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Communicating the diagnosis of Down Syndrome to a couple of parents is never easy, whether before or after birth. As doctors, we must certainly rely on our own relational skills, but it is also necessary to be confident in some general indications, which are often overlooked in the strict hospital routine. This article is intended as a summary of the main articles published on this subject in the international literature, collecting and summarising the most important indications that have emerged in years of medical practice all over the world as well as in our personal experience. The diffusion of these guidelines is essential to help the doctor in this difficult task, on which there is often little training, and above all to guarantee to the parents the least traumatic communication possible.

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.

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.

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.

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.

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.

Trisomy 21 (Down syndrome, DS) is the main human genetic cause of intellectual disability (ID). Lejeune hypothesized that DS could be considered a metabolic disease, and we found that subjects with DS have a specific plasma and urinary metabolomic profile. In this work we confirmed the alteration of mitochondrial metabolism in DS and also investigated if metabolite levels are related to cognitive aspects of DS. We analyzed the metabolomic profiles of plasma samples from 129 subjects with DS and 46 healthy control (CTRL) subjects by 1H Nuclear Magnetic Resonance (NMR). Multivariate analysis of the NMR metabolomic profiles showed a clear discrimination (up to 94% accuracy) between the two groups. The univariate analysis revealed a significant alteration in 7 metabolites out of 28 assigned unambiguously. Correlations among the metabolite levels in DS and CTRL groups were separately investigated and statistically significant relationships appeared. On the contrary, statistically significant correlations among the NMR-detectable part of DS plasma metabolome and the different intelligence quotient ranges obtained by Griffiths-III or WPPSI-III tests were not found. Even if metabolic imbalance provides a clear discrimination between DS and CTRL groups, it appears that the investigated metabolomic profiles cannot be associated with the degree of ID.

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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13104-019-4137-z) contains supplementary material, which is available to authorized users.

Background: 5,10-Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR-RFLP method for the detection of this polymorphism. Methods: In order to retrieve all published articles possibly describing any PCR-RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer-BLAST software was used to check primer pair's biological specificity. Results: The analysis of previous literature showed that PCR-RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA-fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by HinfI enzyme in two pieces of 146bp and 367bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out. Conclusion: Our original PCR-RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR-RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date.

Down Syndrome (DS) is the most frequent form of intellectual disability (ID) of genetic origin, whose main features include craniofacial dysmorphisms and cardiovascular defects. In 1959, Lejeune and coll. described an extra copy of chromosome 21 (Hsa21) in children with DS (trisomy 21, or T21). We first review how different biological mechanisms may lead to the gain of genetic material of Hsa21 in the cells, originating from different combinations of genetic conditions, including a free or translocated extra copy of Hsa21, distributed in all cells or only in a part of them (mosaicism), with a complete or partial representation of the Hsa21 long arm (21q). Although it is broadly agreed that the DS phenotype originates from the altered expression of the genes located on Hsa21, its molecular pathogenesis is still unknown. We therefore illustrate how recent genomic science may be useful in the elucidation of the genotype-phenotype relationship in DS.

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.

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.

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 Jerome 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.

Background A noninvasive blood test for the early detection of colorectal cancer (CRC) is highly required. We evaluated a panel of 4 mRNAs as putative markers of CRC. Materials and Methods We tested LGALS4, CEACAM6, TSPAN8, and COL1A2, referred to as the CELTiC panel, using quantitative reverse transcription polymerase chain reaction, on subjects with positive fecal immunochemical test (FIT) results and undergoing colonoscopy. Using a nonparametric test and multinomial logistic model, FIT-positive subjects were compared with CRC patients and healthy individuals. Results All the genes of the CELTiC panel displayed statistically significant differences between the healthy subjects (n = 67), both low-risk (n = 36) and high-risk/CRC (n = 92) subjects, and those in the negative-colonoscopy, FIT-positive group (n = 36). The multinomial logistic model revealed LGALS4 was the most powerful marker discriminating the 4 groups. When assessing the diagnostic values by analysis of the areas under the receiver operating characteristic curves (AUCs), the CELTiC panel reached an AUC of 0.91 (sensitivity, 79%; specificity, 94%) comparing normal subjects to low-risk subjects, and 0.88 (sensitivity, 75%; specificity, 87%) comparing normal and high-risk/CRC subjects. The comparison between the normal subjects and the negative-colonoscopy, FIT-positive group revealed an AUC of 0.93 (sensitivity, 82%; specificity, 97%). Conclusion The CELTiC panel could represent a useful tool for discriminating subjects with positive FIT findings and for the early detection of precancerous adenomatous lesions and CRC.

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. © 2015 Wiley Periodicals, Inc.

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. Electronic supplementary material The online version of this article (doi:10.1186/s12864-017-4049-z) contains supplementary material, which is available to authorized users.

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.

The ideal reference, or control, gene for the study of gene expression in a given organism should be expressed at a medium‑high level for easy detection, should be expressed at a constant/stable level throughout different cell types and within the same cell type undergoing different treatments, and should maintain these features through as many different tissues of the organism. From a biological point of view, these theoretical requirements of an ideal reference gene appear to be best suited to housekeeping (HK) genes. Recent advancements in the quality and completeness of human expression microarray data and in their statistical analysis may provide new clues toward the quantitative standardization of human gene expression studies in biology and medicine, both cross‑ and within‑tissue. The systematic approach used by the present study is based on the Transcriptome Mapper tool and exploits the automated reassignment of probes to corresponding genes, intra‑ and inter‑sample normalization, elaboration and representation of gene expression values in linear form within an indexed and searchable database with a graphical interface recording quantitative levels of expression, expression variability and cross‑tissue width of expression for more than 31,000 transcripts. The present study conducted a meta‑analysis of a pool of 646 expression profile data sets from 54 different human tissues and identified actin γ 1 as the HK gene that best fits the combination of all the traditional criteria to be used as a reference gene for general use; two ribosomal protein genes, RPS18 and RPS27, and one aquaporin gene, POM121 transmembrane nucleporin C, were also identified. The present study provided a list of tissue‑ and organ‑specific genes that may be most suited for the following individual tissues/organs: Adipose tissue, bone marrow, brain, heart, kidney, liver, lung, ovary, skeletal muscle and testis; and also provides in these cases a representative, quantitative portrait of the relative, typical gene‑expression profile in the form of searchable database tables.

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.

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

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.

not available

The term "5´ end mRNA artifact" refers to the incorrect assignment of the first AUG codon in an mRNA, due to the incomplete determination of its 5´ end sequence (Casadei et al., 2003). Since the '70s, the amino acid sequence of gene products has been routinely deduced from the nucleotide sequence of the relative cloned cDNA (DNA complementary to mRNA), according to rules for recognition of the start codon (first-AUG rule, optimal sequence context) and the genetic code (Kozak, 2002). All standard methods for the cloning of cDNA are affected by a potential inability to effectively clone the 5´ region of mRNA. This is due to the reverse transcriptase failure to extend first-strand cDNA along the full length of the mRNA template toward its 5´ end (Sambrook, 2001). The identification of a more complete mRNA 5´ end could reveal an additional upstream AUG – in-frame with the previously determined one – thus extending the predicted amino terminus sequence of the product and avoiding subsequent relevant errors in the experimental study of the relative cDNA (Casadei et al., 2003). The continuous incorporation of information derived from individual and large-scale cDNA sequencing projects, including those specifically designed to characterize mRNA 5´ end (Carninci et al., 1996; Suzuki et al., 2000; Porcel et al., 2004), in the last few years led to continuous improvement of completeness of mRNA reference sequences (e.g., RefSeq), and also to the corresponding protein coding sequences. However, genome browsers do not appear to systematically extract useful information from the ever-increasing vast quantity of EST (expressed sequence tag) data. To date, EST data remain invaluable due to significantly longer continuous RNA sequences they may provide in comparison with the very short fragments typically deposited in current high-throughput nucleotide sequencing databases. We previously used individual EST-based gene model refinement by classic in silico sequence analysis to revise the mRNA sequence of 109 human chromosome 21 protein-coding genes (Casadei et al., 2003). The success of this approach encouraged us to develop a piece of software ("5'_ORF_Extender" software) in order to automate the steps that were previously performed manually, applying it to the Danio rerio (zebrafish) genome (Frabetti et al., 2007). In the present work, we present a modified strategy able to analyze the much more numerous human sequences. Firstly, we fully revised the software algorithm by using pre-computed coordinates of the UCSC-downloaded RefSeqs and ESTs genome alignment data and specific UCSC-downloaded EST sequence entries. Furthermore, we adopted an original quality filter which was able to test if each single EST candidate with sequence information of possible use for extending a known mRNA, was attributed to the same locus of that mRNA by an updated, complete and embedded version of UniGene. Lastly, we automated data summarization for an analyzed genome. Following these improvements, parsing more than 7 million BLAT alignment, 5'_ORF_Extender 2.0 recognized a total of 477 loci, out of the 18,665 human loci represented in the mRNA reference set, as bona fide candidates for extension. Proof-of-concept confirmation was obtained by in vitro cloning and sequencing for GNB2L1 (guanine nucleotide binding protein (G protein), beta polypeptide 2-like 1), QARS (glutaminyl-tRNA synthetase) and TDP2 (tyrosyl-DNA phosphodiesterase 2) cDNAs, and the consequences for the functional studies of these loci are discussed. In addition, we generated a list of 20,775 human mRNAs in which 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. Bibliografia: R. Casadei et al., mRNA 5’ region sequence incompleteness: a potential source of systematic errors in translation initiation codon assignment in human mRNAs, Gene 321 (2003) 185–193. M. Kozak, Pushing the limits of the scanning mechanism for initiation of translation, Gene 99 (2002) 1–34. P. Carninci et al., High-efficiency fulllength cDNA cloning by biotinylated CAP trapper, Genomics 37 (1996) 327–336. F. Frabetti et al., Systematic analysis of mRNA 5’ coding sequence incompleteness in Danio rerio: an automated EST-based approach, Biol. Direct 2 (2007) 34.

We have developed GeneBase, a full parser of the National Center for Biotechnology Information (NCBI) Gene database, which generates a fully structured local database with an intuitive user-friendly graphic interface for personal computers. Features of all the annotated eukaryotic genes are accessible through three main software tables, including for each entry details such as the gene summary, the gene exon/intron structure and the specific Gene Ontology attributions. The structuring of the data, the creation of additional calculation fields and the integration with nucleotide sequences allow users to make many types of comparisons and calculations that are useful for data retrieval and analysis. We provide an original example analysis of the existing introns across all the available species, through which the classic biological problem of the 'minimal intron' may find a solution using available data. Based on all currently available data, we can define the shortest known eukaryotic GT-AG intron length, setting the physical limit at the 30 base pair intron belonging to the human MST1L gene. This 'model intron' will shed light on the minimal requirement elements of recognition used for conventional splicing functioning. Remarkably, this size is indeed consistent with the sum of the splicing consensus sequence lengths.

Down Syndrome (DS) is the most frequent human chromosomal disorder. Main symptoms include intellectual disability (ID), cardiovascular defects and craniofacial dysmorphisms. Despite ID being measured by a test of symbolic logic skills, it is common for children with DS to arouse a climate of affective intensity greater than the norm. In 1959, Jérôme Lejeune (1926-1994) and coll. described an additional chromosome 21 (Hsa21) in children with DS (trisomy 21), giving origin to the field of medical genetics. Remarkably, the discovery of trisomy 21 had relevant social consequences for the affected children, in that their parents were no longer suspected to be alcoholics or infected with syphilis. Although it is broadly agreed that the DS phenotype originates from the altered expression of the genes located on Hsa21, its molecular pathogenesis is still unknown. To date, no therapy is recognized and recommended by guidelines as being effective in improving the cognitive abilities of persons with DS. The aim of this article is to categorize main therapeutical approaches or pathways to new approaches reported in the biomedical literature, to extract critical methodological points from the works of Lejeune and then to propose a new research project aimed to generate and integrate clinical, biochemical, genetic and bioinformatic data in order to identify novel therapeutic targets for this form of trisomy. We show here that nearly all the current lines of research were pursued, theorized or foreseen by Lejeune, and that central points of his method remain current: positive hypothesis about the existence of a solution, envision of systematic investigation of cell machinery, anchoring of clinical and biochemical finding to the chromosome physical map, and continuing clinical observation of the affected children. We therefore propose a project aimed at producing both experimentally and by meta-analysis state-of-the-art maps and databases related to clinical/phenotype, cytogenetics, exome, transcriptome, methylome, molecular biology, metabolome and mutations data. The primary expected outcome of this research project is the identification of a restricted list of strong candidate genes and mechanisms for ID in persons with DS in order to devise new rational therapeutic approaches.

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 1012 and 1016 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 × 1013. 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.

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.

Down syndrome critical region gene 1-like 2 (DSCR1L2) belongs to the human DSCR1-like gene family, which also includes DSCR1 and DSCR1L1. Both DSCR1 and DSCR1L1 proteins interact with calcineurin, a calcium/calmodulin-dependent phosphatase. To date, no interactor has been described for DSCR1L2. The aim of this work was to perform a first functional study of DSCR1L2 using yeast two-hybrid analysis conducted on a human heart cDNA library. Here, we report the interaction between DSCR1L2 and the human cardiac troponin I (TNNI3), the heart-specific inhibitory subunit of the troponin complex, a central component of the contractile apparatus. This interaction was confirmed by both yeast cotransformation and GST (glutathione-sepharose transferase) fusion protein assay. Moreover, a new DSCR1L2 mRNA isoform, generated by alternative splicing, was identified and cloned in different tissues: it lacks two central exons, encoding the most conserved domains among the DSCR1-like protein family. A quantitative relative reverse transcription-polymerase chain reaction (RT-PCR) assay showed that in heart tissue the normalized expression level ratio for DSCR1L2 and DSCR1L2-E2E5 mRNA isoforms is 3.5 : 1, respectively. The yeast cotransformation and GST fusion protein assay demonstrated the interaction between this new DSCR1L2 variant and the human cardiac troponin I and the prominent role of DSCR1L2 exon 2 in determining binding between both DSCR1L2 isoforms and TNNI3. These data indicate an entirely new role for a DSCR1-like family gene, suggesting a possible involvement of DSCR1L2 in cardiac contraction.

A novel human gene has been identified by in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.1), with no coding region predicted in any previous analysis. Brain-derived DNA complementary to RNA (cDNA) sequencing predicts a 154-amino acid product with no similarity to any known protein. The gene has been named cysteine and tyrosine-rich protein 1 gene (symbol cysteine and tyrosine-rich 1, CYYR1). The CYYR1 messenger RNA was found by Northern blot analysis in a broad range of tissues (two transcripts of 3.4 and 2.2 kb). The gene consists of four exons and spans about 107 kb, including a very large intron of 85.8 kb. Analysis of expressed sequence tags shows high CYYR1 expression in cells belonging to the amine precursor uptake and decarboxylation system. We also cloned the cDNA of the murine ortholog Cyyr1, which was mapped by a radiation hybrid panel on chromosome 16 within the region corresponding to that containing the respective human homolog on chromosome 21. Sequence and phylogenetic analysis led to identification of several genes encoding CYYR1 homologous proteins. The most prominent feature identified in the protein family is a central, unique cysteine and tyrosine-rich domain, which is strongly conserved from lower vertebrates (fishes) to humans but is absent in bacteria and invertebrates.

Background The incidence of Acute Megakaryoblastic Leukemia (AMKL) is 500-fold higher in children with Down Syndrome (DS) compared with non-DS children, but the relevance of trisomy 21 as a specific background of AMKL in DS is still an open issue. Several Authors have determined gene expression profiles by microarray analysis in DS and/or non-DS AMKL. Due to the rarity of AMKL, these studies were typically limited to a small group of samples. Methods We generated integrated quantitative transcriptome maps by systematic meta-analysis from any available gene expression profile dataset related to AMKL in pediatric age. This task has been accomplished using a tool recently described by us for the generation and the analysis of quantitative transcriptome maps, TRAM (Transcriptome Mapper), which allows effective integration of data obtained from different experimenters, experimental platforms and data sources. This allowed us to explore gene expression changes involved in transition from normal megakaryocytes (MK, n=19) to DS (n=43) or non-DS (n=45) AMKL blasts, including the analysis of Transient Myeloproliferative Disorder (TMD, n=20), a pre-leukemia condition. Results We propose a biological model of the transcriptome depicting progressive changes from MK to TMD and then to DS AMKL. The data indicate the repression of genes involved in MK differentiation, in particular the cluster on chromosome 4 including PF4 (platelet factor 4) and PPBP (pro-platelet basic protein); the gene for the mitogen-activated protein kinase MAP3K10 and the thrombopoietin receptor gene MPL. Moreover, comparing both DS and non-DS AMKL with MK, we identified three potential clinical markers of progression to AMKL: TMEM241 (transmembrane protein 241) was the most over-expressed single gene, while APOC2 (apolipoprotein C-II) and ZNF587B (zinc finger protein 587B) appear to be the most discriminant markers of progression, specifically to DS AMKL. Finally, the chromosome 21 (chr21) genes resulted to be the most over-expressed in DS and non-DS AMKL, as well as in TMD, pointing out a key role of chr21 genes in differentiating AMKL from MK. Conclusions Our study presents an integrated original model of the DS AMLK transcriptome, providing the identification of genes relevant for its pathophysiology which can potentially be new clinical markers. Electronic supplementary material The online version of this article (doi:10.1186/s12920-014-0063-z) contains supplementary material, which is available to authorized users.

Down syndrome (DS) is the most frequent constitutional form of intellectual disability in humans. In 1958 Jerome Lejeune discovered that children with DS have one extra copy of chromosome 21 (trisomy 21) in their cells. Lejeune considered the trisomic cells as ‘‘drug addicted’’, intoxicated by the excess of gene products that are formed due to the presence of the additional chromosome. If we knew the pathogenetic mechanisms we could devise a more specific therapy. Lejeune, a genial geneticist and biochemist, was also a pediatrician who visited thousands of children with intellectual disability, encouraging their families, highlighting the enrichment in humanity experienced by so many families because of the presence of a person with DS and not only the problems. He dedicated all of his life and so much love to these children, and at the same time he would correct the biochemical errors which hamper the full expression of their human abilities and he used to say: ‘‘Hate the disease, love the patient: that is the practice of medicine’’. To date, no therapy is recognized as being effective in improving the cognitive abilities of persons with DS. Research was slowed by the fact that so many studies have been focused on prenatal diagnosis of the syndrome rather than on its treatment. Following the encounter with Lejeune’s history and work, we have started a systematic study of DS to integrate clinical, biochemical, genetic and bioinformatic data in order to identify novel therapeutic targets for this form of trisomy.

A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C. elegans single members (Strippoli P., Lenzi L., Petrini M., Carinci P., Zannotti M., 2000. A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member. Genomics 64, 252-263). The proposed family labels were a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif. We have used a bioinformatics-driven molecular biology approach to characterize the murine members of DSCR1-like gene family. Systematic expressed-sequence-tags (EST) database search and reverse-transcription polymerase chain rection (RT-PCR) product sequencing allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2. The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins. The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes. Using a radiation hybrid panel, we mapped the murine DSCR1-like family members. The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region. DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1. Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members.

The c-mpl ligand, thrombopoietin (TPO), is a physiologic regulator of platelet and megakaryocytic production, acting synergistically on thrombopoiesis with the growth factors interleukin 11 (IL-11), stem cell factor, interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte-macrophage colony-stimulating factor. Because some of these growth factors, especially TPO and IL-11, are now being evaluated clinically to reduce chemotherapy-associated thrombocytopenia in cancer patients, we evaluated 25 acute myeloid leukemia (AML) samples to test whether TPO, IL-11, and other early-acting megakaryocyte growth factors can affect leukemic cell proliferation, cell cycle activation, and programmed cell death (PCD) protection. TPO induced proliferation in the majority of AML samples from an overall mean proportion of S-phase cells of 7.8% +/-1.5% to 14.5% +/- 2.1% (p = 0.0006). Concurrent G0 cell depletion was found in 47.3% of AML samples. TPO-supported leukemic cell precursor (CFU-L) proliferation was reported in 5 of 17 (29.4%) of the samples with a mean colony number of 21.4 +/- 9.6 x 10(5) cells plated. In 13 of 19 samples, a significant protection from PCD (from an overall mean value of 13% +/-0.7% to 8.8% +/- 1.8%;p = 0.05) was detected after TPO exposure. Conversely, IL-11-induced cell cycle changes (recruitment from G0 to S phase) were detected in only 2 of 14 samples (14.2%). In addition, IL-11 showed little, if any, effect on CFU-L growth (mean colony number = 17.5 9.5) or apoptosis. Combination of TPO with IL-11 resulted in only a slight increase in the number of CFU-L, whereas IL-3 and stem cell factor significantly raised the mean colony numbers up to 119.2 +/- 68.3 and 52.9 +/- 22.1 x 10(5) cells plated, respectively. We conclude that TPO induces cell cycle activation in a significant proportion of cases and generally protects the majority of AML blast cells from PCD. On the other hand, IL-11 has little effect on the cell cycle or PCD. Combination of both TPO and IL-11 is rarely synergistic in stimulating AML clonogenic growth. These findings may be useful for designing clinical studies aimed at reducing chemotherapy-associated thrombocytopenia in AML patients.

A human megakaryocyte cell line (B1647) has been established from bone marrow cells obtained from a patient with acute myelogenous leukaemia (FAB M2). The cells were CD34-, CD33+, HLA-DR+, CD38+, and expressed the immunophenotypic markers of the megakaryocyte lineage (CD41 and von Willebrand factor). Moreover the cells expressed the c-mpl (thrombopoietin receptor) mRNA and protein. On the other hand, the B1647 cells also possessed erythroid lineage characteristics: the vast majority of cells were glycophorin positive, and about 10% of unstimulated cells stained with an anti-globin gamma chain MoAb. In addition, S1 protection analysis demonstrated expression of beta-globin mRNA, and Epo receptor (Epo-R) protein was detected by cytofluorimetric assay. Several growth factors, when tested alone or in combination, failed to influence the B1647 cell growth. A significant increase of cell proliferation was observed only after the addition, in serum-free culture, of recombinant human megakaryocyte growth development factor (MGDF), a recombinant c-mpl ligand encompassing the receptor-binding domain and identical to thrombopoietin (TPO), at concentrations ranging from 0.01 to 1 ng/ml. Interestingly, MGDF failed to induce megakaryocytic differentiation of the B1647 cells, but significantly increased the synthesis of the globin gamma-chain. B1647 cells could be a useful model for studying the biological effect of TPO on common megakaryocyte and erythroid progenitors.

The aim of this study was to test the in vitro cytokine production by peripheral blood mononuclear cells (PBMCs) in patients with Fanconi's anemia (FA). Phytohemagglutinin (PHA)-stimulated PBMCs from 21 patients with FA were studied for their ability to produce interleukin 6 (IL-6), leukemia inhibitory factor (LIF) and granulocyte-macrophage colony stimulating factor (GM-CSF). Enzymatic immunoassay (EIA) was used for both IL-6 and LIF, while GM-CSF was evaluated in a highly sensitive biological assay provided by GM-CSF-dependent M-07e cells. A significant decrease of IL-6 was detected in 9 out of 11 FA patients compared with five normal donors, while similar amounts of LIF were produced from 21 FA patients and 21 healthy subjects. A drastic increase of active GM-CSF was documented in PHA-stimulated PBMC-conditioned medium in all 18 FA patients tested. Since IL-6 and GM-CSF play an important role in maintaining basal hemopoiesis, our results suggest that an abnormal cytokine network may be involved in the pathogenesis of FA pancytopenia.

Summary: Extracting the desired data from a database entry for later analysis is a constant need in the biological sequence analysis community; GeneRecords 1.0 is a solution for GenBank biological flat file parsing, as it implements a structured representation of each feature and feature qualifier in GenBank following import in a common database managing system usable in a personal computer (Macintosh and Windows environments). This collection of related databases enables the local management of GenBank records, allowing indexing, retrieval and analysis of both information and sequences on a personal computer. Availability: the current release, including the FileMaker Pro runtime application (built for Windows and Macintosh environments), is freely available at http://apollo11.isto.unibo.it/software/

Granulocyte macrophage colony-stimulating factor (GM-CSF) has emerged as an important growth factor for trophoblast and other placental cells, leading to improved placental functioning and fetal survival. Recent observations have indicated that GM-CSF is synthesized by epithelial cells in the murine pregnant and non-pregnant uterus. In this study, the production of GM-CSF by cells derived from human endometrium is assessed using a sensitive bioassay and specific neutralization of the cytokine bioactivity with a monoclonal antibody to GM-CSF. Originally, GM-CSF was assayed in the culture supernatants of explant cultures of human endometria. Concentrations of GM-CSF up to 4440 pg/ml were detected. Subsequently, enriched epithelial cell cultures were prepared from glands isolated from human endometrium. The purity of epithelial cultures was demonstrated by the expression of cytokeratin, a weak immunoreactivity for vimentin and a lack of immunoreactivity for leukocyte common antigen, CD68, a macrophage-specific protein and endothelial marker (factor VIII-related antigens). Detected concentrations of GM-CSF were as high as 18,800 pg/ml. Furthermore, pure epithelial cells of a neoplastic endometrial cell line ECC1 secreted GM-CSF, confirming the ability of endometrial epithelial cells to secrete this cytokine. The immunostaining of dated endometria from proliferative and secretory phases showed primarily that epithelial cells, and to a lesser extent stromal cells, exhibited immunoreactivity for GM-CSF. A Western blot analysis, performed to validate the immunohistochemical data, confirmed the presence of an immunoreactive gene product for GM-CSF in human endometrium throughout the menstrual cycle. These findings indicate that human endometrium synthesizes GM-CSF and that epithelial cells are a major contributor to its production.

The SR-1 monoclonal antibody (MoAb) recognizes an epitope of the c-kit receptor (KR), present on normal hemopoietic CD34+ stem cells as well as on blasts from patients with acute leukemia. Cytometric analysis by indirect immunofluorescence with the SR-1 MoAb was performed in 98 patients with acute myeloblastic leukemia (AML) and in 37 patients with acute lymphoblastic leukemia (ALL) in order to detect the presence of the KR and to examine its prognostic significance. Sixty-nine of 98 (70%) AML patients were SR-1 positive independently of the FAB subtype, although a higher incidence of SR-1 positive cases was observed in M4 and M5 AML and in those cases that also coexpressed lymphoid antigens. Fourteen AML samples were studied by Northern blot analysis and the KR mRNA was detected in the majority of SR-1 positive cases and also in 2 of 3 SR-1 negative samples. Furthermore, "in vitro" cultures from 15 cases showed that recombinant human Stem cell factor (rhSCF) induced an increased proliferative activity in most tested cases (11/15); this was further enhanced when rhSCF was combined with rhIL-3 + rhGM-CSF (p = 0.007) and with the GM-CSF/IL-3 fusion protein PIXY321 (p = 0.003). Thirty-seven ALL cases were also studied and all but one were SR-1 negative. Interestingly, the only SR-1 positive case also coexpressed myeloid antigens and showed an "in vitro" response when stimulated with rhSCF. Finally, the complete remission (CR) rate, survival and event-free survival were evaluated in 75 AML patients who received standard and identical chemotherapy; unlike previous studies which utilized a different anti-KR MoAb (YB5.B8) and which showed a poor prognosis for KR positive patients, we were unable to document any significant difference in CR rate, survival and event-free survival.

Recent genomic research has identified interleukin-23 receptor (IL23R), nucleotide-binding oligomerization domain containing 2 caspase-activation recruitment domain 15 (NOD2/CARD15), autophagy related 16-like 1 (ATG16L1) and paired-like homeobox 2b (PHOX2B) as susceptibility loci for Crohn's Disease (CD). Our aim was to investigate these gene variants in a group of CD patients and to analyse the correlation to sub-phenotypes such as gender, smoking habits, disease behaviour at diagnosis, severity of disease and extra-intestinal manifestations. Nineteen patients with CD and 20 healthy controls were included in the study. The gene variants IL23R rs7517847 and rs11209026, NOD2/CARD15 rs2066845, PHOX2B rs16853571, ATG16L1 rs2241879 and rs2241880 were genotyped by PCR followed by sequencing. The frequency of the G risk allele of IL23R rs7517847 was found to be increased in patients with CD (42%) compared to that in control subjects (20%) [odds ratio (OR), 2.9; 95% confidence interval [CI], 1.06-7.9; P=0.03]. In addition, the homozygous condition GG was also associated with CD (OR, 8.70; 95% CI, 0.9-81.6; P=0.038). The analysis of correlation of genotype to sub-phenotypes showed an association of ATG16L1 rs2241879 with the lack of extra-intestinal manifestations (OR, 0.03; 95% CI, 0.002-0.45; P=0.006), and the patients defined as non-smokers displayed an increased frequency of the risk allele C (P=0.03). The present study confirms the association of the heterozygous and homozygous IL23R rs7517847 variant with CD and suggests an additive effect of smoking to the ATG16L1 rs2241879 C risk allele SNP, in the context of the multifactorial model established for the development of CD and a protective effect of the same allele against extra-intestinal manifestations.

Housekeeping (HK) genes are constitutively expressed in order to maintain cellular function. They produce the minimal essential transcripts necessary for normal cellular physiology. Wide range expression, stable expression level and high expression level are independent features of a single gene expression and are all desirable for the definition of an “ideal” HK. Recent studies have questioned the possible existence of “ideal” HK mRNAs, mainly because of the wide expression conditions variability. This would imply that for each investigated organism the suitability of a putative HK should be verified. We perform a systematic analysis to identify “optimal” HK genes in Danio rerio (zebrafish), to be used in expression analyses conducted on embryos/larvae at different developmental stages, as well as on differentiated adult tissues from single donors. The expression pattern of candidate genes, selected on the basis of the literature available and of ad hoc bioinformatics analysis, was assessed by quantitative relative RT-PCR in an RNA panel, including six different embryo/larvae developmental stages and six adult tissues. Statistical analysis was performed to identify genes with the lowest expression standard deviation in the studied panel. Our results showed that beta-actin 2 (bactin2) is the mRNA with the lowest variability of expression.

Evidence from the literature widely supports the efficacy of screening for colorectal cancer (CRC) in reducing mortality. A blood-based assay, potentially, represents a more accessible early detection tool for the identification of circulating tumour cells originating from a primary tumour site in the body. The present work aimed at identifying a set of specific mRNAs expressed in colon tissue but not in blood cells. These mRNAs may represent useful markers for early detection of circulating colon cancer cells by a simple, qualitative RT-PCR assay, following RNA extraction from peripheral blood samples. Using a data-mining tool called cDNA digital gene expression displayer (DGED), based on serial analysis of gene expression (SAGE) from the Cancer Genome Anatomy Project (CGAP) database, 4-colon and 14-blood cDNA libraries were analyzed. We selected 7 genes expressed in colon tissue but not in blood and were able to test 6 of them by RT-PCR in peripheral blood of CRC patients and healthy controls. We present a relatively easy and highly reproducible technique for the detection of mRNA expression of genes as candidate markers of malignancy in blood samples of patients with colon cancer. SAGE DGED provided a list of the best candidate mRNAs predicted to detect colon cells in the blood, namely those encoding the following proteins: hypothetical protein LOC644844 (LOC644844, whose cDNA was not amplifiable), fatty acid binding protein 1 (FABP1), carcinoembryonic antigen-related cell adhesion molecule 5 (CEACAM5), mucin 13 cell surface associated (MUC13), guanylate cyclase activator 2A (GUCA2A), amiloride binding protein 1 (ABP1), galactoside-binding, solute carrier family 26, member 3 (SLC26A3). The mRNA expression of these genes was evaluated in 8 samples from subjects diagnosed with CRC and 9 from healthy controls. We observed the expression of 2 of the 6 investigated genes in the blood samples of the vast majority of patients considered, but also in a subset of the controls. Our data confirm the extreme sensitivity of RT-PCR, making this technique able to detect minimal amounts of mRNA expressed in a non-tissue-specific manner. Moreover, DGED remains a powerful tool to identify candidate epithelial markers in blood, such as colon related mRNAs. However, to date, none of these qualified as tumour markers.

Background EGFR is frequently overexpressed in colon cancer. We characterized HT-29 and Caco-2, human colon cancer cell lines, untreated and treated with cetuximab or gefitinib alone and in combination with EGF. Methods Cell growth was determined using a variation on the MTT assay. Cell-cycle analysis was conducted by flow cytometry. Immunohistochemistry was performed to evaluate EGFR expression and scanning electron microscopy (SEM) evidenced the ultrastructural morphology. Gene expression profiling was performed using hybridization of the microarray Ocimum Pan Human 40 K array A. Results Caco-2 and HT-29 were respectively 66.25 and 59.24 % in G0/G1. They maintained this level of cell cycle distribution after treatment, suggesting a predominantly differentiated state. Treatment of Caco-2 with EGF or the two EGFR inhibitors produced a significant reduction in their viability. SEM clearly showed morphological cellular transformations in the direction of cellular death in both cell lines treated with EGFR inhibitors. HT-29 and Caco-2 displayed an important reduction of the microvilli (which also lose their erect position in Caco-2), possibly invalidating microvilli absorption function. HT-29 treated with cetuximab lost their boundary contacts and showed filipodi; when treated with gefitinib, they showed some vesicles: generally membrane reshaping is evident. Both cell lines showed a similar behavior in terms of on/off switched genes upon treatment with cetuximab. The gefitinib global gene expression pattern was different for the 2 cell lines; gefitinib treatment induced more changes, but directly correlated with EGF treatment. In cetuximab or gefitinib plus EGF treatments there was possible summation of the morphological effects: cells seemed more weakly affected by the transformation towards apoptosis. The genes appeared to be less stimulated than for single drug cases. Conclusion This is the first study to have systematically investigated the effect of cetuximab or gefitinib, alone and in combination with EGF, on human colon cancer cell lines. The EGFR inhibitors have a weaker effect in the presence of EGF that binds EGFR. Cetuximab treatment showed an expression pattern that inversely correlates with EGF treatment. We found interesting cyto-morphological features closely relating to gene expression profile. Both drugs have an effect on differentiation towards cellular death.

Human RCAN3 (Regulator of calcineurin 3; previously known as DSCR1L2, Down syndrome critical region gene 1-like 2) is a five-exon gene mapped on chromosome 1 and belongs to the human RCAN gene family which also includes RCAN1 and RCAN2. The novel denomination RCAN for genes and proteins, instead of DSCR1L (Down syndrome critical region gene 1-like) has recently been widely discussed. The aim of the present work was to perform a multiple approach analysis of five RCAN3 mRNA and encoded protein isoforms, two of which have been identified for the first time in this research. The two new RCAN3 mRNA isoforms, RCAN3-2,4,5, which lacks exon 3, and RCAN3-2,3,5, which lacks exon 4, were identified during RCAN3 RT-PCR (reverse transcription-polymerase chain reaction) cloning, the product of which unexpectedly revealed the presence of five isoforms as opposed to the three previously known. In order to analyze the expression pattern of the five RCAN3 mRNA isoforms in seven different human tissues, a quantitative relative RT-PCR was performed: interestingly, all isoforms are present in all tissues investigated, with a statistically significant constant prevalence of RCAN3 isoform (the most complete, "reference" isoform). The RCAN3 locus expression level was comparable in all seven tissues analyzed, considering all isoforms, which indicates a ubiquitous expression of this human RCAN family member. To date two possible interactors have been described for this protein: human cardiac troponin I (TNNI3) and calcineurin. Here we report the interaction between the new RCAN3 variants and TNNI3, demonstrated by both yeast cotransformation and by the GST (glutathione-sepharose transferase) fusion protein assay, as was to be expected from the presence of exon 2 whose product has been seen to be sufficient for binding to TNNI3.

Kelvin J. A. Davies,* Gennady Ermak,* Beverley A. Rothermel, Melanie Pritchard, Joseph Heitman, Joohong Ahnn, Flavio Henrique-Silva, Dana Crawford, Silvia Canaider,** Pierluigi Strippoli,** Paolo Carinci,** Kyung-Tai Min, Deborah S. Fox, Kyle W. Cunningham, Rhonda Bassel-Duby, Eric N. Olson, Zhuohua Zhang, R. Sanders Williams, Hans-Peter Gerber,*** Merce Perez-Riba, Hisao Seo, Xia Cao, Claude B. Klee, Juan Miguel Redondo, Lois J. Maltais, Elspeth A. Bruford, Sue Povey, Jeffery D. Molkentin,**** Frank D. McKeon, Elia J. Duh, Gerald R. Crabtree,§§§§ Martha S. Cyert, Susana de la Luna, and Xavier Estivill