Biomedical subjects

Annemarie Poustka

Publications and source records attributed to Annemarie Poustka.

At least 19 recordsLinked to original sources

PubMed2007

Allelic variants of CAMTA1 and FLJ10737 within a commonly deleted region at 1p36 in neuroblastoma.

Deletion of a distal portion of 1p is seen in a wide range of human malignancies, including neuroblastoma. Here, a 1p36.3 commonly deleted region of 216 kb has been defined encompassing two genes, CAMTA1 and FLJ10737. Low expression of CAMTA1 has been recently shown to be an independent predictor of poor outcome in neuroblastoma patients. The present study surveys CAMTA1 and FLJ10737 for genetic alterations by fluorescence-based single strand conformation polymorphism (SSCP) using a panel of DNAs from 88 neuroblastomas, their matching blood samples and 97 unaffected individuals. Nucleotide variants encoding amino acid substitutions were found in both genes. One CAMTA1 variant (T1336I) was not detected in 97 unaffected individuals, another (N1177K) resides in a conserved domain of the CAMTA1 protein and was found hemizygous in six neuroblastomas. We found no evidence for somatic mutations in FLJ10737 or CAMTA1. Further investigations are needed to address the functional impact of the identified variants and their possible significance for neuroblastoma.

Alleles↗

PubMed2006

Identification and meta-analysis of a small gene expression signature for the diagnosis of estrogen receptor status in invasive ductal breast cancer.

In breast cancer, the determination of estrogen receptor (ER) expression is crucial for the decision on therapeutic strategies. Current ER expression analysis is based on immunohistochemical (IHC) staining of ER on formalin fixed tissue sections. However, low levels of ER expression frequently escape detection because of varying sensitivities of routine histopathological laboratories. Moreover, in estimating ER by IHC the receptor protein only is tested instead of the complex underlying ER pathway, which reflects its biological activity. To overcome this limitation, we have used the microarray technology to study 56 samples of invasive ductal carcinoma. We infer a robust and reliable signature of 10 genes, which is associated with ER expression and presumably therapeutically relevant biological processes. In a meta-analysis, the signature was tested on 3 further independent microarray gene expression data sets, covering different laboratories, array platforms, and clinics. The classification based on the signature showed a very low misclassification rate. In summary, the expression of few genes is sufficient to determine ER status. Future decisions on antiestrogen based therapy in breast cancer could be based on this signature rather than on immunostaining alone.

Adult↗

PubMed2006

CAFTAN: a tool for fast mapping, and quality assessment of cDNAs.

BACKGROUND: The German cDNA Consortium has been cloning full length cDNAs and continued with their exploitation in protein localization experiments and cellular assays. However, the efficient use of large cDNA resources requires the development of strategies that are capable of a speedy selection of truly useful cDNAs from biological and experimental noise. To this end we have developed a new high-throughput analysis tool, CAFTAN, which simplifies these efforts and thus fills the gap between large-scale cDNA collections and their systematic annotation and application in functional genomics. RESULTS: CAFTAN is built around the mapping of cDNAs to the genome assembly, and the subsequent analysis of their genomic context. It uses sequence features like the presence and type of PolyA signals, inner and flanking repeats, the GC-content, splice site types, etc. All these features are evaluated in individual tests and classify cDNAs according to their sequence quality and likelihood to have been generated from fully processed mRNAs. Additionally, CAFTAN compares the coordinates of mapped cDNAs with the genomic coordinates of reference sets from public available resources (e.g., VEGA, ENSEMBL). This provides detailed information about overlapping exons and the structural classification of cDNAs with respect to the reference set of splice variants. The evaluation of CAFTAN showed that is able to correctly classify more than 85% of 5950 selected "known protein-coding" VEGA cDNAs as high quality multi- or single-exon. It identified as good 80.6 % of the single exon cDNAs and 85 % of the multiple exon cDNAs. The program is written in Perl and in a modular way, allowing the adoption of this strategy to other tasks like EST-annotation, or to extend it by adding new classification rules and new organism databases as they become available. We think that it is a very useful program for the annotation and research of unfinished genomes. CONCLUSION: CAFTAN is a high-throughput sequence analysis tool, which performs a fast and reliable quality prediction of cDNAs. Several thousands of cDNAs can be analyzed in a short time, giving the curator/scientist a first quick overview about the quality and the already existing annotation of a set of cDNAs. It supports the rejection of low quality cDNAs and helps in the selection of likely novel splice variants, and/or completely novel transcripts for new experiments.

Chromosome Mapping↗

PubMed2006

Identification of a common gene expression signature in dilated cardiomyopathy across independent microarray studies.

OBJECTIVES: This study was designed to identify a common gene expression signature in dilated cardiomyopathy (DCM) across different microarray studies. BACKGROUND: Dilated cardiomyopathy is a common cause of heart failure in Western countries. Although gene expression arrays have emerged as a powerful tool for delineating complex disease patterns, differences in platform technology, tissue heterogeneity, and small sample sizes obscure the underlying pathophysiologic events and hamper a comprehensive interpretation of different microarray studies in heart failure. METHODS: We accounted for tissue heterogeneity and technical aspects by performing 2 genome-wide expression studies based on cDNA and short-oligonucleotide microarray platforms which comprised independent septal and left ventricular tissue samples from nonfailing (NF) (n = 20) and DCM (n = 20) hearts. RESULTS: Concordant results emerged for major gene ontology classes between cDNA and oligonucleotide microarrays. Notably, immune response processes displayed the most pronounced down-regulation on both microarray types, linking this functional gene class to the pathogenesis of end-stage DCM. Furthermore, a robust set of 27 genes was identified that classified DCM and NF samples with >90% accuracy in a total of 108 myocardial samples from our cDNA and oligonucleotide microarray studies as well as 2 publicly available datasets. CONCLUSIONS: For the first time, independent microarray datasets pointed to significant involvement of immune response processes in end-stage DCM. Moreover, based on 4 independent microarray datasets, we present a robust gene expression signature of DCM, encouraging future prospective studies for the implementation of disease biomarkers in the management of patients with heart failure.

Antibody Formation↗

PubMed2006

Statistical methods and software for the analysis of highthroughput reverse genetic assays using flow cytometry readouts.

Highthroughput cell-based assays with flow cytometric readout provide a powerful technique for identifying components of biologic pathways and their interactors. Interpretation of these large datasets requires effective computational methods. We present a new approach that includes data pre-processing, visualization, quality assessment, and statistical inference. The software is freely available in the Bioconductor package prada. The method permits analysis of large screens to detect the effects of molecular interventions in cellular systems.

Databases, Factual↗

PubMed2006

Molecular characterization of the porcine deleted in malignant brain tumors 1 gene (DMBT1).

The human gene deleted in malignant brain tumors 1 (DMBT1) is considered to play a role in tumorigenesis and pathogen defense. It encodes a protein with multiple scavenger receptor cysteine-rich (SRCR) domains, which are involved in recognition and binding of a broad spectrum of bacterial pathogens. The SRCR domains are encoded by highly homologous repetitive exons, whose number in humans may vary from 8 to 13 due to genetic polymorphism. Here, we characterized the porcine DMBT1 gene on the mRNA and genomic level. We assembled a 4.5 kb porcine DMBT1 cDNA sequence from RT-PCR amplified seminal vesicle RNA. The porcine DMBT1 cDNA contains an open reading frame of 4050 nt. The transcript gives rise to a putative polypeptide of 1349 amino acids with a calculated mass of 147.9 kDa. Compared to human DMBT1, it contains only four N-terminal SRCR domains. Northern blotting revealed transcripts of approximately 4.7 kb in size in the tissues analyzed. Analysis of ESTs suggested the existence of secreted and transmembrane variants. The porcine DMBT1 gene spans about 54 kb on chromosome 14q28-q29. In contrast to the characterized cDNA, the genomic BAC clone only contained 3 exons coding for N-terminal SRCR domains. In different mammalian DMBT1 orthologs large interspecific differences in the number of SRCR exons and utilization of the transmembrane exon exist. Our data suggest that the porcine DMBT1 gene may share with the human DMBT1 gene additional intraspecific variations in the number of SRCR-coding exons.

Amino Acid Sequence↗

PubMed2006

An anthropoid-specific segmental duplication on human chromosome 1q22.

Segmental duplications (SDs) play a key role in genome evolution by providing material for gene diversification and creation of variant or novel functions. They also mediate recombinations, resulting in microdeletions, which have occasionally been associated with human genetic diseases. Here, we present a detailed analysis of a large genomic region (about 240 kb), located on human chromosome 1q22, that contains a tandem SD, SD1q22. This duplication occurred about 37 million years ago in a lineage leading to anthropoid primates, after their separation from prosimians but before the Old and New World monkey split. We reconstructed the hypothetical unduplicated ancestral locus and compared it with the extant SD1q22 region. Our data demonstrate that, as a consequence of the duplication, new anthropoid-specific genetic material has evolved in the resulting paralogous segments. We describe the emergence of two new genes, whose new functions could contribute to the speciation of anthropoid primates. Moreover, we provide detailed information regarding structure and evolution of the SD1q22 region that is a prerequisite for future studies of its anthropoid-specific functions and possible linkage to human genetic disorders.

Animals↗

PubMed2006

The 3of5 web application for complex and comprehensive pattern matching in protein sequences.

BACKGROUND: The identification of patterns in biological sequences is a key challenge in genome analysis and in proteomics. Frequently such patterns are complex and highly variable, especially in protein sequences. They are frequently described using terms of regular expressions (RegEx) because of the user-friendly terminology. Limitations arise for queries with the increasing complexity of patterns and are accompanied by requirements for enhanced capabilities. This is especially true for patterns containing ambiguous characters and positions and/or length ambiguities. RESULTS: We have implemented the 3of5 web application in order to enable complex pattern matching in protein sequences. 3of5 is named after a special use of its main feature, the novel n-of-m pattern type. This feature allows for an extensive specification of variable patterns where the individual elements may vary in their position, order, and content within a defined stretch of sequence. The number of distinct elements can be constrained by operators, and individual characters may be excluded. The n-of-m pattern type can be combined with common regular expression terms and thus also allows for a comprehensive description of complex patterns. 3of5 increases the fidelity of pattern matching and finds ALL possible solutions in protein sequences in cases of length-ambiguous patterns instead of simply reporting the longest or shortest hits. Grouping and combined search for patterns provides a hierarchical arrangement of larger patterns sets. The algorithm is implemented as internet application and freely accessible. The application is available at http://dkfz.de/mga2/3of5/3of5.html. CONCLUSION: The 3of5 application offers an extended vocabulary for the definition of search patterns and thus allows the user to comprehensively specify and identify peptide patterns with variable elements. The n-of-m pattern type offers an improved accuracy for pattern matching in combination with the ability to find all solutions, without compromising the user friendliness of regular expression terms.

Algorithms↗

PubMed2006

The systematic functional characterisation of Xq28 genes prioritises candidate disease genes.

BACKGROUND: Well known for its gene density and the large number of mapped diseases, the human sub-chromosomal region Xq28 has long been a focus of genome research. Over 40 of approximately 300 X-linked diseases map to this region, and systematic mapping, transcript identification, and mutation analysis has led to the identification of causative genes for 26 of these diseases, leaving another 17 diseases mapped to Xq28, where the causative gene is still unknown. To expedite disease gene identification, we have initiated the functional characterisation of all known Xq28 genes. RESULTS: By using a systematic approach, we describe the Xq28 genes by RNA in situ hybridisation and Northern blotting of the mouse orthologs, as well as subcellular localisation and data mining of the human genes. We have developed a relational web-accessible database with comprehensive query options integrating all experimental data. Using this database, we matched gene expression patterns with affected tissues for 16 of the 17 remaining Xq28 linked diseases, where the causative gene is unknown. CONCLUSION: By using this systematic approach, we have prioritised genes in linkage regions of Xq28-mapped diseases to an amenable number for mutational screens. Our database can be queried by any researcher performing highly specified searches including diseases not listed in OMIM or diseases that might be linked to Xq28 in the future.

Animals↗

PubMed2006

The LIFEdb database in 2006.

LIFEdb (http://www.LIFEdb.de) integrates data from large-scale functional genomics assays and manual cDNA annotation with bioinformatics gene expression and protein analysis. New features of LIFEdb include (i) an updated user interface with enhanced query capabilities, (ii) a configurable output table and the option to download search results in XML, (iii) the integration of data from cell-based screening assays addressing the influence of protein-overexpression on cell proliferation and (iv) the display of the relative expression ('Electronic Northern') of the genes under investigation using curated gene expression ontology information. LIFEdb enables researchers to systematically select and characterize genes and proteins of interest, and presents data and information via its user-friendly web-based interface.

Cell Proliferation↗

PubMed2006

Analysis of pigmented villonodular synovitis with genome-wide complementary DNA microarray and tissue array technology reveals insight into potential novel therapeutic approaches.

OBJECTIVE: To characterize the gene expression profile and determine potential diagnostic markers and therapeutic targets in pigmented villonodular synovitis (PVNS). METHODS: Gene expression patterns in 11 patients with PVNS, 18 patients with rheumatoid arthritis (RA), and 19 patients with osteoarthritis (OA) were investigated using genome-wide complementary DNA microarrays. Validation of differentially expressed genes was performed by real-time quantitative polymerase chain reaction and immunohistochemical analysis on tissue arrays (80 patients with PVNS, 51 patients with RA, and 20 patients with OA). RESULTS: The gene expression profile in PVNS was clearly distinct from those in RA and OA. One hundred forty-one up-regulated genes and 47 down-regulated genes were found in PVNS compared with RA, and 153 up-regulated genes and 89 down-regulated genes were found in PVNS compared with OA (fold change > or = 1.5; Q < or = 0.001). Genes differentially expressed in PVNS were involved in apoptosis regulation, matrix degradation, and inflammation (ALOX5AP, ATP6V1B2, CD53, CHI3L1, CTSL, CXCR4, HSPA8, HSPCA, LAPTM5, MMP9, MOAP1, and SPP1). CONCLUSION: The gene expression signature in PVNS is similar to that of activated macrophages and is consistent with the local destructive course of the disease. The gene and protein expression patterns suggest that the ongoing proliferation in PVNS is sustained by apoptosis resistance. This result suggests the possibility of a potential novel therapeutic intervention against PVNS.

Arthritis, Rheumatoid↗

PubMed2005

Haplotype analysis of the COMT-ARVCF gene region in Israeli anorexia nervosa family trios.

Anorexia nervosa (AN) is a severe and complex psychiatric disorder with a significant genetic contribution. Previously, we found an association between AN and the 158Val/Met polymorphism of the catechol-O-methyltransferase (COMT) gene in a family-based study of 51 Israeli AN trios. In the present study, we extended the original sample to include 85 family trios [66 AN restricting (AN-R) and 19 bingeing/purging (AN-BP) subtype] and performed a family-based transmission disequilibrium test (TDT) analysis for five SNPs in the COMT and two in the adjacent ARVCF gene. Association was found between AN-R and several SNPs in the COMT-ARVCF region including the 158Val/Met polymorphism. TDT analysis of 5-SNP haplotypes in AN-R trios revealed an overall statistically significant transmission disequilibrium (P < 0.001). Specifically, haplotype B [COMT-186C-408G-472G(158Val)-ARVCF-659C(220Pro)-524T(175Val)] was preferentially transmitted (P < 0.001) from parents of AN-R patients to their affected daughters, while haplotype A [COMT-186T-408C-472A(158Met)-ARVCF-659T(220Leu)-524C(175Ala)] was preferentially (P = 0.01) not transmitted. Haplotype B was associated with increased risk (RR 3.38; 0.95CI 1.98-6.43) while haplotype A exhibited a protective effect (RR 0.40; 0.95CI 0.21-0.70) for AN-R. Preferential transmission of the risk alleles and haplotypes from the parents was mostly contributed by the fathers. No significant transmission disequilibrium of alleles or haplotypes was found for AN-BP trios. The risk and protective haplotypes may carry molecular variations in the COMT gene or its vicinity that are relevant to the pathophysiology of restrictive anorexia nervosa in the Israeli-Jewish population.

Adolescent↗

PubMed2005

Primary cutaneous T-cell lymphomas show a deletion or translocation affecting NAV3, the human UNC-53 homologue.

Multicolor fluorescent in situ hybridization (FISH) was used to identify acquired chromosomal aberrations in 12 patients with mycosis fungoides or Sézary syndrome, the most common forms of primary cutaneous T-cell lymphoma (CTCL). The most frequently affected chromosome was 12, which showed clonal deletions or translocations with a break point in 12q21 or 12q22 in five of seven consecutive Sézary syndrome patients and a clonal monosomy in the sixth patient. The break point of a balanced translocation t(12;18)(q21;q21.2), mapped in the minimal common region of two deletions, fine mapped to 12q2. By locus-specific FISH, the translocation disrupted one gene, NAV3 (POMFIL1), a human homologue of unc-53 in Caenorhabditis elegans. A missense mutation in the remaining NAV3 allele was found in one of six cases with a deletion or translocation. With locus-specific FISH, NAV3 deletions were found in the skin lesions of four of eight (50%) patients with early mycosis fungoides (stages IA-IIA) and in the skin or lymph node of 11 of 13 (85%) patients with advanced mycosis fungoides or Sézary syndrome. Preliminary functional studies with lentiviral small interfering RNA-based NAV3 silencing in Jurkat cells and in primary lymphocytes showed enhanced interleukin 2 expression (but not CD25 expression). Thus, NAV3 may contribute to the growth, differentiation, and apoptosis of CTCL cells as well as to the skewing from Th1-type to Th2-type phenotype during disease progression. NAV3, a novel putative haploinsufficient tumor suppressor gene, is disrupted in most cases of the commonest types of CTCL and may thus provide a new diagnostic tool.

Alleles↗

PubMed2005

Functional profiling: from microarrays via cell-based assays to novel tumor relevant modulators of the cell cycle.

Cancer transcription microarray studies commonly deliver long lists of "candidate" genes that are putatively associated with the respective disease. For many of these genes, no functional information, even less their relevance in pathologic conditions, is established as they were identified in large-scale genomics approaches. Strategies and tools are thus needed to distinguish genes and proteins with mere tumor association from those causally related to cancer. Here, we describe a functional profiling approach, where we analyzed 103 previously uncharacterized genes in cancer relevant assays that probed their effects on DNA replication (cell proliferation). The genes had previously been identified as differentially expressed in genome-wide microarray studies of tumors. Using an automated high-throughput assay with single-cell resolution, we discovered seven activators and nine repressors of DNA replication. These were further characterized for effects on extracellular signal-regulated kinase 1/2 (ERK1/2) signaling (G1-S transition) and anchorage-independent growth (tumorigenicity). One activator and one inhibitor protein of ERK1/2 activation and three repressors of anchorage-independent growth were identified. Data from tumor and functional profiling make these proteins novel prime candidates for further in-depth study of their roles in cancer development and progression. We have established a novel functional profiling strategy that links genomics to cell biology and showed its potential for discerning cancer relevant modulators of the cell cycle in the candidate lists from microarray studies.

Animals↗

PubMed2005

Alternative pre-mRNA processing regulates cell-type specific expression of the IL4l1 and NUP62 genes.

BACKGROUND: Given the complexity of higher organisms, the number of genes encoded by their genomes is surprisingly small. Tissue specific regulation of expression and splicing are major factors enhancing the number of the encoded products. Commonly these mechanisms are intragenic and affect only one gene. RESULTS: Here we provide evidence that the IL4I1 gene is specifically transcribed from the apparent promoter of the upstream NUP62 gene, and that the first two exons of NUP62 are also contained in the novel IL4I1_2 variant. While expression of IL4I1 driven from its previously described promoter is found mostly in B cells, the expression driven by the NUP62 promoter is restricted to cells in testis (Sertoli cells) and in the brain (e.g., Purkinje cells). Since NUP62 is itself ubiquitously expressed, the IL4I1_2 variant likely derives from cell type specific alternative pre-mRNA processing. CONCLUSION: Comparative genomics suggest that the promoter upstream of the NUP62 gene originally belonged to the IL4I1 gene and was later acquired by NUP62 via insertion of a retroposon. Since both genes are apparently essential, the promoter had to serve two genes afterwards. Expression of the IL4I1 gene from the "NUP62" promoter and the tissue specific involvement of the pre-mRNA processing machinery to regulate expression of two unrelated proteins indicate a novel mechanism of gene regulation.

Alternative Splicing↗

PubMed2005

Gamma-BAR, a novel AP-1-interacting protein involved in post-Golgi trafficking.

A novel peripheral membrane protein (2c18) that interacts directly with the gamma 'ear' domain of the adaptor protein complex 1 (AP-1) in vitro and in vivo is described. Ultrastructural analysis demonstrates a colocalization of 2c18 and gamma1-adaptin at the trans-Golgi network (TGN) and on vesicular profiles. Overexpression of 2c18 increases the fraction of membrane-bound gamma1-adaptin and inhibits its release from membranes in response to brefeldin A. Knockdown of 2c18 reduces the steady-state levels of gamma1-adaptin on membranes. Overexpression or downregulation of 2c18 leads to an increased secretion of the lysosomal hydrolase cathepsin D, which is sorted by the mannose-6-phosphate receptor at the TGN, which itself involves AP-1 function for trafficking between the TGN and endosomes. This suggests that the direct interaction of 2c18 and gamma1-adaptin is crucial for membrane association and thus the function of the AP-1 complex in living cells. We propose to name this protein gamma-BAR.

Adaptor Protein Complex 1↗

PubMed2005

Gene expression in kidney cancer is associated with cytogenetic abnormalities, metastasis formation, and patient survival.

Current diagnosis of renal cancer consists of histopathologic examination of tissue sections and classification into tumor stages and grades of malignancy. Until recently, molecular differences between tumor types were largely unknown. To examine such differences, we did gene expression measurements of 112 renal cell carcinoma and normal kidney samples on renal cell carcinoma-specific cDNA microarrays containing 4,207 genes and expressed sequence tags. The gene expression patterns showed deregulation of complete biological pathways in the tumors. Many of the molecular changes corresponded well to the histopathologic tumor types, and a set of 80 genes was sufficient to classify tumors with a very low error rate. Distinct gene expression signatures were associated with chromosomal abnormalities of tumor cells, metastasis formation, and patient survival. The data highlight the benefit of microarrays to detect novel tumor classes and to identify genes that are associated with patient variables and tumor properties.

Adult↗

PubMed2005

Multiplex reverse transcription-polymerase chain reaction combined with on-chip electrophoresis as a rapid screening tool for candidate gene sets.

Combining multiplex reverse transcription-polymerase chain reaction (mRT-PCR) with microfluidic amplicon analysis, we developed an assay for the rapid and reliable semiquantitative expression screening of 11 candidate genes for drug resistance in human malignant melanoma. The functionality of this approach was demonstrated by low interexperimental variations of amplicon quantities after endpoint analysis. When applied to RNA samples derived from drug-sensitive and -resistant melanoma cell lines, mRT-PCR delivered results qualitatively concordant with data obtained from Northern blot and array analyses. The screening of additional melanoma cell lines resulted in distinct expression patterns for ten candidate genes. Our approach reveals a rapid and easy-to-handle alternative for candidate gene set evaluation from limited amounts of RNA.

Cell Line, Tumor↗