NT-proBNP and stem cell factor plasma concentrations are independently associated with cardiovascular outcomes in end-stage renal disease hemodialysis patients.

Aims: End-stage renal disease (ESRD) treated by chronic hemodialysis (HD) is associated with poor cardiovascular (CV) outcomes, with no available evidence-based therapeutics. A multiplexed proteomic approach may identify new pathophysiological pathways associated with CV outcomes, potentially actionable for precision medicine. Methods and results: The AURORA trial was an international, multicentre, randomized, double-blind trial involving 2776 patients undergoing maintenance HD. Rosuvastatin vs. placebo had no significant effect on the composite primary endpoint of death from CV causes, nonfatal myocardial infarction or nonfatal stroke. We first compared CV risk-matched cases and controls (n = 410) to identify novel biomarkers using a multiplex proximity extension immunoassay (276 proteomic biomarkers assessed with OlinkTM). We replicated our findings in 200 unmatched cases and 200 controls. External validation was conducted from a multicentre real-life Danish cohort [Aarhus-Aalborg (AA), n = 331 patients] in which 92 OlinkTM biomarkers were assessed. In AURORA, only N-terminal pro-brain natriuretic peptide (NT-proBNP, positive association) and stem cell factor (SCF) (negative association) were found consistently associated with the trial's primary outcome across exploration and replication phases, independently from the baseline characteristics. Stem cell factor displayed a lower added predictive ability compared with NT-ProBNP. In the AA cohort, in multivariable analyses, BNP was found significantly associated with major CV events, while higher SCF was associated with less frequent CV deaths. Conclusions: Our findings suggest that NT-proBNP and SCF may help identify ESRD patients with respectively high and low CV risk, beyond classical clinical predictors and also point at novel pathways for prevention and treatment.

Gene-disease relationship discovery based on model-driven data integration and database view definition.

MOTIVATION: Computational methods are widely used to discover gene-disease relationships hidden in vast masses of available genomic and post-genomic data. In most current methods, a similarity measure is calculated between gene annotations and known disease genes or disease descriptions. However, more explicit gene-disease relationships are required for better insights into the molecular bases of diseases, especially for complex multi-gene diseases. RESULTS: Explicit relationships between genes and diseases are formulated as candidate gene definitions that may include intermediary genes, e.g. orthologous or interacting genes. These definitions guide data modelling in our database approach for gene-disease relationship discovery and are expressed as views which ultimately lead to the retrieval of documented sets of candidate genes. A system called ACGR (Approach for Candidate Gene Retrieval) has been implemented and tested with three case studies including a rare orphan gene disease.

Refined localization of twenty-one genes in subregion p13.1 of human chromosome 1.

In this report, we describe a refinement of the human transcript map of chromosome 1p13.1, a subregion undergoing many aberrations in various types of human cancers. Publicly available genetic linkage, radiation hybrid and physical maps, as well as cytogenetic and sequence data were used to establish the relative order and orientation of ten known intragenic markers. The complete sequence of genomic clones of the region, available at the Sanger Centre, provided the tool for further studies performed by BLAST analysis against all cDNA sequences registered in the Genexpress Index2. This allowed us to assign to subband 1p13.1 nine of the ten known genes, an additional member of the gene family of one of these genes and eleven new transcripts. The remaining known gene and one additional new transcript map at the 1p13.1 and 1p13.2 boundary. The corresponding genes may be responsible for disorders related to this region. The resulting transcript map of 1p13.1 is presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/CARTO.

The genexpress IMAGE knowledge base of the human muscle transcriptome: a resource of structural, functional, and positional candidate genes for muscle physiology and pathologies.

Sequence, gene mapping, and expression data corresponding to 910 genes transcribed in human skeletal muscle have been integrated to form the muscle module of the Genexpress IMAGE Knowledge Base. Based on cDNA array hybridization, a set of 14 transcripts preferentially or specifically expressed in muscle have been selected and characterized in more detail: Their pattern of expression was confirmed by Northern blot analysis; their structure was further characterized by full-insert cDNA sequencing and cDNA extension; the map location of the corresponding genes was refined by radiation hybrid mapping. Five of the 14 selected genes appear as interesting positional and functional candidate genes to study in relation with muscle physiology and/or specific orphan muscular pathologies. One example is discussed in more detail. The expression profiling data and the associated Genexpress Index2 entries for the 910 genes and the detailed characterization of the 14 selected transcripts are available from a dedicated Web server at. The database has been organized to provide the users with a working space where they can find curated, annotated, integrated data for their genes of interest. Different navigation routes to exploit the resource are discussed.

Detailed transcript map of a 810-kb region at 11p14 involving identification of 10 novel human 3' exons.

A limited number of genes, including the human brain-derived neutrotrophic factor (BDNF) gene, have been identified in the human chromosome 11p14 region. Since this area is involved in a genetic disorder (WAGR syndrome) and because of interest in studying the regulation of the human BDNF gene, we have established a detailed transcript map of a 810-kb region clone in a yeast artificial chromosome (YAC), corresponding to a portion of this genomic locus. A set of nested deletion mutants has been generated to map genes at a mean resolution of 75kb. Four genic markers from available mapping databases have been mapped on the YAC. Ten potential novel human exons have been isolated by a 3' terminal exon trapping procedure directly applied to purified YAC DNA. Most of these exons display polyadenylation signals and they all yield positive signals in RT-PCR experiments, confirming their status of transcribed sequences. The BDNF gene is now co-localised with three other genes on a 120 kb DNA fragment.

The Genexpress IMAGE knowledge base of the human brain transcriptome: a prototype integrated resource for functional and computational genomics.

Expression profiles of 5058 human gene transcripts represented by an array of 7451 clones from the first IMAGE Consortium cDNA library from infant brain have been collected by semiquantitative hybridization of the array with complex probes derived by reverse transcription of mRNA from brain and five other human tissues. Twenty-one percent of the clones corresponded to transcripts that could be classified in general categories of low, moderate, or high abundance. These expression profiles were integrated with cDNA clone and sequence clustering and gene mapping information from an upgraded version of the Genexpress Index. For seven gene transcripts found to be transcribed preferentially or specifically in brain, the expression profiles were confirmed by Northern blot analyses of mRNA from eight adult and four fetal tissues, and 15 distinct regions of brain. In four instances, further documentation of the sites of expression was obtained by in situ hybridization of rat-brain tissue sections. A systematic effort was undertaken to further integrate available cytogenetic, genetic, physical, and genic map informations through radiation-hybrid mapping to provide a unique validated map location for each of these genes in relation to the disease map. The resulting Genexpress IMAGE Knowledge Base is illustrated by five examples presented in the printed article with additional data available on a dedicated Web site at the address http://idefix.upr420.vjf.cnrs.fr/EXPR++ +/ welcome.html.

The complete cDNA sequence encoding dog gastric lipase.

Oligodeoxyribonucleotide ligation to single-stranded cDNA (SLIC) and polymerase chain reaction (PCR) techniques were used to clone an entire dog gastric lipase (DGL) cDNA. The size of the cDNA is confirmed by Northern blot analysis. The DGL is synthesized as a 379-amino acid mature polypeptide with a molecular mass of 43176 Da which is preceded by a 19-amino acid signal sequence located at the NH2-terminus. Comparison of the signal sequences reveals a high degree of similitude between the DGL, the human gastric lipase (HGL), the rabbit gastric lipase (RGL) and the rat lingual lipase (RLL).

Integrated physical, genetic, and genic map covering 3 Mb around the human NGF gene (NGFB) at 1p13.

We have characterized 11 overlapping yeast artificial chromosomes (YACs) in the 1p13 region, 8 of them containing the human nerve growth factor (NGF) gene (HGMW-approved symbol NGFB). Sequence-tagged sites (STSs) corresponding to YAC extremities have been designed and used for chromosome assignment on a panel of monochromosomic somatic cell hybrids to check for YAC chimerism, in parallel with analyses by fluorescence in situ hybridization. Determination of end STS content and restriction mapping of the YACs led to the construction of a 3-Mb YAC contig. Four microsatellite markers from the Généthon collection and seven genes known to map to the 1p13 region have been ordered on the contig around the NGF gene. A new gene transcript from the Genexpress catalog has been localized on the contig. This work provides an integrated physical, genetic, and genic map of this chromosome 1 region. It constitutes a basis for determining the structure of the NGF gene and for further characterizing its genic environment.

Mapping of 22 YACs on human chromosomes by fish using yeast DNA Alu-PCR products for competition.

Chromosomal assignment and analysis of chimerism of 22 YACs was performed by FISH. Probes were obtained by PCR amplification of the human YAC inserts with Alu primers. Maximum amplification of various inter-Alu elements was obtained when the primer annealing temperature was below the optimal temperature needed for high specificity. In these conditions, yeast DNA contributed to the amplification of various Alu-PCR products and, since strong competition was required for the suppression of all Alu sequences, yeast Alu-PCR products fulfilled this purpose efficiently.

Regional assignment of 68 new human gene transcripts on chromosome 11.

We have tested 80 expressed sequence-tagged site (eSTS) markers assigned to human chromosome 11 by the Genexpress program on a panel of somatic cell hybrids containing parts of this chromosome, characterized by cytogenetic data, reference markers, and with respect to the Généthon microsatellite genetic map. Sixty-eight new gene transcripts have been assigned to 25 subregions, one of which was newly defined by five of the eSTS markers. The markers are distributed on the short and long arms in agreement with their physical length. The genic map thus obtained has been integrated with the cytogenetic, genetic, and disease maps. Two eSTS markers have been further mapped with respect to a yeast artificial chromosome (YAC) contig close to the brain-derived neurotrophic factor (BDNF) gene and thus provide potential candidate genes for the mental retardation phenotype of WAGR (Wilms' tumor, aniridia, genitourinary abnormalities and mental retardation) syndrome. Altogether, the 68 new gene transcripts localized here represent more than a threefold increase in the number of unknown regionalized genes that could reveal potential candidate genes for the numerous orphan pathologies associated with chromosome 11.

[IMAGE: molecular integration of the analysis of the human genome and its expression]. / IMAGE: intégration au niveau moléculaire de l'analyse du génome humain et de son expression.

We have developed an integrated approach for the analysis of human cDNA libraries from neuromuscular tissues, based on the acquisition of primary structural, expression and mapping data. 26,938 sequence signatures (over 7 million bases) have been derived from both ends of skeletal muscle and brain cDNA clones. Primary redundancy analysis and classification of database similarities made it possible to characterize by structural data about 8,000 human gene transcripts, the majority of which is catalogued for the first time. Collecting hybridization signatures of complex cDNA probes derived from the tissues of origin to cDNA clones arrayed on high density filters provided a global and quantifiable view of the complexity and level of expression of the different transcripts. The development of 2,792 eSTS markers amplifiable by PCR defined the chromosomal localization of some 2,500 genes corresponding to the transcripts sequenced. The data collected are part of the corpus of the human gene transcript catalog and the genic map of the human genome.

A 1.7-Mb YAC contig around the human BDNF gene (11p13): integration of the physical, genetic, and cytogenetic maps in relation to WAGR syndrome.

WAGR (Wilms tumor, aniridia, genito-urinary abnormalities, mental retardation) syndrome in humans is associated with deletions of the 11p13 region. The brain-derived neurotrophic factor (BDNF) gene maps to this region, and its deletion seems to contribute to the severity of the patients' mental retardation. Yeast artificial chromosomes (YACs) carrying the BDNF gene have been isolated and characterized. Localization of two known exons of this gene leads to a minimal estimation of its size of about 40 kb. Chimerism of the BDNF YACs has been investigated by fluorescence in situ hybridization and chromosome assignment on somatic cell hybrids. Using the BDNF gene, YAC end sequence tagged sites (STS), and Généthon microsatellite markers, we constructed a 1.7-Mb contig and refined the cytogenetic map at 11p13. The resulting integrated physical, genetic, and cytogenetic map constitutes a resource for the characterization of genes that may be involved in the WAGR syndrome.

Chicken tyrosine hydroxylase gene: isolation and functional characterization of the 5' flanking region.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in the biosynthesis of catecholamines. We describe here the isolation of the chicken TH gene and the analysis of 3 kb of its 5' flanking region. The chicken TH transcription unit spans 19 kb. The 60-bp proximal promoter contains a TATA box and a cyclic AMP response element (CRE) sequence. The 5' flanking region contains several AP1-, AP2-, and octamer-like sequences as well as a glucocorticoid response element at position -1.4 kb. A construct containing the 3-kb 5' flanking DNA fused to the chloramphenicol acetyltransferase (CAT) gene was transiently transfected into PC12 cells, and the effect of various effectors was tested. Only forskolin increased the CAT activity, likely owing to the presence of the CRE sequence. Constructs prepared by progressively deleting the 5' flanking DNA were transfected into PC12 and QT6 (quail transformed fibroblasts) cells. In both cell types, the transcriptional activity increased with deletion of the 5' flanking region. These results show that the 60-bp region containing the TATA box and the CRE is sufficient to act as a constitutive promoter for the chicken TH gene and that this region appears to be negatively controlled by upstream sequences.

In vitro transcripts of turnip yellow mosaic virus encompassing a long 3' extension or produced from a full-length cDNA clone harbouring a 2 kb-long PCR-amplified segment are infectious.

Two types of full-length cDNA clones have been constructed corresponding to the entire genome of turnip yellow mosaic virus (TYMV), from which infectious transcripts devoid of 5' non-viral extensions can be synthesized in vitro. The first type of transcript (tTYFL7) harbours 75 non-viral nucleotides at its 3' end, whereas the second type (tTYFL84) possesses only 2 non-viral nucleotides at its 3' end. The 2 kilobase-long 3' region of tTYFL84 derives from amplification by the polymerase chain reaction of the corresponding TYMV cDNA. Both tTYFL7 and tTYFL84 are infectious in rapeseed protoplasts and plants. tTYFL7 is far less infectious than wild-type TYMV RNA and somewhat less infectious than tTYFL84. The possible effects of the 3' extraviral sequences of tTYFL7 and the heterogeneity observed in the infectivity of other transcripts prepared as was tTYFL84 are discussed.

Cloning and mapping of 5' exons from the gene encoding chicken beta nerve growth factor.

An NGF cDNA containing the 5' exons of the nerve growth factor (NGF) messenger was obtained from chicken heart mRNA using the anchored polymerase chain reaction technique. Alignment of the chicken with the corresponding murine and human sequences reveals interspecies similarities. A sequence corresponding to an exon found only in the NGF messenger, which is abundant in the submaxillary gland of the male mouse, is present in the chicken NGF cDNA. The first non-coding exons of the NGF gene are much less conserved between chicken and mouse or human than the region of the last exon encoding the mature protein. After the cloning of the chicken NGF gene from a cosmid library, the chicken NGF exons have been located within 20 kb of DNA. The chicken NGF gene is therefore shorter than its murine counterpart which spans more than 43 kb. Furthermore, the organization of the chicken and murine NGF genes markedly differs in their 5' portion.