Copy number variants, diseases and gene expression.

Copy number variation (CNV) has recently gained considerable interest as a source of genetic variation likely to play a role in phenotypic diversity and evolution. Much effort has been put into the identification and mapping of regions that vary in copy number among seemingly normal individuals in humans and a number of model organisms, using bioinformatics or hybridization-based methods. These have allowed uncovering associations between copy number changes and complex diseases in whole-genome association studies, as well as identify new genomic disorders. At the genome-wide scale, however, the functional impact of CNV remains poorly studied. Here we review the current catalogs of CNVs, their association with diseases and how they link genotype and phenotype. We describe initial evidence which revealed that genes in CNV regions are expressed at lower and more variable levels than genes mapping elsewhere, and also that CNV not only affects the expression of genes varying in copy number, but also have a global influence on the transcriptome. Further studies are warranted for complete cataloguing and fine mapping of CNVs, as well as to elucidate the different mechanisms by which they influence gene expression.

Gene expression patterns unveil a new level of molecular heterogeneity in colorectal cancer.

The recognition that colorectal cancer (CRC) is a heterogeneous disease in terms of clinical behaviour and response to therapy translates into an urgent need for robust molecular disease subclassifiers that can explain this heterogeneity beyond current parameters (MSI, KRAS, BRAF). Attempts to fill this gap are emerging. The Cancer Genome Atlas (TGCA) reported two main CRC groups, based on the incidence and spectrum of mutated genes, and another paper reported an EMT expression signature defined subgroup. We performed a prior free analysis of CRC heterogeneity on 1113 CRC gene expression profiles and confronted our findings to established molecular determinants and clinical, histopathological and survival data. Unsupervised clustering based on gene modules allowed us to distinguish at least five different gene expression CRC subtypes, which we call surface crypt-like, lower crypt-like, CIMP-H-like, mesenchymal and mixed. A gene set enrichment analysis combined with literature search of gene module members identified distinct biological motifs in different subtypes. The subtypes, which were not derived based on outcome, nonetheless showed differences in prognosis. Known gene copy number variations and mutations in key cancer-associated genes differed between subtypes, but the subtypes provided molecular information beyond that contained in these variables. Morphological features significantly differed between subtypes. The objective existence of the subtypes and their clinical and molecular characteristics were validated in an independent set of 720 CRC expression profiles. Our subtypes provide a novel perspective on the heterogeneity of CRC. The proposed subtypes should be further explored retrospectively on existing clinical trial datasets and, when sufficiently robust, be prospectively assessed for clinical relevance in terms of prognosis and treatment response predictive capacity. Original microarray data were uploaded to the ArrayExpress database (http://www.ebi.ac.uk/arrayexpress/) under Accession Nos E-MTAB-990 and E-MTAB-1026. © 2013 Swiss Institute of Bioinformatics. Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Leishmania major: histone H1 gene expression from the sw3 locus.

Histone H1 in the parasitic protozoan Leishmania is a developmentally regulated protein encoded by the sw3 gene. Here we report that histone H1 variants exist in different Leishmania species and strains of L. major and that they are encoded by polymorphic genes. Amplification of the sw3 gene from the genome of three strains of L. major gave rise to different products in each strain, suggesting the presence of a multicopy gene family. In L. major, these genes were all restricted to a 50-kb Bg/II fragment found on a chromosomal band of 1.3 Mb (chromosome 27). The detection of RFLPs in this locus demonstrated its heterogeneity within several species and strains of Leishmania. Two different copies of sw3 (sw3.0 and sw3.1) were identified after screening a cosmid library containing L. major strain Friedlin genomic DNA. They were identical in their 5' UTRs and open reading frames, but differed in their 3' UTRs. With respect to the originally cloned copy of sw3 from L. major strain LV39, their open reading frames lacked a repeat unit of 9 amino acids. Immunoblots of L. guyanensis parasites transfected with these cosmids revealed that both copies could give rise to the histone H1 protein. The characterization of this locus will now make possible a detailed analysis of the function of histone H1 in Leishmania, as well as permit the dissection of the molecular mechanisms governing the developmental regulation of the sw3 gene.

Differential gene expression, induced by salicylic acid and Fusarium oxysporum f. sp. lycopersici infection, in tomato

The objective of this work was to determine the transcript profile of tomato plants (Lycopersicon esculentum Mill.), during Fusarium oxysporum f. sp. lycopersici infection and after foliar application of salicylic acid. The suppression subtractive hybridization (SSH) technique was used to generate a cDNA library enriched for transcripts differentially expressed. A total of 307 clones was identified in two subtractive libraries, which allowed the isolation of several defense-related genes that play roles in different mechanisms of plant resistance to phytopathogens. Genes with unknown roles were also isolated from the two libraries, which indicates the possibility of identifying new genes not yet reported in studies of stress/defense response. The SSH technique is effective for identification of resistance genes activated by salicylic acid and F. oxysporum f. sp. lycopersici infection. Not only the application of this technique enables a cost effective isolation of differentially expressed sequences, but also it allows the identification of novel sequences in tomato from a relative small number of sequences.

Low-P tolerance mechanisms and differential gene expression in contrasting wheat genotypes

The objectives of this study were to determine low-P tolerance mechanisms in contrasting wheat genotypes and to evaluate the association of these mechanisms to differential gene expression. Wheat seedlings of cultivars Toropi (tolerant to low-P availability) and Anahuac (sensitive) were evaluated. Seedlings were hydroponically grown in the absence or presence of P (1.0 mmol L-1) during three different time periods: 24, 120 and 240 hours. Free phosphate (Pi) and total P contents were measured in shoots and roots. The experiment's design was in randomized blocks with three replicates, each formed by ten plants. The relative expression of genes encoding the malate transporter TaALMT1 and the transcription factor PTF1 was evaluated. Phosphorus starvation beyond ten days increased the expression of TaALMT1 only in 'Toropi'. PTF1's expression was early induced in both genotypes under P starvation, but remained significant after ten days only in 'Toropi'. Shoot Pi concentration in 'Toropi' was independent from P availability; under starvation, 'Toropi' favored the maintenance of shoot Pi concentration. The low-P tolerance of Toropi cultivar at initial growth stages is mainly due to its ability to maintain constant the Pi shoot level.

Fusaric acid-producing strains of Fusarium oxysporum alter 2,4-diacetylphloroglucinol biosynthetic gene expression in Pseudomonas fluorescens CHA0 in vitro and in the rhizosphere of wheat.

The phytotoxic pathogenicity factor fusaric acid (FA) represses the production of 2,4-diacetylphloroglucinol (DAPG), a key factor in the antimicrobial activity of the biocontrol strain Pseudomonas fluorescens CHA0. FA production by 12 Fusarium oxysporum strains varied substantially. We measured the effect of FA production on expression of the phlACBDE biosynthetic operon of strain CHA0 in culture media and in the wheat rhizosphere by using a translational phlA'-'lacZ fusion. Only FA-producing F. oxysporum strains could suppress DAPG production in strain CHA0, and the FA concentration was strongly correlated with the degree of phlA repression. The repressing effect of FA on phlA'-'lacZ expression was abolished in a mutant that lacked the DAPG pathway-specific repressor PhlF. One FA-producing strain (798) and one nonproducing strain (242) of F. oxysporum were tested for their influence on phlA expression in CHA0 in the rhizosphere of wheat in a gnotobiotic system containing a sand and clay mineral-based artificial soil. F. oxysporum strain 798 (FA(+)) repressed phlA expression in CHA0 significantly, whereas strain 242 (FA(-)) did not. In the phlF mutant CHA638, phlA expression was not altered by the presence of either F. oxysporum strain 242 or 798. phlA expression levels were seven to eight times higher in strain CHA638 than in the wild-type CHA0, indicating that PhlF limits phlA expression in the wheat rhizosphere.

Apparent versus true gene expression changes of three hypoxia-related genes in autopsy derived tissue and the importance of normalisation.

The aim of our work was to show how a chosen normal-isation strategy can affect the outcome of quantitative gene expression studies. As an example, we analysed the expression of three genes known to be upregulated under hypoxic conditions: HIF1A, VEGF and SLC2A1 (GLUT1). Raw RT-qPCR data were normalised using two different strategies: a straightforward normalisation against a single reference gene, GAPDH, using the 2(-ΔΔCt) algorithm and a more complex normalisation against a normalisation factor calculated from the quantitative raw data from four previously validated reference genes. We found that the two different normalisation strategies revealed contradicting results: normalising against a validated set of reference genes revealed an upregulation of the three genes of interest in three post-mortem tissue samples (cardiac muscle, skeletal muscle and brain) under hypoxic conditions. Interestingly, we found a statistically significant difference in the relative transcript abundance of VEGF in cardiac muscle between donors who died of asphyxia versus donors who died from cardiac death. Normalisation against GAPDH alone revealed no upregulation but, in some instances, a downregulation of the genes of interest. To further analyse this discrepancy, the stability of all reference genes used were reassessed and the very low expression stability of GAPDH was found to originate from the co-regulation of this gene under hypoxic conditions. We concluded that GAPDH is not a suitable reference gene for the quantitative analysis of gene expression in hypoxia and that validation of reference genes is a crucial step for generating biologically meaningful data.

IFN Stimulated Gene Expression in the Liver is a Better Predictor of Treatment Response in Chronic Hepatitis C than the IL28B (IFN lambda 3) Genotype

BACKGROUND: Therapy of chronic hepatitis C (CHC) with pegIFNα/ribavirin achieves a sustained virologic response (SVR) in ∼55%. Pre-activation of the endogenous interferon system in the liver is associated with non-response (NR). Recently, genome-wide association studies described associations of allelic variants near the IL28B (IFNλ3) gene with treatment response and with spontaneous clearance of the virus. We investigated if the IL28B genotype determines the constitutive expression of IFN stimulated genes (ISGs) in the liver of patients with CHC. METHODS: We genotyped 93 patients with CHC for 3 IL28B single nucleotide polymorphisms (SNPs, rs12979860, rs8099917, rs12980275), extracted RNA from their liver biopsies and quantified the expression of IL28B and of 8 previously identified classifier genes which discriminate between SVR and NR (IFI44L, RSAD2, ISG15, IFI22, LAMP3, OAS3, LGALS3BP and HTATIP2). Decision tree ensembles in the form of a random forest classifier were used to calculate the relative predictive power of these different variables in a multivariate analysis. RESULTS: The minor IL28B allele (bad risk for treatment response) was significantly associated with increased expression of ISGs, and, unexpectedly, with decreased expression of IL28B. Stratification of the patients into SVR and NR revealed that ISG expression was conditionally independent from the IL28B genotype, i.e. there was an increased expression of ISGs in NR compared to SVR irrespective of the IL28B genotype. The random forest feature score (RFFS) identified IFI27 (RFFS = 2.93), RSAD2 (1.88) and HTATIP2 (1.50) expression and the HCV genotype (1.62) as the strongest predictors of treatment response. ROC curves of the IL28B SNPs showed an AUC of 0.66 with an error rate (ERR) of 0.38. A classifier with the 3 best classifying genes showed an excellent test performance with an AUC of 0.94 and ERR of 0.15. The addition of IL28B genotype information did not improve the predictive power of the 3-gene classifier. CONCLUSIONS: IL28B genotype and hepatic ISG expression are conditionally independent predictors of treatment response in CHC. There is no direct link between altered IFNλ3 expression and pre-activation of the endogenous system in the liver. Hepatic ISG expression is by far the better predictor for treatment response than IL28B genotype.

Gene expression in cultured endometrium from women with different outcomes following IVF.

Estradiol and progesterone are crucial for the acquisition of receptivity and the change in transcriptional activity of target genes in the implantation window. The aim of this study was to differentiate the regulation of genes in the endometrium of patients with recurrent implantation failure (IF) versus those who became pregnant after in vitro fertilization (IVF) treatment. Moreover, the effect of embryo-derived factors on endometrial transcriptional activity was studied. Nine women with known IVF outcome (IF, M, miscarriage, OP, ongoing pregnancy) and undergoing hysteroscopy with endometrial biopsy were enrolled. Biopsies were taken during the midluteal phase. After culture in the presence of embryo-conditioned IVF media, total RNA was extracted and submitted to reverse transcription, target cDNA synthesis, biotin labelling, fragmentation and hybridization using the Affymetrix Human Genome U133A 2.0 Chip. Differential expression of selected genes was re-analysed by quantitative PCR, in which the results were calculated as threshold cycle differences between the groups and normalized to Glyceraldehyde phosphate dehydrogenase and beta-actin. Differences were seen for several genes from endometrial tissue between the IF and the pregnancy groups, and when comparing OP with M, 1875 up- and 1807 down-regulated genes were returned. Real-time PCR analysis confirmed up-regulation for somatostatin, PLAP-2, mucin 4 and CD163, and down-regulation of glycodelin, IL-24, CD69, leukaemia inhibitory factor and prolactin receptor between Op and M. When the different embryo-conditioned media were compared, no significant differential regulation could be demonstrated. Although microarray profiling may currently not be sensitive enough for studying the effects of embryo-derived factors on the endometrium, the observed differences in gene expression between M and OP suggest that it will become an interesting tool for the identification of fertility-relevant markers produced by the endometrium.

Gene expression profiling reveals consistent differences between clinical samples of human leukaemias and their model cell lines.

Microarray gene expression profiles of fresh clinical samples of chronic myeloid leukaemia in chronic phase, acute promyelocytic leukaemia and acute monocytic leukaemia were compared with profiles from cell lines representing the corresponding types of leukaemia (K562, NB4, HL60). In a hierarchical clustering analysis, all clinical samples clustered separately from the cell lines, regardless of leukaemic subtype. Gene ontology analysis showed that cell lines chiefly overexpressed genes related to macromolecular metabolism, whereas in clinical samples genes related to the immune response were abundantly expressed. These findings must be taken into consideration when conclusions from cell line-based studies are extrapolated to patients.

Differential gene expression in the pathogenic dermatophyte Arthroderma benhamiae in vitro versus during infection.

Although dermatophytes are the most common agents of superficial mycoses in humans and animals, the molecular basis of the pathogenicity of these fungi is largely unknown. In vitro digestion of keratin by dermatophytes is associated with the secretion of multiple proteases, which are assumed to be responsible for their particular specialization to colonize and degrade keratinized host structures during infection. To investigate the role of individual secreted proteases in dermatophytosis, a guinea pig infection model was established for the zoophilic dermatophyte Arthroderma benhamiae, which causes highly inflammatory cutaneous infections in humans and rodents. By use of a cDNA microarray covering approximately 20-25 % of the A. benhamiae genome and containing sequences of at least 23 protease genes, we revealed a distinct in vivo protease gene expression profile in the fungal cells, which was surprisingly different from the pattern elicited during in vitro growth on keratin. Instead of the major in vitro -expressed proteases, others were activated specifically during infection. These enzymes are therefore suggested to fulfil important functions that are not exclusively associated with the degradation of keratin. Most notably, the gene encoding the serine protease subtilisin 6, which is a known major allergen in the related dermatophyte Trichophyton rubrum and putatively linked to host inflammation, was found to be the most strongly upregulated gene during infection. In addition, our approach identified other candidate pathogenicity-related factors in A. benhamiae, such as genes encoding key enzymes of the glyoxylate cycle and an opsin-related protein. Our work provides what we believe to be the first broad-scale gene expression profile in human pathogenic dermatophytes during infection, and points to putative virulence-associated mechanisms that make these micro-organisms the most successful aetiological agents of superficial mycoses.

Gene expression profile during coffee fruit development and identification of candidate markers for phenological stages

The objective of this work was to identify genes that could be used as suitable markers for molecular recognition of phenological stages during coffee (Coffea arabica) fruit development. Four cultivars were evaluated as to their differential expression of genes associated to fruit development and maturation processes. Gene expression was characterized by both semi-quantitative and quantitative RT-PCR, in fruit harvested at seven different developmental stages, during three different seasons. No size polymorphisms or differential expression were observed among the cultivars for the evaluated genes; however, distinct expression profiles along fruit development were determined for each gene. Four out of the 28 evaluated genes exhibited a regular expression profile in all cultivars and harvest seasons, and, therefore, they were validated as candidate phenological markers of coffee fruit. The gene α-galactosidase can be used as a marker of green stage, caffeine synthase as a marker of transition to green and yellowish-green stages, and isocitrate lyase and ethylene receptor 3 as markers of late maturation.