The tandem duplicator phenotype as a distinct genomic configuration in cancer.

Next-generation sequencing studies have revealed genome-wide structural variation patterns in cancer, such as chromothripsis and chromoplexy, that do not engage a single discernable driver mutation, and whose clinical relevance is unclear. We devised a robust genomic metric able to identify cancers with a chromotype called tandem duplicator phenotype (TDP) characterized by frequent and distributed tandem duplications (TDs). Enriched only in triple-negative breast cancer (TNBC) and in ovarian, endometrial, and liver cancers, TDP tumors conjointly exhibit tumor protein p53 (TP53) mutations, disruption of breast cancer 1 (BRCA1), and increased expression of DNA replication genes pointing at rereplication in a defective checkpoint environment as a plausible causal mechanism. The resultant TDs in TDP augment global oncogene expression and disrupt tumor suppressor genes. Importantly, the TDP strongly correlates with cisplatin sensitivity in both TNBC cell lines and primary patient-derived xenografts. We conclude that the TDP is a common cancer chromotype that coordinately alters oncogene/tumor suppressor expression with potential as a marker for chemotherapeutic response.

Functional chromatin features are associated with structural mutations in cancer.

BACKGROUND: Structural mutations (SMs) play a major role in cancer development. In some cancers, such as breast and ovarian, DNA double-strand breaks (DSBs) occur more frequently in transcribed regions, while in other cancer types such as prostate, there is a consistent depletion of breakpoints in transcribed regions. Despite such regularity, little is understood about the mechanisms driving these effects. A few works have suggested that protein binding may be relevant, e.g. in studies of androgen receptor binding and active chromatin in specific cell types. We hypothesized that this behavior might be general, i.e. that correlation between protein-DNA binding (and open chromatin) and breakpoint locations is common across divergent cancers. RESULTS: We investigated this hypothesis by comprehensively analyzing the relationship among 457 ENCODE protein binding ChIP-seq experiments, 125 DnaseI and 24 FAIRE experiments, and 14,600 SMs from 8 diverse cancer datasets covering 147 samples. In most cancers, including breast and ovarian, we found enrichment of protein binding and open chromatin in the vicinity of SM breakpoints at distances up to 200 kb. Furthermore, for all cancer types we observed an enhanced enrichment in regions distant from genes when compared to regions proximal to genes, suggesting that the SM-induction mechanism is independent from the bias of DSBs to occur near transcribed regions. We also observed a stronger effect for sites with more than one protein bound. CONCLUSIONS: Protein binding and open chromatin state are associated with nearby SM breakpoints in many cancer datasets. These observations suggest a consistent mechanism underlying SM locations across different cancers.

Scotty: a web tool for designing RNA-Seq experiments to measure differential gene expression.

MOTIVATION: A common question arises at the beginning of every experiment where RNA-Seq is used to detect differential gene expression between two conditions: How many reads should we sequence? RESULTS: Scotty is an interactive web-based application that assists biologists to design an experiment with an appropriate sample size and read depth to satisfy the user-defined experimental objectives. This design can be based on data available from either pilot samples or publicly available datasets. AVAILABILITY: Scotty can be freely accessed on the web at http://euler.bc.edu/marthlab/scotty/scotty.php

Copy Number Variation detection from 1000 Genomes Project exon capture sequencing data.

BACKGROUND: DNA capture technologies combined with high-throughput sequencing now enable cost-effective, deep-coverage, targeted sequencing of complete exomes. This is well suited for SNP discovery and genotyping. However there has been little attention devoted to Copy Number Variation (CNV) detection from exome capture datasets despite the potentially high impact of CNVs in exonic regions on protein function. RESULTS: As members of the 1000 Genomes Project analysis effort, we investigated 697 samples in which 931 genes were targeted and sampled with 454 or Illumina paired-end sequencing. We developed a rigorous Bayesian method to detect CNVs in the genes, based on read depth within target regions. Despite substantial variability in read coverage across samples and targeted exons, we were able to identify 107 heterozygous deletions in the dataset. The experimentally determined false discovery rate (FDR) of the cleanest dataset from the Wellcome Trust Sanger Institute is 12.5%. We were able to substantially improve the FDR in a subset of gene deletion candidates that were adjacent to another gene deletion call (17 calls). The estimated sensitivity of our call-set was 45%. CONCLUSIONS: This study demonstrates that exonic sequencing datasets, collected both in population based and medical sequencing projects, will be a useful substrate for detecting genic CNV events, particularly deletions. Based on the number of events we found and the sensitivity of the methods in the present dataset, we estimate on average 16 genic heterozygous deletions per individual genome. Our power analysis informs ongoing and future projects about sequencing depth and uniformity of read coverage required for efficient detection.

Specific residues of the cytoplasmic domains of cardiac inward rectifier potassium channels are effective antifibrillatory targets.

Atrial and ventricular tachyarrhythmias can be perpetuated by up-regulation of inward rectifier potassium channels. Thus, it may be beneficial to block inward rectifier channels under conditions in which their function becomes arrhythmogenic (e.g., inherited gain-of-function mutation channelopathies, ischemia, and chronic and vagally mediated atrial fibrillation). We hypothesize that the antimalarial quinoline chloroquine exerts potent antiarrhythmic effects by interacting with the cytoplasmic domains of Kir2.1 (I(K1)), Kir3.1 (I(KACh)), or Kir6.2 (I(KATP)) and reducing inward rectifier potassium currents. In isolated hearts of three different mammalian species, intracoronary chloroquine perfusion reduced fibrillatory frequency (atrial or ventricular), and effectively terminated the arrhythmia with resumption of sinus rhythm. In patch-clamp experiments chloroquine blocked I(K1), I(KACh), and I(KATP). Comparative molecular modeling and ligand docking of chloroquine in the intracellular domains of Kir2.1, Kir3.1, and Kir6.2 suggested that chloroquine blocks or reduces potassium flow by interacting with negatively charged amino acids facing the ion permeation vestibule of the channel in question. These results open a novel path toward discovering antiarrhythmic pharmacophores that target specific residues of the cytoplasmic domain of inward rectifier potassium channels.

A single-cell model of phase-driven control of ventricular fibrillation frequency.

The mechanisms controlling the rotation frequency of functional reentry in ventricular fibrillation (VF) are poorly understood. It has been previously shown that Ba2+ at concentrations up to 50 mumol/L slows the rotation frequency in the intact guinea pig (GP) heart, suggesting a role of the inward rectifier current (I(K1)) in the mechanism governing the VF response to Ba2+. Given that other biological (e.g., sinoatrial node) and artificial systems display phase-locking behavior, we hypothesized that the mechanism for controlling the rotation frequency of a rotor by I(K1) blockade is phase-driven, i.e., the phase shift between transmembrane current and voltage remains constant at varying levels of I(K1) blockade. We measured whole-cell admittance in isolated GP myocytes and in transfected human embryonic kidney (HEK) cells stably expressing Kir 2.1 and 2.3 channels. The admittance phase, i.e., the phase difference between current and voltage, was plotted versus the frequency in control conditions and at 10 or 50 micromol/L Ba2+ (in GP heart cells) or 1 mM Ba2+ (in HEK cells). The horizontal distance between plots was called the "frequency shift in a single cell" and analyzed. The frequency shift in a single cell was -14.14 +/- 5.71 Hz (n = 14) at 10 microM Ba2+ and -18.51 +/- 4.00 Hz (n = 10) at 50 microM Ba2+, p < 0.05. The values perfectly matched the Ba2+-induced reduction of VF frequency observed previously in GP heart. A similar relationship was found in the computer simulations. The phase of Ba2+-sensitive admittance in GP cells was -2.65 +/- 0.32 rad at 10 Hz and -2.79 +/- 0.26 rad at 30 Hz. In HEK cells, the phase of Ba2+-sensitive admittance was 3.09 +/- 0.03 rad at 10 Hz and 3.00 +/- 0.17 rad at 30 Hz. We have developed a biological single-cell model of rotation-frequency control. The results show that although rotation frequency changes as a result of I(K1) blockade, the phase difference between transmembrane current and transmembrane voltage remains constant, enabling us to quantitatively predict the change of VF frequency resulting from I(K1) blockade, based on single-cell measurement.

Adenoviral expression of IKs contributes to wavebreak and fibrillatory conduction in neonatal rat ventricular cardiomyocyte monolayers.

Previous studies have shown that the gating kinetics of the slow component of the delayed rectifier K(+) current (I(Ks)) contribute to postrepolarization refractoriness in isolated cardiomyocytes. However, the impact of such kinetics on arrhythmogenesis remains unknown. We surmised that expression of I(Ks) in rat cardiomyocyte monolayers contributes to wavebreak formation and facilitates fibrillatory conduction by promoting postrepolarization refractoriness. Optical mapping was performed in 44 rat ventricular myocyte monolayers infected with an adenovirus carrying the genomic sequences of KvLQT1 and minK (molecular correlates of I(Ks)) and 41 littermate controls infected with a GFP adenovirus. Repetitive bipolar stimulation was applied at increasing frequencies, starting at 1 Hz until loss of 1:1 capture or initiation of reentry. Action potential duration (APD) was significantly shorter in I(Ks)-infected monolayers than in controls at 1 to 3 Hz (P<0.05), whereas differences at higher pacing frequencies did not reach statistical significance. Stable rotors occurred in both groups, with significantly higher rotation frequencies, lower conduction velocities, and shorter action potentials in the I(Ks) group. Wavelengths in the latter were significantly shorter than in controls at all rotation frequencies. Wavebreaks leading to fibrillatory conduction occurred in 45% of the I(Ks) reentry episodes but in none of the controls. Moreover, the density of wavebreaks increased with time as long as a stable source sustained the fibrillatory activity. These results provide the first demonstration that I(Ks)-mediated postrepolarization refractoriness can promote wavebreak formation and fibrillatory conduction during pacing and sustained reentry and may have important implications in tachyarrhythmias.

Mechanisms of fractionated electrograms formation in the posterior left atrium during paroxysmal atrial fibrillation in humans.

OBJECTIVES: The aim of this paper was to study mechanisms of formation of fractionated electrograms on the posterior left atrial wall (PLAW) in human paroxysmal atrial fibrillation (AF). BACKGROUND: The mechanisms responsible for complex fractionated atrial electrogram formation during AF are poorly understood. METHODS: In 24 patients, we induced sustained AF by pacing from a pulmonary vein. We analyzed transitions between organized patterns and changes in electrogram morphology leading to fractionation in relation to interbeat interval duration (systolic interval [SI]) and dominant frequency. Computer simulations of rotors helped in the interpretation of the results. RESULTS: Organized patterns were recorded 31 ± 18% of the time. In 47% of organized patterns, the electrograms and PLAW activation sequence were similar to those of incoming waves during pulmonary vein stimulation that induced AF. Transitions to fractionation were preceded by significant increases in electrogram duration, spike number, and SI shortening (R(2) = 0.94). Similarly, adenosine infusion during organized patterns caused significant SI shortening leading to fractionated electrograms formation. Activation maps during organization showed incoming wave patterns, with earliest activation located closest to the highest dominant frequency site. Activation maps during transitions to fragmentation showed areas of slowed conduction and unidirectional block. Simulations predicted that SI abbreviation that heralds fractionated electrograms formation might result from a Doppler effect on wave fronts preceding an approaching rotor or by acceleration of a stationary or meandering, remotely located source. CONCLUSIONS: During induced AF, SI shortening after either drift or acceleration of a source results in intermittent fibrillatory conduction and formation of fractionated electrograms at the PLAW.

Complex fractionated atrial electrograms: properties of time-domain versus frequency-domain methods.

BACKGROUND: Complex fractionated atrial electrograms (CFAEs) are thought to identify high-frequency electrical sources and have become an important target for radiofrequency ablation of atrial fibrillation (AF). Methods used to identify CFAEs and locate suitable ablation sites usually depend on subjective analysis of the electrograms but may also involve objective, computer-based paradigms through either time- or frequency-domain approaches. METHODS: We generated a set of simulated test electrograms, which were defined by a combination of a basic cycle length, phase-resetting noise, and phase-preserving noise, accounting for far-field effects. The simulated electrograms were analyzed separately by well-known basic time-domain (complex fractionated electrogram [CFE]) and frequency-domain (dominant frequency [DF]) methods, and the results were compared with each other to determine objectively the potential reliability of either method to accurately estimate the cycle length of the atrial electrogram. RESULTS: The behavior of the time-domain method depends on the assumed amplitude-sensitivity threshold and can be tuned to its optimal performance but only for signals having stable (and known a priori) amplitude. When the signal amplitude varies randomly (with +/-20% standard deviation range), the time-domain method loses performance. By contrast, the performance of the frequency-domain method remains stable. CONCLUSION: Despite potentially good performance of time-domain methods to estimate the cycle length during AF and localize ablation sites, their performance is easily prone to degradation. The frequency-domain method seems to be much more robust.