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BACKGROUND: Recent studies have indicated that the implementation of international guidelines for the management of renal patients is suboptimal in Italy. The Italian Society of Nephrology (SIN) decided to undertake a multicentre study to obtain a clear picture of clinical policies on chronic kidney disease (CKD) in Italy. METHODS: A 76-item structured questionnaire, designed to evaluate the organization of clinical care, was administered to the director of each participating centre, within the context of a large observational trial in 100 Italian nephrology centres, collecting information on newly diagnosed CKD patients (K/DOQI stage 3-5) on conservative treatment. This paper reports the questionnaire results related to management of anaemia and bone metabolism disorders; assessment of renal function; creation of a vascular access for dialysis and referral of patients to a nephrologist. RESULTS: Clinical policies at the centre level deviated from guideline recommendations in 70% (timing of vascular access creation) to 25% (assessment of iron deficiency) of centres. Assessment of renal function differed from the recommended approach in 30% of centres; clinical policies related to anaemia and bone disease did not coincide with guideline standards in 50 and 40% of centres, respectively. Directors of renal unit estimates indicate that the creation of a vascular access occurs very late in 38% of patients and that referral to a nephrologist is late in approximately 40% of cases. CONCLUSION: This survey in Italy highlights important deviations of clinical policies at the centre level from guideline recommendations.

Background and aims The prognostic role of LDL in non-dialysis chronic kidney disease (CKD) is still undefined. We addressed this question in a multicenter prospective study including patients referred to nephrologist for management. Methods and results 1306 patients with CKD stage III–V were studied at basal visit in 79 Italian nephrology clinics in 2004–2006, and then followed for survival analyses. Study endpoints were incident cardiovascular -CV events (fatal and major non-fatal) and renal events (start of renal replacement therapy or eGFR halving). Mean age was 67.6 ± 11.8 years, male 65%, diabetes 25%, CV disease 27%, and eGFR 35.8 ± 12.5 mL/min/1.73 m 2 . LDL was 119 ± 40 mg/dL, with high levels in 50.1% and 82.8% defined on the basis of the individual CV risk profile estimated according to ATPIII 2001 and ESC 2012 guidelines (LDL 100 to 160, and >70 or >100 mg/dL, respectively). Over a median follow up of 2.87 years, 178 CV and 181 renal events occurred. At multivariable Cox analyses, CV risk linearly increased with higher LDL (hazard ratio-HR per 40 mg/dL higher LDL: 1.20, 95% confidence intervals-CI 1.03–1.39); risk doubled when considering high LDL defined according to ESC 2012 (HR 2.37, 95%CI 1.39–4.03) while this association was not significant when considering the higher threshold levels of ATPIII 2001 (HR 1.10, 95%CI 0.82–1.49). No association emerged between LDL and renal risk. Conclusion In non-dialysis CKD patients, CV risk increases linearly with higher LDL and is more than doubled when considering the lower threshold values currently indicated for defining optimal LDL level.

Exposure of human neutrophils to conventional, acidic, lactate-based peritoneal dialysis solutions for 5 minutes results in a depression of superoxide generation. In spite of restoration of extracellular pH to 7.4, these stunned cells failed to recover their ability to generate the anion after a period of an hour.

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.

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.

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.

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.

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.

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.

Alkaline phosphatases (ALPs) are a family of cell surface glycoproteins that catalyze the hydrolysis of phosphomonoesters with release of inorganic phosphate. Liver/bone/kidney (L/B/K) ALP participates in bone mineralization, but its other physiological and pathological functions remain obscure. In human osteosarcoma, an inverse relationship has been found between cellular L/B/K ALP expression and aggressiveness. To explore this relationship, we employed cDNA microarray technology to characterize and compare the gene expression profile of two U-2 OS osteosarcoma clones with high L/B/K ALP activity (U-2/ALP28 and U-2/ALP40) and one with contrasting characteristics (U-2/ALP23). We identified 79 differentially expressed genes (58 upregulated in U-2/ALP28 and U-2/ALP40 compared to U-2/ALP23). Using GenMAPP/MAPPFinder, we highlighted nine functional groups strictly related to high L/B/K ALP activity, including microtubule-based movement and cell adhesion groups, two functions well related to tumor invasiveness. Notably, cadherin 13 ( CDH13 ) and caveolin 1 ( CAV1 ) genes were upregulated in our cells. Since these two genes are involved in cell–cell adhesion and cell growth, their co-expression with L/B/K ALP could help explain the lower levels of malignancy found in osteosarcoma cells with high L/B/K ALP activity. Although functional studies are needed to better define the role of CDH13 and CAV1 in the malignant behavior of osteosarcoma cells, the data presented here provide an aid to understanding the biological functions of L/B/K ALP in bone tumors.

Background : The ADARB1 gene is located in 21q22.3 region, previously linked to familial bipolar disorder, and its product has a documented action in the editing of the pre-mRNA of glutamate receptor B subunit. Dysfunction of glutamatergic neurotransmission could play an important role in the patophysiology of bipolar disorder (BD). Glutamate excitatory neurotransmission regulation is a possible mechanism of the initial effect of anticonvulsants in regulating mood. Methods : To investigate the hypothesis of an involvement of ADARB1 gene in the BD, the ADARB1 cDNA has been cloned and sequenced in seven selected bipolar I disorder patients with evidence of familiarity of mood disorders. A detailed investigation of the gene nucleotide sequence in the open reading frame has been performed. Results : No alteration in the sequence of the ADARB1 gene cDNA was found in any patient, except a common neutral polymorphism in three out of seven patients. Conclusions : Mutations in ADARB1 gene are not commonly associated with bipolar I disorder, therefore other genes in the 21q22 region could be associated with bipolar illness in some families, likely in the context of a multifactorial transmission model.

Thrombocytopenia with absent radii (TAR) is a rare autosomal recessive disease characterized by hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. We performed mutational screening of coding and promoter regions of the c-mpl gene, encoding thrombopoietin (TPO) receptor, by sequence analysis in four unrelated patients affected by TAR syndrome. Our results indicate that c-mpl gene mutations are not a common cause of thrombocytopenia in TAR syndrome.

Summary Fanconi anemia (FA) is an autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. At least five complementation groups (FA-A–FA-E) have been identified. The relative prevalence of FA-A has been estimated at an average of ∼65% but may widely vary according to ethnic background. In Italy, 11 of 12 patients analyzed by cell-fusion studies were assigned to group FA-A, suggesting an unusually high relative prevalence of this FA subtype in patients of Italian ancestry. We have screened the 43 exons of the FAA gene and their flanking intronic sequences in 38 Italian FA patients, using RNA-SSCP. Ten different mutations were detected: three nonsense and one missense substitutions, four putative splice mutations, an insertion, and a duplication. Most of the mutations are expected to cause a premature termination of the FAA protein at various sites throughout the molecule. Four protein variants were also found, three of which were polymorphisms. The missense mutation D1359Y, not found in chromosomes from healthy unrelated individuals, was responsible for a local alteration of hydrophobicity in the FAA protein, and it was likely to be pathogenic. Thus, the mutations so far encountered in the FAA gene are essentially all different. Since screening based on the analysis of single exons by genomic DNA amplification apparently detects only a minority of the mutations, methods designed to detect alterations in the genomic structure of the gene or in the FAA polypeptide may be helpful in the identification of FAA mutations.

Background The efficacy of screening for colorectal cancer using a simple blood-based assay for the detection of tumor cells disseminated in the circulation at an early stage of the disease is gaining positive feedback from several lines of research. This method seems able to reduce colorectal cancer mortality and may replace colonoscopy as the most effective means of detecting colonic lesions. Methods In this work, we present a new microarray-based high-throughput screening method to identifying candidate marker mRNAs for the early detection of epithelial cells diluted in peripheral blood cells. This method includes 1. direct comparison of different samples of colonic mucosa and of blood cells to identify consistent epithelial-specific mRNAs from among 20,000 cDNA assayed by microarray slides; 2. identification of candidate marker mRNAs by data analysis, which allowed selection of only 10 putative differentially expressed genes; 3. Selection of some of the most suitable mRNAs (TMEM69, RANBP3 and PRSS22) that were assayed in blood samples from normal subjects and patients with colon cancer as possible markers for the presence of epithelial cells in the blood, using reverse transcription – polymerase chain reaction (RT-PCR). Results Our present results seem to provide an indication, for the first time obtained by genome-scale screening, that a suitable and consistent colon epithelium mRNA marker may be difficult to identify. Conclusion The design of new approaches to identify such markers is warranted.