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Differential expression of a new isoform of DLG2 in renal oncocytoma.

ABSTRACT: Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type.In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expressed differentially in renal oncocytomas. Subsequently, we used RACE and Northern blot hybridization to characterize the potential candidates for molecular diagnosis.We have identified new isoform of DLG2 gene, which contains 3'-end exons of the known DLG2 gene along with the hypothetical gene FLJ37266. The new isoform is specifically upregulated in renal oncocytoma, whereas the known DLG2 gene is downregulated in this type of kidney tumour.The new isoform of DLG2 is the promising candidate gene for molecular differential diagnostics of renal oncocytoma.

SUBMITTER: Zubakov D

PROVIDER: S-EPMC1524971 | biostudies-literature | 2006 Apr

REPOSITORIES: biostudies-literature

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Differential expression of a new isoform of DLG2 in renal oncocytoma.

Zubakov Dmitry D Stupar Zorica Z Kovacs Gyula G

BMC cancer 20060426

<h4>Background</h4>Renal oncocytoma, a benign tumour of the kidney, may pose a differential diagnostic problem due to overlapping phenotype with chromophobe renal cell carcinoma or other types of renal cell tumours. Therefore, identification of molecular markers would be of great value for molecular diagnostics of this tumour type.<h4>Methods</h4>In the current study we applied various techniques, including Affymetrix microarray hybridization and semiquantitative RT-PCR, to identify genes expres ...[more]

PMID: 16640776

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Project description:BACKGROUND: Chromophobe renal cell carcinoma (chRCC) and renal oncocytoma are two distinct but closely related entities with strong morphologic and genetic similarities. While chRCC is a malignant tumor, oncocytoma is usually regarded as a benign entity. The overlapping characteristics are best explained by a common cellular origin, and the biologic differences between chRCC and oncocytoma are therefore of considerable interest in terms of carcinogenesis, diagnosis and clinical management. Previous studies have been relatively limited in terms of examining the differences between oncocytoma and chromophobe RCC. METHODS: Gene expression profiling using the Affymetrix HGU133Plus2 platform was applied on chRCC (n = 15) and oncocytoma specimens (n = 15). Supervised analysis was applied to identify a discriminatory gene signature, as well as differentially expressed genes. High throughput single-nucleotide polymorphism (SNP) genotyping was performed on independent samples (n = 14) using Affymetrix GeneChip Mapping 100 K arrays to assess correlation between expression and gene copy number. Immunohistochemical validation was performed in an independent set of tumors. RESULTS: A novel 14 probe-set signature was developed to classify the tumors internally with 93% accuracy, and this was successfully validated on an external data-set with 94% accuracy. Pathway analysis highlighted clinically relevant dysregulated pathways of c-erbB2 and mammalian target of rapamycin (mTOR) signaling in chRCC, but no significant differences in p-AKT or extracellular HER2 expression was identified on immunohistochemistry. Loss of chromosome 1p, reflected in both cytogenetic and expression analysis, is common to both entities, implying this may be an early event in histogenesis. Multiple regional areas of cytogenetic alterations and corresponding expression biases differentiating the two entities were identified. Parafibromin, aquaporin 6, and synaptogyrin 3 were novel immunohistochemical markers effectively discriminating the two pathologic entities. CONCLUSIONS: Gene expression profiles, high-throughput SNP genotyping, and pathway analysis effectively distinguish chRCC from oncocytoma. We have generated a novel transcript predictor that is able to discriminate between the two entities accurately, and which has been validated both in an internal and an independent data-set, implying generalizability. A cytogenetic alteration, loss of chromosome 1p, common to renal oncocytoma and chRCC has been identified, providing the opportunities for identifying novel tumor suppressor genes and we have identified a series of immunohistochemical markers that are clinically useful in discriminating chRCC and oncocytoma.

Project description:BackgroundThe murine discs large homolog 2 (DLG2; post synaptic density 93 (PSD-93); Chapsyn-110) is a member of the membrane-associated guanylate kinase (MAGUK) protein family involved in receptor assembly and associated with signaling enzymes on cell membranes. In neurons, DLG2 protein isoforms derived from alternatively spliced transcripts have been described to bind to NMDA (N-methyl-aspartate) receptors and K channels and to mediate clustering of these channels in the postsynaptic membrane. In myeloid cells of the immune system, such as dendritic cells (DCs), a lack of data exists on the expression or function of DLG2. In cDNA microarray transcriptome analyses, we found Dlg2 highly expressed in a subpopulation of plasmacytoid DCs (pDCs) stimulated to produce type I interferons (IFNs) such as IFNβ.ResultsUsing RACE- and RT-PCR as well as immunoprecipitation followed by Western blotting we characterised the differential expression of the Dlg2 splice variants in IFNβ-producing pDCs. Besides Dlg2ɣ this cell population expressed a novel short Dlg2η transcript we termed Dlg2η3. Our expression data were integrated into information from genome databases to obtain a novel and comprehensive overview of the mouse Dlg2 gene architecture. To elucidate the intracellular localisation pattern of protein isoforms, ectopical expression analysis of fluorescently tagged DLG2 splice variants was performed. Here we found an enrichment of the larger isoform DLG2α1 at the plasma membrane while the newly identified shorter (DLG2η) isoform as well as DLG2ɣ were equally distributed throughout the cytoplasm. Additionally, DLG2η was also found in the nucleus. Analysis of Dlg2-knockout mice previously generated by deleting exon 9 surprisingly revealed that the protein for the novel DLG2η isoform was still expressed in the brain and in bone marrow-derived pDCs from mice carrying the homozygous deletion (Dlg2 ΔE9/ΔE9 ).ConclusionWe describe a novel splice variant of the mouse Dlg2 gene termed Dlg2η and define the differential expression pattern of DLG2 isoforms in IFNβ-producing pDCs. The presence of DLG2η protein in the CNS of Dlg2 ΔE9/ΔE9 mice might influence the phenotype of these mice and has to be taken into account in the interpretation of results regarding the functional role of DLG2 in neuronal postsynaptic membranes.

Project description:Background: While subtyping of the majority of malignant chromophobe renal cell carcinoma (cRCC) and benign renal oncocytoma (rO) is possible on morphology alone, additional histochemical, immunohistochemical or molecular investigations are required in a subset of cases. As currently used histochemical and immunohistological stains as well as genetic aberrations show considerable overlap in both tumors, additional techniques are required for differential diagnostics. Mass spectrometry imaging (MSI) combining the detection of multiple peptides with information about their localization in tissue may be a suitable technology to overcome this diagnostic challenge. Patients and Methods: Formalin-fixed paraffin embedded (FFPE) tissue specimens from cRCC (n=71) and rO (n=64) were analyzed by MSI. Data were classified by linear discriminant analysis (LDA), classification and regression trees (CART), k-nearest neighbors (KNN), support vector machine (SVM), and random forest (RF) algorithm with internal cross validation and visualized by t-distributed stochastic neighbor embedding (t-SNE). Most important variables for classification were identified and the classification algorithm was optimized. Results: Applying different machine learning algorithms on all m/z peaks, classification accuracy between cRCC and rO was 85%, 82%, 84%, 77% and 64% for RF, SVM, KNN, CART and LDA. Under the assumption that a reduction of m/z peaks would lead to improved classification accuracy, m/z peaks were ranked based on their variable importance. Reduction to six most important m/z peaks resulted in improved accuracy of 89%, 85%, 85% and 85% for RF, SVM, KNN, and LDA and remained at the level of 77% for CART. t-SNE showed clear separation of cRCC and rO after algorithm improvement. Conclusion: In summary, we acquired MSI data on FFPE tissue specimens of cRCC and rO, performed classification and detected most relevant biomarkers for the differential diagnosis of both diseases. MSI data might be a useful adjunct method in the differential diagnosis of cRCC and rO.

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Differential expression of a new isoform of DLG2 in renal oncocytoma.

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Differential expression of a new isoform of DLG2 in renal oncocytoma.

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