Eivind Hovig

Somatic point mutations in the p53 gene of human tumors and cell lines: Updated compilation

In 1994 we described a list of approximately 2500 point mutations in the p53 gene of human tumors and cell lines which we had compiled from the published literature and made available electronically through the file server at the EMBL Data Library. This database, updated twice a year, now contains records on 4496 published mutations (July 1995 release) and can be obtained from the EMBL Outstation-the European Bioinformatics Institute (EBI) through the network or on CD-ROM. This report describes the criteria for inclusion of data in this database, a description of the current format and a brief discussion of the current relevance of p53 mutation analysis to clinical and biological questions.

Ten Simple Rules for Reproducible Computational Research

Replication is the cornerstone of a cumulative science [1]. However, new tools and technologies, massive amounts of data, interdisciplinary approaches, and the complexity of the questions being asked are complicating replication efforts, as are increased pressures on scientists to advance their research [2]. As full replication of studies on independently collected data is often not feasible, there has recently been a call for reproducible research as an attainable minimum standard for assessing the value of scientific claims [3]. This requires that papers in experimental science describe the results and provide a sufficiently clear protocol to allow successful repetition and extension of analyses based on original data [4]. The importance of replication and reproducibility has recently been exemplified through studies showing that scientific papers commonly leave out experimental details essential for reproduction [5], studies showing difficulties with replicating published experimental results [6], an increase in retracted papers [7], and through a high number of failing clinical trials [8], [9]. This has led to discussions on how individual researchers, institutions, funding bodies, and journals can establish routines that increase transparency and reproducibility. In order to foster such aspects, it has been suggested that the scientific community needs to develop a “culture of reproducibility” for computational science, and to require it for published claims [3]. We want to emphasize that reproducibility is not only a moral responsibility with respect to the scientific field, but that a lack of reproducibility can also be a burden for you as an individual researcher. As an example, a good practice of reproducibility is necessary in order to allow previously developed methodology to be effectively applied on new data, or to allow reuse of code and results for new projects. In other words, good habits of reproducibility may actually turn out to be a time-saver in the longer run. We further note that reproducibility is just as much about the habits that ensure reproducible research as the technologies that can make these processes efficient and realistic. Each of the following ten rules captures a specific aspect of reproducibility, and discusses what is needed in terms of information handling and tracking of procedures. If you are taking a bare-bones approach to bioinformatics analysis, i.e., running various custom scripts from the command line, you will probably need to handle each rule explicitly. If you are instead performing your analyses through an integrated framework (such as GenePattern [10], Galaxy [11], LONI pipeline [12], or Taverna [13]), the system may already provide full or partial support for most of the rules. What is needed on your part is then merely the knowledge of how to exploit these existing possibilities. In a pragmatic setting, with publication pressure and deadlines, one may face the need to make a trade-off between the ideals of reproducibility and the need to get the research out while it is still relevant. This trade-off becomes more important when considering that a large part of the analyses being tried out never end up yielding any results. However, frequently one will, with the wisdom of hindsight, contemplate the missed opportunity to ensure reproducibility, as it may already be too late to take the necessary notes from memory (or at least much more difficult than to do it while underway). We believe that the rewards of reproducibility will compensate for the risk of having spent valuable time developing an annotated catalog of analyses that turned out as blind alleys. As a minimal requirement, you should at least be able to reproduce the results yourself. This would satisfy the most basic requirements of sound research, allowing any substantial future questioning of the research to be met with a precise explanation. Although it may sound like a very weak requirement, even this level of reproducibility will often require a certain level of care in order to be met. There will for a given analysis be an exponential number of possible combinations of software versions, parameter values, pre-processing steps, and so on, meaning that a failure to take notes may make exact reproduction essentially impossible. With this basic level of reproducibility in place, there is much more that can be wished for. An obvious extension is to go from a level where you can reproduce results in case of a critical situation to a level where you can practically and routinely reuse your previous work and increase your productivity. A second extension is to ensure that peers have a practical possibility of reproducing your results, which can lead to increased trust in, interest for, and citations of your work [6], [14]. We here present ten simple rules for reproducibility of computational research. These rules can be at your disposal for whenever you want to make your research more accessible—be it for peers or for your future self.

Identification of a novel cytokeratin 19 pseudogene that may interfere with reverse transcriptase-polymerase chain reaction assays used to detect micrometastatic tumor cells

In many recent publications, it has been claimed that reverse transcriptase-polymerase chain reaction (RT-PCR) assays involving genes with tissue-restricted expression can be used for specific and sensitive detection of cancer cells in blood, bone marrow and lymph nodes. Many different target mRNAs have been evaluated for such purposes. One of the most extensively studied genes, CK19, is predominantly expressed in cells of epithelial origin and normally not at detectable levels in hematopoietic or lymphatic tissues. Based on previous reports on CK19 we wanted to establish a useful assay for detection of micrometastatic cells. RNA and DNA specimens extracted from peripheral blood nucleated cells of healthy volunteers, as well as cell lines positive and negative for CK19 expression, were used in nested RT-PCR assays. Using previously published primers, we found a novel pseudogene that shows a high degree of identity with the CK19 gene sequence, except for differences caused by 3 small deletions and a number of point mutations, resulting in termination codons and frameshifts. The gene has therefore no coding potential. Importantly, published primer sequences and reaction conditions used by several other groups to detect CK19 mRNA may have led to the amplification of this pseudogene. The data illustrate one of the problems that must be addressed in validating RT-PCR assays for micrometastasis detection, and it is suggested that previous work using CK19 as a marker should be reassessed in view of the present finding.

Differential expression patterns of S100a2, S100a4 and S100a6 during progression of human malignant melanoma

Three members of the S100 gene family, S100A2, S100A4 and S100A6, have been suggested to be associated with cancer development and metastasis. To study their involvement in the tumorigenesis of human melanoma, we examined the mRNA expression levels of the 3 genes in 45 melanoma metastases and in 20 benign nevi. Interestingly, whereas none of the metastases expressed S100A2 mRNA, and the expression level was low in 6 cell lines established from primary melanomas, all nevi showed moderate to high expression levels. Our results suggest that loss of S100A2 gene expression may be an early event in melanoma development. A significant correlation was found between the expression of S100A6 in melanoma metastases and both the survival time of the patients and the thickness of the corresponding primary tumors. For the S100A4 gene, however, no relationship was found between gene expression and clinical parameters of melanoma malignancy. The observed differences in expression patterns of the 3 S100 genes suggest distinct roles of their products in melanoma tumorigenesis and/or metastasis, and the results encourage studies to evaluate the potential value of using S100A2 and S100A6 expression levels as markers in the clinical management of melanoma. Int. J. Cancer 74:464–469, 1997.

Options available for profiling small samples: a review of sample amplification technology when combined with microarray profiling

The possibility of performing microarray analysis on limited material has been demonstrated in a number of publications. In this review we approach the technical aspects of mRNA amplification and several important implicit consequences, for both linear and exponential procedures. Amplification efficiencies clearly allow profiling of extremely small samples. The conservation of transcript abundance is the most important issue regarding the use of sample amplification in combination with microarray analysis, and this aspect has generally been found to be acceptable, although demonstrated to decrease in highly diluted samples. The fact that variability and discrepancies in microarray profiles increase with minute sample sizes has been clearly documented, but for many studies this does appear to have affected the biological conclusions. We suggest that this is due to the data analysis approach applied, and the consequence is the chance of presenting misleading results. We discuss the issue of amplification sensitivity limits in the light of reports on fidelity, published data from reviewed articles and data analysis approaches. These are important considerations to be reflected in the design of future studies and when evaluating biological conclusions from published microarray studies based on extremely low input RNA quantities.

Disentangling the perturbational effects of amino acid substitutions in the DNA-binding domain of p53

The spectrum of somatic cancer-associated missense mutations in the human TP53 gene was studied in order to assess the potential structural and functional importance of various intra-molecular properties associated with these substitutions. Relating the observed frequency of particular amino acid substitutions in the p53 DNA-binding domain to their expected frequency, as calculated from DNA sequence-dependent mutation rates, yielded estimates of their relative clinical observation likelihood (RCOL). Several biophysical properties were found to display significant covariation with RCOL values. Thus RCOL values were observed to decrease with increasing solvent accessibility of the substituted residue and with increasing distance from the p53 DNA-binding and Zn2+-binding sites. The number of adverse steric interactions introduced by an amino acid replacement was found to be positively correlated with its RCOL value, irrespective of the magnitude of the interactions. A gain in hydrogen bond number was found to be only half as likely to come to clinical attention as mutations involving either a reduction or no change in hydrogen bond number. When the difference in potential energy between the wild-type and mutant DNA-binding domains was considered, RCOL values exhibited a minimum around changes of zero. Finally, classification of mutated residues in terms of their protein/solvent environment yielded, for somatic p53 mutations, RCOL values that resembled those previously determined for inherited mutations of human factor IX causing haemophilia B, suggesting that similar mechanisms may be responsible for the mutation-related perturbation of biological function in different protein folds.

CONSTANT DENATURANT GEL ELECTROPHORESIS, A MODIFICATION OF DENATURING GRADIENT GEL ELECTROPHORESIS, IN MUTATION DETECTION

Abstract Denaturing gradient gel electrophoresis (DGGE) is increasingly being utilized in mutational detection, both in characterization of variations in genomic DNA and in the generation of mutational spectra after in vitro and in vivo mutagenesis. The basis for this electrophoretic separation technique is strand dissociation of DNA fragments in discrete, sequence-dependent melting domains followed by an abrupt decrease in mobility. We have modified the DGGE by using constant denaturant gels corresponding to the specific melting domains of certain DNA fragments. This leads to increased resolution of mutants as fragments differing in as little as 1 base pair migrate with a consistently different mobility through the whole gel allowing separations of several centimeters. By using a set of constant denaturant gels it is also possible to obtain a better approximation of the location of the different mutations as each denaturant concentration will correspond to specific melting domains. We have used this technique to separate 6 out of 7 exon-3 hypoxanthine phosphoribosyltransferase (HPRT) mutants while using conventional DGGE we were only able to separate 3.

A Uniform System for the Annotation of Vertebrate microRNA Genes and the Evolution of the Human microRNAome

Although microRNAs (miRNAs) are among the most intensively studied molecules of the past 20 years, determining what is and what is not a miRNA has not been straightforward. Here, we present a uniform system for the annotation and nomenclature of miRNA genes. We show that less than a third of the 1,881 human miRBase entries, and only approximately 16% of the 7,095 metazoan miRBase entries, are robustly supported as miRNA genes. Furthermore, we show that the human repertoire of miRNAs has been shaped by periods of intense miRNA innovation and that mature gene products show a very different tempo and mode of sequence evolution than star products. We establish a new open access database--MirGeneDB ( http://mirgenedb.org )--to catalog this set of miRNAs, which complements the efforts of miRBase but differs from it by annotating the mature versus star products and by imposing an evolutionary hierarchy upon this curated and consistently named repertoire.

Involvement of the pRb/p16/cdk4/cyclin D1 pathway in the tumorigenesis of sporadic malignant melanomas.

Biopsies from 61 sporadic metastatic malignant melanomas and five melanoma cell lines were examined for homozygous deletions and mutations in the CDKN2 gene (p16). As the p16 protein is involved in a cell cycle regulatory pathway consisting of at least pRb, cdk4 and cyclin D1, the tumours were also screened for amplifications of the last two genes. Moreover, the transcript levels of the genes were determined and the results compared with the immunohistochemically assessed expression of pRb. Altogether, homozygous deletions of CDKN2 were found in seven tumours (11%) and two of five cell lines, whereas a mutation was detected in only one biopsy, indicating that in sporadic melanomas the former mechanism is predominant for inactivating this gene. Notably, in total 59% of the metastatic lesions lacked detectable expression of p16 mRNA, whereas all the biopsies were found to express pRb. In accordance with the postulated negative feedback loop between p16 and pRb, one melanoma cell line showed overexpression of CDKN2 mRNA together with very low levels of the Rb protein. Amplification of the other two genes may not be important in the tumorigenesis of melanomas, as only one CDK4 and no CCND1 amplification was observed. However, highly elevated CDK4 mRNA levels, compared with that seen in a panel of normal tissues, were observed in 76% of the tumours, accompanied in 71% of the cases by high expression of the CCND1 cyclin activator. Although a low frequency of CDKN2 DNA aberrations was observed, the high number of tumours that lacked CDKN2 expression but showed overexpression of CDK4 and/or CCND1, suggest that functional inactivation of pRb through this pathway may be involved in the development or progression of sporadic human melanomas.

Homozygous deletion frequency and expression levels of the CDKN2 gene in human sarcomas--relationship to amplification and mRNA levels of CDK4 and CCND1.

Homozygous deletions of the putative tumour-suppressor gene CDKN2, which encodes an inhibitor of cdk4, have been detected in a high percentage of cancer cell lines of various histological types. In the present study, 109 human sarcomas were examined for homozygous deletions and for mRNA expression levels of the CDKN2 gene. Altogether, deletions were found in only eight (7%) of the cases, but, interestingly, in two (of eight) malignant Schwannomas and in two (of five) rhabdomyosarcomas. In comparison, such deletions were seen in only one (of 21) osteosarcomas and in none of 20 MFHs and 21 liposarcomas. Notably, highly elevated CDKN2 mRNA levels were found in 33% of the sarcomas, whereas no detectable transcript was present in 12 normal tissues. Amplifications of CDK4 and CCND1 (cyclin D1) were observed in 11% and 4% of the sarcomas respectively, but never in tumours with CDKN2 deletions. The level of CDK4 mRNA expression was increased in nine tumours in addition to the 12 samples with CDK4 amplification. Increased levels of the cyclin D1 transcript was found in 37 cases, four with and 33 without amplification. The data indicate that aberrations of these functionally related genes, or in regulation of the expression of the kinase, the activator or the inhibitor, may participate in sarcoma development. Furthermore, the data suggest that homozygous CDKN2 deletions may be of dissimilar significance in different sarcoma subtypes.