Author Correction:Conservation of copy number profiles during engraftment and passaging of patient-derived cancer xenografts (Nature Genetics, (2021), 53, 1, (86-99), 10.1038/s41588-020-00750-6)

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Exploiting orthologue diversity for systematic detection of gain-of-function phenotypes

<p>Abstract</p> <p>Background</p> <p>Systematic search for genes whose gain-of-function by exogenous expression confers an advantage in cell-based selective screenings is a powerful method for unbiased functional exploration of the genome, and has the potential to disclose new targets for cancer therapy. A major limit of this approach resides in the labor-intensive cloning of resistant cells, identification of the integrated genes and validation of their ability to confer a selective advantage. Moreover, the selection has to be drastic and genes conferring a limited advantage are typically missed.</p> <p>Results</p> <p>We developed a new functional screening strategy based on transduction of mammalian cells of a given species with an expression library from another species, followed by one-shot quantitative tracing with DNA microarrays of all library-derived transcripts before and after selection. In this way, exogenous transcripts enriched after selection, and therefore likely to confer resistance, are readily detected. We transduced a retroviral cDNA expression library from mouse testis into human and canine cells, and optimized the use of commercial murine gene expression arrays for species-specific detection of library-derived transcripts. We then conducted a functional screening by growing library-transduced canine MDCK cells in suspension, to enrich for cDNAs conferring anchorage independence. Notably, these cells show partial resistance to loss of anchorage, and the selection can be of limited stringency, compromising approaches based on clonal selection or anyway requiring high stringency. Microarray analysis revealed reproducible enrichment after three weeks of growth on polyhema for seven genes, among which the Hras proto-oncogene and Sox5. When individually transduced into MDCK cells, Sox5 specifically promoted anchorage-independent growth, thereby confirming the validity and specificity of the approach.</p> <p>Conclusion</p> <p>The procedure described here brings substantial advantages to the field of expression cloning, being faster, more systematic and more sensitive. Indeed, this strategy allowed identification and validation of genes promoting anchorage-independent growth of epithelial cells under selection conditions not amenable to conventional expression cloning.</p

Mulcom: a multiple comparison statistical test for microarray data in Bioconductor

Many microarray experiments compare a common control group with several ”test ” groups, like in the case, for example of a time-course experiments where time zero serves as a common reference point. The MulCom package described here implements the Dunnett’s t-test, which has been specifically developed to handle multiple comparisons against a common reference, in a version tailored for genomic data analysis that we named MulCom (Multiple Comparisons) test. The implementation includes two test parameters, namely the t value and an optional minimal fold-change value, m, with automated, permutation-based estimation of False Discovery Rate (FDR) for parameter combinations of choice. The package permits automated optimization of the test parameters to obtain the maximum number of significant genes at a given FDR value. In this vignette we present the rationale, implementation and usage of the MulCom package, plus a practical application on a time-course microarra

Formalin Fixation at Low Temperature Better Preserves Nucleic Acid Integrity

Fixation with formalin, a widely adopted procedure to preserve tissue samples, leads to extensive degradation of nucleic acids and thereby compromises procedures like microarray-based gene expression profiling. We hypothesized that RNA fragmentation is caused by activation of RNAses during the interval between formalin penetration and tissue fixation. To prevent RNAse activation, a series of tissue samples were kept under-vacuum at 4°C until fixation and then fixed at 4°C, for 24 hours, in formalin followed by 4 hours in ethanol 95%. This cold-fixation (CF) procedure preserved DNA and RNA, so that RNA segments up to 660 bp were efficiently amplified. Histological and immunohistochemical features were fully comparable with those of standard fixation. Microarray-based gene expression profiles were comparable with those obtained on matched frozen samples for probes hybridizing within 700 bases from the reverse transcription start site. In conclusion, CF preserves tissues and nucleic acids, enabling reliable gene expression profiling of fixed tissues

Transcriptional Hallmarks of Noonan Syndrome and Noonan-Like Syndrome with Loose Anagen Hair

Noonan syndrome (NS) is among the most common nonchromosomal disorders affecting development and growth. NS is genetically heterogeneous, being caused by germline mutations affecting various genes implicated in the RAS signaling network. This network transduces extracellular signals into intracellular biochemical and transcriptional responses controlling cell proliferation, differentiation, metabolism, and senescence. To explore the transcriptional consequences of NS-causing mutations, we performed global mRNA expression profiling on peripheral blood mononuclear cells obtained from 23 NS patients carrying heterozygous mutations in PTPN11 or SOS1. Gene expression profiling was also resolved in five subjects with Noonan-like syndrome with loose anagen hair (NS/LAH), a condition clinically related to NS and caused by an invariant mutation in SHOC2. Robust transcriptional signatures were found to specifically discriminate each of the three mutation groups from 21 age- and sex-matched controls. Despite the only partial overlap in terms of gene composition, the three signatures showed a notable concordance in terms of biological processes and regulatory circuits affected. These data establish expression profiling of peripheral blood mononuclear cells as a powerful tool to appreciate differential perturbations driven by germline mutations of transducers involved in RAS signaling and to dissect molecular mechanisms underlying NS and other RASopathies. Hum Mutat 33:703–709, 2012. © 2012 Wiley Periodicals, Inc