematic retrospective reanalysis of 125 PT21 cases described up to 2015 allowed the creation of the most comprehensive PT21 map and the identification of a 34‐kb highly restricted DS critical region (HR‐DSCR) as the minimal region whose duplication is shared by all PT21 subjects diagnosed with DS. We reanalyzed at higher resolution three cases previously published and we accurately searched for any new PT21 reports in order to verify whether HR‐DSCR limits could prospectively be confirmed and possibly refined. Methods Hsa21 partial duplications of three PT21 subjects were refined by adding array‐based comparative genomic hybridization data. Seven newly described PT21 cases fulfilling stringent cytogenetic and clinical criteria have been incorporated into the PT21 integrated map. Results The PT21 map now integrates fine structure of Hsa21 sequence intervals of 132 subjects onto a common framework fully consistent with the presence of a duplicated HR‐DSCR, on distal 21q22.13 sub‐band, only in DS subjects and not in non‐DS individuals. No documented exception to the HR‐DSCR model was found. Conclusions The findings presented here further support the association of the HR‐DSCR with the diagnosis of DS, representing an unbiased validation of the original model. Further studies are needed to identify and characterize genetic determinants presumably located in the HR‐DSCR and functionally associated to the critical manifestations of DS.

This article contains further data and information from our published manuscript [1]. We aim to identify significant transcriptome alterations of total normal human thyroid vs. histologically normal thyroid adjacent to papillary thyroid carcinoma. We performed a systematic meta-analysis of all the available gene expression profiles for the whole organ also collecting gene expression data for the normal thyroid adjacent to papillary thyroid carcinoma. A differential quantitative transcriptome reference map was generated by using TRAM (Transcriptome Mapper) software able to combine, normalize and integrate a total of 35 datasets from total normal thyroid and 40 datasets from histologically normal thyroid adjacent to papillary thyroid carcinoma from different sources. This analysis identified genes and genome segments that significantly discriminated the two groups of samples. Differentially expressed genes were grouped and enrichment function analyses were performed identifying the main features of the differentially expressed genes between total normal thyroid and histologically normal thyroid adjacent to papillary thyroid carcinoma. The search for housekeeping genes retrieved 414 loci.

Down Syndrome (DS) is the most common genetic alteration responsible for intellectual disability, which refers to deficits in both intellectual and adaptive functioning. According to this, individuals with Down Syndrome (DS) reach developmental milestones (e.g., sitting, walking, and babbling) in the same order as their typically developing peers, but later in life. Since developmental milestones are the first blocks on which development builds, the aims of the current study are to: (i) expand the knowledge of developmental milestone acquisition; and (ii) explore the relationship between developmental milestone acquisition and later development. For this purpose 105 children/adolescents with DS were involved in this study, divided in two groups, Preschoolers (n = 39) and School-age participants (n = 66). Information on the age of acquisition of Sitting, Walking, Babbling, and Sphincter Control was collected, together with cognitive, motor, and adaptive functioning. Sitting predicted later motor development, but, with age, it became less important in predicting motor development in everyday life. Babbling predicted later language development in older children. Finally, Sphincter Control emerged as the strongest predictor of motor, cognitive, language, and adaptive skills, with its role being more evident with increasing age. Our data suggest that the age of reaching the milestones considered in the study has an influence on successive development, a role that can be due to common neural substrates, the environment, and the developmental cascade effect.

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.

Human RCAN3 (regulator of calcineurin 3) belongs to the human RCAN gene family.In this study we provide, with in silico and in vitro analyses, the first detailed description of the human multi-transcript RCAN3 locus. Its analysis revealed that it is composed of a multigene system that includes at least 21 RCAN3 alternative spliced isoforms (16 of them identified here for the first time) and a new RCAN3 antisense gene (RCAN3AS). In particular, we cloned RCAN3-1,3,4,5 (lacking exon 2), RCAN3-1a,2,3,4,5, RCAN3-1a,3,4,5, RCAN3-1b,2,3,4,5, RCAN3-1c,2,3,4,5, RCAN3-1c,2,4,5 and RCAN3-1c,3,4,5, isoforms that present a different 5' untranslated region when compared to RCAN3. Moreover, in order to verify the possible 5' incompleteness of previously identified cDNA isoforms with the reference exon 1, ten more alternative isoforms were retrieved. Bioinformatic searches allowed us to identify RCAN3AS, which overlaps in part with exon 1a, on the opposite strand, for which four different RCAN3AS isoforms were cloned.In order to analyze the different expression patterns of RCAN3 alternative first exons and of RCAN3AS mRNA isoforms, RT-PCR was performed in 17 human tissues. Finally, analyses of RCAN3 and RCAN3AS genomic sequences were performed to identify possible promoter regions, to examine donor and acceptor splice sequences and to compare evolutionary conservation, in particular of alternative exon 1 or 1c--exon 2 junctions in different species.The description of its number of transcripts, of their expression patterns and of their regulatory regions can be important to clarify the functions of RCAN3 gene in different pathways and cellular processes.