Computational approaches to identify functional genetic variants in cancer genomes

González-Pérez, Abel; Mustonen, Ville; Reva, Boris; Ritchie, Graham R. S.; Creixell, Pau; Karchin, Rachel; Vázquez, Miguél; Fink, J. Lynn; Kassahn, Karin S.; Pearson, John V.; Bader, Gary D.; Boutros, Paul C.; Muthuswamy, Lakshmi; Ouellette, B. F. Francis; Reimand, Jüri; Linding, Rune; Shibata, Tatsuhiro; Valencia, Alfonso; Butler, Adam; Dronov, Serge; Flicek, Paul; Shannon, Nicholas B.; Carter, Hannah; Li, Ding; Sander, Chris; Stuart, Joshua M.; Stein, Lincoln

doi:10.1038/nmeth.2562

Cited by 148 publications

(100 citation statements)

References 66 publications

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“…Each of these methods makes predictions about whether a mutation is likely to affect protein function but does not attempt to predict whether the mutation induces gain or loss of function. To compare these approaches, we used the term “functional variant” to denote both gain-of-function and loss-of-function alleles (50) and “neutral variant” for all other alleles. The concordance rates between each of these methods and our approach ranged from 66% to 77% (Methods; Supplementary Table S6; Supplementary Fig.…”

Section: Resultsmentioning

confidence: 99%

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles

et al. 2016

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Cancer genome characterization efforts now provide an initial view of the somatic alterations in primary tumors. However, most point mutations occur at low frequency. and the function of these alleles remain undefined. We have developed a scalable systematic approach to interrogate the function of cancer-associated gene variants. We subjected 474 mutant alleles curated from 5,338 tumors to pooled in vivo tumor formation assays and gene expression profiling. We identified 12 transforming alleles including two in genes (PIK3CB, POT1) that have not been shown to be tumorigenic. One rare KRAS allele, D33E, displayed tumorigenicity and constitutive activation of known RAS effector pathways. By comparing gene expression changes induced upon expression of wild type and mutant alleles, we inferred the activity of specific alleles. Since alleles found to be mutated only once in 5,338 tumors rendered cells tumorigenic, these observations underscore the value of integrating genomic information with functional studies.

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Section: Resultsmentioning

confidence: 99%

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles

et al. 2016

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“…20,40,41 At our median sequencing depth (265-fold), the estimated sensitivity for detecting somatic mutations with an atrial somatic fraction of 10% is 99.999%. When the atrial somatic fraction drops to 5%, 2%, and 1%, our sensitivity for detection correspondingly falls to 99.2%, 76.8%, and 26.9%, respectively.…”

Section: Discussionmentioning

confidence: 97%

Targeted Deep Sequencing Reveals No Definitive Evidence for Somatic Mosaicism in Atrial Fibrillation

Roberts

Longoria

Poon

et al. 2015

Circ Cardiovasc Genet

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Background Studies of ≤15 atrial fibrillation (AF) patients have identified atrial-specific mutations within connexin genes, suggesting that somatic mutations may account for sporadic cases of the arrhythmia. We sought to identify atrial somatic mutations among patients with and without AF using targeted deep next-generation sequencing of 560 genes, including genetic culprits implicated in AF, the Mendelian cardiomyopathies and channelopathies, and all ion channels within the genome. Methods and Results Targeted gene capture and next generation sequencing were performed on DNA from lymphocytes and left atrial appendages of 34 patients (25 with AF). Twenty AF patients had undergone cardiac surgery exclusively for pulmonary vein isolation, and 17 had no structural heart disease. Sequence alignment and variant calling were performed for each atrial-lymphocyte pair using the Burrows-Wheeler Aligner, the Genome Analysis Toolkit, and MuTect packages. Next generation sequencing yielded a median 265-fold coverage depth (IQR 164–369). Comparison of the 3 million base pairs from each atrial-lymphocyte pair revealed a single potential somatic missense mutation in 3 AF patients and 2 in a single control (12 vs. 11%; p=1). All potential discordant variants had low allelic fractions (range: 2.3–7.3%) and none were detected with conventional sequencing. Conclusions Using high-depth next generation sequencing and state-of-the art somatic mutation calling approaches, no pathogenic atrial somatic mutations could be confirmed among 25 AF patients in a comprehensive cardiac arrhythmia genetic panel. These findings indicate that atrial specific mutations are rare and that somatic mosaicism is unlikely to exert a prominent role in AF pathogenesis.

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“…The mechanisms by which the driver variants may affect protein stability, interactions, and function remain largely unknown. Various computational methods have been developed to estimate the impacts of disease mutations on proteins but most of them exclusively use sequence features and do not explicitly utilize the protein three-dimensional structures, their physico-chemical properties and dynamics (2, 3). Many cancers are characterized by (de)activation of certain proteins which may be a result of missense mutations (4).…”

Section: Introductionmentioning

confidence: 99%

Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation

Kales

et al. 2016

Cancer Research

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Oncogenic mutations in the monomeric Casitas B-lineage lymphoma (Cbl) gene have been found in many tumors, but their significance remains largely unknown. Several human c-Cbl (CBL) structures have recently been solved depicting the protein at different stages of its activation cycle and thus provide mechanistic insight underlying how stability-activity tradeoffs in cancer-related proteins may influence disease onset and progression. In this study, we computationally modeled the effects of missense cancer mutations on structures representing four stages of the CBL activation cycle to identify driver mutations that affect CBL stability, binding, and activity. We found that recurrent, homozygous, and leukemia-specific mutations had greater destabilizing effects on CBL states than did random non-cancer mutations. We further tested the ability of these computational models assessing the changes in CBL stability and its binding to ubiquitin conjugating enzyme E2, by performing blind CBL-mediated EGFR ubiquitination assays in cells. Experimental CBL ubiquitin ligase activity was in agreement with the predicted changes in CBL stability and, to a lesser extent, with CBL-E2 binding affinity. Two-thirds of all experimentally tested mutations affected the ubiquitin ligase activity by either destabilizing CBL or disrupting CBL-E2 binding, whereas about one-third of tested mutations were found to be neutral. Collectively, our findings demonstrate that computational methods incorporating multiple protein conformations and stability and binding affinity evaluations can successfully predict the functional consequences of cancer mutations on protein activity, and provide a proof of concept for mutations in CBL.

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Computational approaches to identify functional genetic variants in cancer genomes

Cited by 148 publications

References 66 publications

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles

Systematic Functional Interrogation of Rare Cancer Variants Identifies Oncogenic Alleles

Targeted Deep Sequencing Reveals No Definitive Evidence for Somatic Mosaicism in Atrial Fibrillation

Balancing Protein Stability and Activity in Cancer: A New Approach for Identifying Driver Mutations Affecting CBL Ubiquitin Ligase Activation

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