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Posts Tagged ‘Polymerase chain reaction’


Deciphering Mode of Action of Functionally Important Regions

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase)

Intrinsically disordered proteins (IDPs) play a major role in various cellular functions ranging from transcription to cell migration. Mutations/modifications in such IDPs are shown to be associated with various diseases. Current strategies to study the mode of action and regulatory mechanisms of disordered proteins at the structural level are time consuming and challenging. Therefore, using simple and swift strategies for identifying functionally important regions in unstructured segments and understanding their underlying mechanisms is critical for many applications. Here we propose a simple strategy that employs dissection of human paxillin (residues 1–313) that comprises intrinsically disordered regions, followed by its interaction study using FAT (Focal adhesion targeting domain of focal adhesion kinase) as its binding partner to retrace structural behavior. Our findings show that the paxillin interaction with FAT exhibits a masking and unmasking effect by a putative intra-molecular regulatory region. This phenomenon suggests how cancer associated mutations in paxillin affect its interactions with Focal Adhesion Kinase (FAK). The strategy could be used to decipher the mode of regulations and identify functionally relevant constructs for other studies.
Neerathilingam M, Bairy SG, Mysore S (2016) Deciphering Mode of Action of Functionally Important Regions in the Intrinsically Disordered Paxillin (Residues 1-313) Using Its Interaction with FAT (Focal Adhesion Targeting Domain of Focal Adhesion Kinase). PLoS ONE 11(2): e0150153. doi:10.1371/journal.pone.0150153

Genomic data suggests that a large proportion of eukaryotic proteins appear to adopt disordered structures in physiological conditions [1, 2]. Mutations/modifications in such IDPs are shown to be associated with various diseases (like cancer) [3]; therefore, understanding their structural behavior is critical for various applications like drug-targeting, mapping protein interactions, deciphering mode of action and finding functional relevance. However, deciphering mode of action in IDPs has been challenging given that unstructured segments render poor chemical shift dispersions and electron density in major techniques like NMR and X-ray, respectively [4]. For example, it took almost 10 years to decipher the mode of action of Sic1, a disordered protein involved in inhibition of a cyclin-dependent kinase [5]. One way to map and study the functional regions is to make truncated constructs by dissecting the whole construct rationally. A limited number of dissection constructs are usually generated; this is due to the time-consuming and challenging process of generating soluble and functionally relevant constructs when studies are performed in-vivo and constructs are prepared and tested sequentially. Here we present a simple high throughput (HTP) screening strategy (Fig 1a), which focuses on finding functionally relevant regions in IDPs based upon its interaction with a binding partner. Close to thirty dissection constructs of the IDP were generated and studied in parallel to understand the importance and functionality of the various regions of the protein. We perform cell-free expression followed by solubility check and GST pull-down interaction study in HTP format. Though both cell-free expression and GST pull-down assay have been individually performed in HTP format [6, 7], we did not find previous studies that combine the two methods in HTP format. Although the nature of interaction of IDPs with respective binding partners may vary, our strategy may be used to derive crucial insights into “structural behavior” of the unstructured segments in modulating the interaction. The strategy can also be used to identify functionally important regions in the IDP that would be suitable for further structural studies.

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Fig 1. Dissection of paxillin constructs (residues 1–313) followed by expression and interaction studies.

(a) Timeline for overall-strategy. (b) Illustration of solubility and activity level of linear dissected human paxillin (residues 1–313). (c) Phosphor screen image of filter assay for optimization of temperature for paxillin constructs (left). Tabular representation of paxillin constructs, negative and positive controls corresponding to each well in filter assay [1].(d) Phosphor screen image of 10% SDS PAGE of 35S labeled cell-free expressed samples after GST pull-down assay of the paxillin constructs A1–E1; The right panel shows fraction of interaction of each construct with respect to B2 (since B2 showed maximum level of interaction) (e) Illustration of solubility and activity of dissected C3 constructs. All experiments were performed in triplicates and averaged. To rule out non-specific interactions that might occur with GST tagged FAT, GFP that was expressed in cell-free system and a reaction without DNA template were used as negative controls.    http://dx.doi.org:/10.1371/journal.pone.0150153.g001

Disorder/Intrinsic disorder seems to be a common feature of hub proteins in eukaryotes [2], thus highlighting the need for studying the mode of action of unstructured segments in such proteins. Here we used paxillin (residues 1–313), an intrinsically disordered construct, for demonstrating this approach. Paxillin (residues 1–313) consists of multiple protein interaction sites that are connected by flexible disordered sequences [8]. The disordered regions in paxillin have been detrimental in efforts to study the complete structure of the protein due to the demerits mentioned previously. This explains the lack of structural details of regulation of paxillin binding. Residues 1–313 of paxillin consist of five leucine-rich sequences LD1-LD5 (with consensus sequence: LDXLLXXL), termed LD motifs, which are highly conserved between species and other family members such as Hic-5, leupaxin and PAXB [8]. Paxillin interacts with multiple proteins involved in cell migration, actin rearrangements and cell proliferation [9]. Mutations in paxillin are shown to be associated with lung cancer [3, 10]; and the differential expression of paxillin is associated with various forms of cancer and other diseases such as Alzheimer’s and inflammation [1113]. This implies the importance of studying the structural and functional characteristics of paxillin. Most paxillin studies focus on interactions of LD motifs with proteins such as focal adhesion kinase (FAK), vinculin and v-crk, providing clues towards their importance in deciphering the functionality of paxillin [8, 14, 15]. Though regions of paxillin that bind to various partners were deciphered through previous studies, the basis of effect of mutations in paxillin on binding its partners was not explained. Mutations in paxillin, some that were observed to be associated with cancer were positioned in the intrinsically disordered regions between the LD motifs and not on the motifs themselves [3, 10]. For example, P30S, G105A and A127T mutations lie between LD1 and LD2 motif; P233L and T255I mutations lie between LD3 and LD4 motifs. This shows that the LD motifs alone do not govern the functionality, but unstructured regions linking the LD motifs could play a major role. In normal conditions, FAT (Focal adhesion targeting domain of FAK) binds hydrophobically through its HP1 (Hydrophobic patch 1) and HP2 (Hydrophobic patch 2) sites to paxillin LD motifs—LD2 and LD4 [16, 17], which lead to activation of binding sites for other proteins on paxillin. LD2 preferentially binds to the HP2 site, whereas LD4 preferentially binds to the HP1 site [18]. In a state of cancer caused by mutations in paxillin, the LD interactions could be hindered, as mutations in the unstructured segments result in abnormal binding of FAK to either of the LD motifs [9]. Here we wanted to locate the region involved in the structural modulation of paxillin-FAT interaction by adopting a simple approach (Fig 1) that involves dissected proteins generated using cell-free protein expression coupled with protein-protein interaction study. We map the disordered proteins’ structural importance to understand the function and modulation of paxillin-FAT interaction in days rather than months (Fig 1a).

Dissection and identification of fragments of paxillin (residues 1–313) with functional relevance

We dissected paxillin (residues 1–313) (Fig 1b) into nested sets using PCR such that each of the constructs had either or both LD2 and LD4 motifs (S1 Fig and S1 Table). Further, these constructs were expressed in soluble form using small-scale cell-free expression system in a 96 well format (Fig 1c). However, all constructs except A6, B6, C4 and C5 expressed detectable amounts of protein (S2a Fig and S2 Table). The failure in expression of the above constructs could be due to the instability of the smaller peptide fragments that might be susceptible to proteolytic cleavage [19]. Soluble protein from small-scale expression of the dissected constructs namely A1, A2, A3, A4, A5, B1, B2, B3, B4, B5, C1, C2, C3, D1, D2, and E1 were pulled down and analysed (Fig 1d). Although constructs A1–A5, B1–B5, C1, C2, D1 and E1 interacted successfully, C3 (containing LD2) and D2 (containing LD4) failed to interact (Fig 2c) despite containing LD motifs. However, based on previous reports [8, 16, 17], we expected all constructs containing either LD2 and/or LD4 to interact with the FAT domain. Therefore, this led us to suspect that intra-molecular auto-inhibition in unstructured segments modulated binding of FAT to LD motifs in paxillin.

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Fig 2. Regulatory and masking regions around paxillin’s LD2 and LD4 and their circular dichroism spectra.

(a) CD spectra of paxillin LD peptides (LD1-LD5) and constructs: B2, C3, C35 and D2. CD spectra of LD2, LD4, C35 and D2 constructs showed negative bands at 222nm and 206nm and a positive band at 192nm that confirms the presence of alpha helical content thus may behave as folded effector binding sites. However, LD1, LD3, LD5, B2 and C3 do not show the characteristic peaks of secondary structures, thus may behave as unfolded effector binding sites. (b) LD2 regulatory region (54–130) and masking region (167–224) evidenced by constructs B3, B4 and B5. (c) LD4 regulatory region (216–257) and masking region (280–313) evidenced by constructs D1, D2 and E1.
http://dx.doi.org:/10.1371/journal.pone.0150153.g002

Identification of regulatory regions and their mechanisms

To investigate the non-interaction of C3, a series of C3 deleted constructs (C31 –C310) (Fig 1e,S1 Table) were generated to determine the internal region that influenced the non-functioning of C3. C36 linear template could not be amplified for expression. As solubility of C3 could play a critical role in determining interaction, the homogeneity of the sample was confirmed by capillary electrophoresis under non-reducing conditions [20] (See S3 Fig). The linear templates—C31, C32, C33, C34 and C35 were successfully expressed in soluble form, The other C3 deleted constructs did not express due to issues related to small size as described earlier. Surprisingly, none of the C3 deleted constructs interacted with FAT despite the presence of the LD2 motif, although constructs such as B3, B4 and B5 that contain regions overlapping with C3 showed interaction (S2b and S2c Fig, Fig 1d and 1e). Here B3 that included the whole of C3 and unstructured segment 54–130 showed interaction (Fig 1b). Constructs B4 and B5 also containing residues 54–130 showed interaction despite differing from B3 by lacking regions 167–224 and 155–224, respectively. Interestingly, the non-interacting constructs C3 and C35 do not contain 54–130 residues, but include the regions 167–224 and 167–189, respectively (Fig 1b). Here constructs containing region 167–189 but lacking 54–130 did not interact with FAT despite LD motif alone showing interaction (switch off) (Fig 3a). Whereas, if 54–130 was included, interaction was reinstated (switch on) (Fig 3a). This clearly shows that interaction of LD2 in construct C35 is masked by residues 167–189 (masking region) (Fig 2b). The constructs B3 and B4 binding to FAT despite the presence of the masking region led us to conclude that the region 54–130 (regulatory region) acts to remove the masking effect (Fig 2b).
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Fig 3. Binding studies of paxillin constructs using Bio-layer Interferometry on OctetRed96.

(a) Switch off in C3 and D2 on LD2 and LD4 respectively; Hypothesis of partial switch on when regulatory region of LD2 is absent, as evidenced in C2. (b) Concentration calibration curves depicting binding of constructs B2, C35, ‘54–189’, ‘79–189’, ‘105–189’ with GST-FAT. The data is representative of a single experiment. Each experiment was performed at-least thrice. (c) Illustrations of C35, C35_1, C35_2 and C35_3.  http://dx.doi.org:/10.1371/journal.pone.0150153.g003

Similar to LD2, LD4 in construct D2 containing 216–257 (masking region) requires additional residues of paxillin 280–313 (regulatory region) for FAT binding (Fig 2c), which was demonstrated by showing the interaction with constructs D1 (spanning region 216–313) (Fig 1b) and E1 (spanning region 258–313). To visualize the non-binding of FAT to C35, in-silico methods were employed to model the C35 construct and docked with the crystal structure of FAT (1K05, residues 916–1050 [21]) (Fig 4). The docking results showed a clear masking effect in the C35 construct by the 167–189 (masking region) residues. The constructs B2, C3 and C35 were also structurally characterized using CD analysis (Large scale cell-free expression was performed for this purpose, see S3 Fig). The percentage of alpha helical content was found to be much higher in C35 (95.32%) as compared to B2 (12.43%) (Fig 2a, S3 Table). Therefore, the dissection(s) of B2 to C35 allowed the identification of structured regions (C35) as compared to the disordered B2. Further, it showed that the LD2 peptide and C35 have significant alpha-helical structures that do not translate into functional similarity as evidenced by the inability of C3, C35 and D2 to bind to FAT. Moreover, LD2 peptide binds to FAT while C35 does not (Fig 1e and S2b and S2c Fig). A similar observation was made when comparing the ability of LD4 peptide and the inability of D2 to bind to FAT despite both having detectable α-helical content (Fig 1b). Thus, these results confirm the existence of masking and regulatory regions (Fig 2b and 2c) that determine switch on and off and in turn, intra-molecular auto-inhibition. C2 showed activity despite missing regulatory regions for both LD2 and LD4 (similar activity observed in C1). This could be because the unfolded nature of LD3 effector binding site that is located between LD2 and LD4 is flexible to mask only a single LD motif but not both (Partial switch on, Fig 3a).

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Fig 4. In-silico analysis of non-binding of C35.

(a) LD2 crystal structure from PDB id: 1K05 (left) being compared with the LD2 structure in the side view and top view of C35 structure showing the masking of the hydrophobic binding region predicted through HMM based SAM-T08 software. The LD2 binding region and the masking regions are depicted by the bracketed region. (b) Docking control showing FAT (co-ordinates from PDB id: 1K05) and LD2 (co-ordinates from PDB id: 2L6F, NMR model # 1) interaction using Hex 6.3 software. (c) Docking of C35 with FAT showing non-interaction due to masking effect. The sidechains of the active residues are shown as red sticks. The hydrophobic patch—HP2 in FAT molecule, which preferentially binds to LD2 is shown as a space filling model in orange (part of helix 1 of FAT) and grey (part of helix 4 of FAT) colors.   http://dx.doi.org:/10.1371/journal.pone.0150153.g004

To predict the influence of this structural modulation, the state of LD motifs structurally before and after binding to FAT had to be understood. CD spectra of LD1, LD3 and LD5 peptides showed characteristics of random coil (Fig 2a, S3 Table) thus validating that the LD1, LD3 and LD5 motifs could exist as unfolded effector binding sites (not available for interaction) in our study and could fold upon undergoing allosteric changes after binding to their respective targets.

Validation of protein-protein interaction study using bio-layer interferometry studies

Bio-layer interferometry studies were performed to further validate the interaction studies and also to get insights into the binding affinities. Here apart from constructs B2 and C35, three other constructs that include different lengths of the regulatory region along with the C35 region were used for the studies, namely—Construct C35_1(54–189); Construct C35_2 (79–189) and Construct C35_3 (105–189) (See Fig 3b and S4 Fig). As seen in Fig 3c and Table 1, B2 shows maximum binding with KD value in the nano-molar range and the curves fit into a 1:1 binding model. C35 shows negligible binding and the rest of the constructs show binding lower than B2 with KD values in micro-molar range and the curves fit into a 2:1 binding model (See S5 Fig).

According to previous reports, FAK has to bind to both LD2 and LD4, failing which phosphorylation during signalling is reduced [8], which is observed in case of cancer [3], thus resulting in abnormal functioning of paxillin. We investigated this by analysing B2, which showed higher interaction than B1, despite missing the regulatory region of LD4 (Fig 1b). Similarly, C2 showed activity despite missing regulatory regions for both LD2 and LD4 and the presence of masking regions (similar activity observed in C1). This suggests that the masking region that is located between LD2 and LD4 is flexible to mask only a single LD motif but not both (Fig 3a). Interestingly, paxillin mutations associated with lung cancer were observed in the unstructured segments, particularly the regulatory region of LD2 and masking region of LD4 [3]. We hypothesize that these mutations prevent proper functioning of the regulatory regions, thus resulting in masking of either of the LD motifs causing abnormal functioning of paxillin. Evidence that these regions regulate FAT-paxillin binding was further provided in our study in the form of the bio-layer interferometry results; where C35 did not show any binding, but the constructs that included different lengths of the regulatory region along with the C35 region showed binding with KD values in the micro-molar range. This suggests that the LD2 region in these constructs is not masked, since it is seen in previous studies that the KD value for FAT binding to a single LD motif of paxillin is in micro-molar range. It also suggests that the region between residues 105–131 is sufficient for preventing the masking of LD2 region, thus allowing interaction with FAT (See illustrations in Fig 3b). Except B2 (that had a 1:1 binding stoichiometry and higher binding affinity), all other constructs (C35_1, C35_2, C35_3) showed a 2:1 binding stoichiometry. This suggests that both LD motifs of B2 engage both the FAT HP sites thus resulting in higher affinity; whereas in the other 3 constructs (C35_1, C35_2, C35_3), each FAT HP site (HP1 and HP2) interacts with individual molecules thus giving a 2:1 stoichiometry. This is in agreement with previous studies where both the LD motifs were found to interact with both HP1 and HP2 hydrophobic patches of FAT [16]. The higher affinity of B2 to FAT could be due to presence of both LD2 and LD4; the proposed intra-molecular regulatory regions could also play a role in the increased affinity. Therefore, we understand that the abnormal modulation in cancer involves redirection of FAK to a single LD motif; and targeting drugs for re-establishing the function at regulatory regions could be critical.

Unlike many existing techniques like array based yeast two hybrid assay, phage display method and tandem affinity purification; the strategy used here (combination of cell-free expression, filter based solubility assay and interaction study in HTP format) facilitated quick identification of the role of unstructured regions involved in paxillin-FAT interaction in HTP format. Particularly, in paxillin-FAK interactions, which determine focal adhesion and cellular signalling, we understood the structural masking and unmasking behaviour of unstructured segments in paxillin to determine FAK interaction. The structure of paxillin is not yet elucidated due to difficulties with respect to its disordered nature. In this study, the templates that we generated using the high throughput dissection strategy allowed us to analyze various regions of paxillin, with respect to structure, solubility and function. To our knowledge, this study is the first report of switch on and off mechanisms working together in controlling allosteric modulation/auto-inhibition in a human hub protein. As many eukaryotic proteins are disordered, our study opens avenues for analyzing novel modulations at allosteric sites using appropriate interaction studies, which could lead to identification of new drug target sites. In this regard, we hope the above strategy will be instrumental in understanding mechanisms of other disordered proteins as well, in days rather than months. This strategy could also be used as an initial screening method for techniques like SAXS, smFRET and others.

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Genomic Pathogen Typing

Larry H. Bernstein, MD, FCAP, Curator

LPBI

 

Genomic Pathogen Typing Using Solid-State Nanopores

Citation: Squires AH, Atas E, Meller A (2015) Genomic Pathogen Typing Using Solid-State Nanopores. PLoS ONE 10(11): e0142944.   http://dx.doi.org:/10.1371/journal.pone.0142944

Editor: Niyaz Ahmed, University of Hyderabad, INDIA

In clinical settings, rapid and accurate characterization of pathogens is essential for effective treatment of patients; however, subtle genetic changes in pathogens which elude traditional phenotypic typing may confer dangerous pathogenic properties such as toxicity, antibiotic resistance, or virulence. Existing options for molecular typing techniques characterize the critical genomic changes that distinguish harmful and benign strains, yet the well-established approaches, in particular those that rely on electrophoretic separation of nucleic acid fragments on a gel, have room for only incremental future improvements in speed, cost, and complexity. Solid-state nanopores are an emerging class of single-molecule sensors that can electrophoretically characterize charged biopolymers, and which offer significant advantages in terms of sample and reagent requirements, readout speed, parallelization, and automation. We present here the first application of nanopores for single-molecule molecular typing using length based “fingerprints” of critical sites in bacterial genomes. This technique is highly adaptable for detection of different types of genetic variation; as we illustrate using prototypical examples including Mycobacterium tuberculosis and methicillin-resistant Streptococcus aureus, the solid-state nanopore diagnostic platform may be used to detect large insertions or deletions, small insertions or deletions, and even single-nucleotide variations in bacterial DNA. We further show that Bayesian classification of test samples can provide highly confident pathogen typing results based on only a few tens of independent single-molecule events, making this method extremely sensitive and statistically robust.

 

Subtle genetic changes in bacteria can produce large variations in factors affecting pathogenicity, such as toxicity, antibiotic resistance, and virulence. These genetic variations are not only used to trace the epidemic and phylogenetic relationships among strains of bacteria, but are also critically important in clinical settings for proper patient diagnosis and treatment. Most existing approaches require sample incubation and growth over the course of multiple days prior to testing, and nearly all require expert handling of samples and interpretation of results. Traditional phenotypic typing techniques such as serotypes, biotypes, phage-types, and antibiograms lack the necessary sensitivity to distinguish between closely related pathogen strains, and therefore fail to adequately capture these critical variations for clinical applications. Gel-based techniques such as restriction fragment length polymorphism (RFLP) or cleaved amplified polymorphic sequences (CAPS) require a large amount of time and results are not easily compared or transferred among labs. Next-generation sequencing is an increasingly popular method of fully characterizing bacterial strains [1] and may be used for typing strains according to the sequences of a panel of housekeeping genes, as in multi-locus sequence typing (MLST) [2], but this approach is more commonly used to trace post hoc epidemic and phylogenetic relationships among clinical isolates. Furthermore, the complexity and quantity of sequencing data far exceeds the minimum information required to efficiently and accurately diagnose a patient. For example, bioinformatics studies suggest that a panel of just 30–50 single nucleotide variations (SNVs) could be used to uniquely identify thousands of strains of Mycobacterium tuberculosis [3, 4]. Yet SNVs are not the only source of variation among pathogens; polymorphisms from SNVs and short indels up to genetic changes as large as whole plasmids or sets of genes may be responsible for critical changes to pathogenicity. Thus there exists a clear clinical need for a novel approach to molecular typing that can quickly and simply screen patient samples for a panel of widely varying known genetic polymorphisms of dangerous pathogens.

Solid-state nanopores may be used to discriminate the lengths of unlabeled individual biopolymers such as DNA molecules across a wide range of lengths [5, 6]. Biopolymers are electrophoretically attracted and threaded through a voltage-biased nanoscale pore drilled in an ultrathin freestanding SiNx membrane [7, 8]. When a DNA molecule is threaded through a nanopore, it partially blocks the flow of ions moving through the pore, allowing real-time detection of the analyte by monitoring changes in the ion current. Nanopore sensing is biochemically simple, as it does not require labeling of the analyte with radioactive or fluorescent probes, yet it can be used to detect minute quantities of nucleic acid molecules, surpassing the sensitivity of bulk methods [8]. Moreover, nanopore sensing involves relatively simple instrumentation (primarily a current amplifier) and may be used to analyze thousands of molecules in just a few minutes, making this technique an ideal candidate for applications such as nucleic acid based diagnostics.

Here we describe and practice a novel detection scheme (Fig 1) for molecular typing of pathogens using solid-state nanopores, and demonstrate its ability to discriminate a wide range of critical genetic polymorphisms in closely related organisms with starkly different pathogenicities. In the first sensing mode of our approach (Mode I), large insertions or deletions are detected by directly classifying the length of DNA in the nanopore. In the second sensing mode (Mode II), small indels down to SNVs may be detected by sequence-specific digestion at the site of the polymorphism to produce either one or two DNA fragments, which are then detected in the nanopore. We first characterize the practical range of our nanopore system for detecting variation in DNA length, and show that fragment length differences are more readily apparent for shorter DNA lengths and for asymmetric cut sites. We then demonstrate that statistical analysis tools such as Bayesian classifiers, commonly used for automated classification, are highly effective for rapid and statistically robust discrimination among different lengths and combinations of DNA fragments translocating through a nanopore, even in cases where significant portions of these distributions overlap. We apply these techniques to demonstrate polymorphism discrimination down to the single nucleotide level in prototypical strains of Mycobacterium tuberculosis (virulent vs. avirulent) and Streptococcus aureus(methicillin-resistant vs. multi-drug resistant). This highly versatile combination of rapid length and digest discrimination, spanning several orders of magnitude of possible genomic variation size, in a single, parallelizable device, could be extended to probe a large panel of critical sites within a genome for point-of-care determination of critical pathogenic properties and sequence typing.

Fig 1.  Two Principal Modes for Nanopore Discrimination of Pathogen Genomic Variation.

http://journals.plos.org/plosone/article/figure/image?size=large&id=info:doi/10.1371/journal.pone.0142944.g001

Fig 1. Two Principal Modes for Nanopore Discrimination of Pathogen Genomic Variation.

Mode I: Direct length detection according to analyte translocation dwell time and depth enables discrimination of longer vs. shorter fragments; i.e: whether or not an insertion or deletion is present (left). Mode II: Prior to translocation, samples are exposed to a restriction enzyme that cuts at the site of a SNV or short indel or mutation. Detection of cleaved vs. uncleaved DNA fragments in the nanopore reveals whether or not the critical genomic variation is present.

http://dx.doi.org:/10.1371/journal.pone.0142944.g001

Detection of DNA Sequence Polymorphisms in Solid-State Nanopores  

The simplest form of nanopore translocation analysis involves the measurement of the depth of each current blockade (ΔIB) and the dwell time of each molecule within the pore (tD). Both parameters have been shown to grow nonlinearly with DNA length, forming the basis for fragment length separation in the nanopore system. The statistical distributions of these independently measured quantities may be used to distinguish between analytes of different lengths, such as DNAs [5, 6, 9], or proteins having identical molecular weight but slightly different charge or 3D structure [1013]. Variation in the translocation dwell-time (tD) in solid-state nanopores measured for different DNA lengths (l), are empirically described by a power law: tDlα where α = 1.38±0.02, which has been reproduced by multiple experimental approaches [5, 9, 14]. Using a log-scale distribution of translocation times to estimate the distribution of tD, note that the difference in log(tD) for two sequences (lengths l0 and l0 + Δl) is more apparent for shorter length l0 as compared with the insertions and deletions Δl (i.e. when Δl/l0 ∼ 1) according to Eq 1:(1)

If the presence of two fragment lengths must be identified from within a single sample, it is desirable that their distributions of ΔIB or tD should be as well-separated as possible. Furthermore, if the presence of a cut sample must be distinguished from an uncut sample, then by Eq 1 the peak produced by the shorter part of a cut sample will appear farther away from the uncut peak than the longer part of a cut sample. To statistically distinguish the samples, it is desirable for the peak of the shorter part to be as dissimilar as possible from the uncut peak. Therefore, asymmetrically cut DNA pieces from a restriction digest are more readily distinguished from the original uncut length than those produced by symmetrically positioned restriction sites, provided that the shorter piece is of sufficient length to be detected by the nanopore. In cases where separation between two similar length biopolymers (Δl/l0 ∼ 1) is required, the measured histograms of either ΔIB or tD may overlap significantly, making discrimination between these molecules difficult. Combinations of multiple fragment lengths within a sample pose additional challenges, as their more complicated distributions may overlap or otherwise preclude simple contour cluster separation.

In the context of sequence typing, identification of fragments by sizing will indicate the presence of specific insertions and deletions that may enhance or reduce pathogenicity or otherwise uniquely identify a pathogenic strain. Upper bounds on Δl are set by: 1) sample preparation parameters and limitations; for example, robust and fast PCR amplification is most easily achieved for fragment lengths of ~102–103 bp [15] and 2) nanopore stability considerations; for example, nanopores are more frequently clogged by very long DNA (>20 kbp). Lower bounds on l0 are set by nanopore sensitivity; while several groups have demonstrated detection of small DNA fragments (<50 bp) [16] we find that a minimum l0 on the order of ~100 bp is more reliable since it is readily detectable in small nanopores with no additional modifications [5], producing an extremely small fraction of missed events due to the finite system bandwidth. Thus a reasonable design range for sequence typing fragments is ~100 bp minimum length forl0, ranging up to a few thousand base pairs maximum length for l0 + Δl. Many types of common genetic variations used for strain typing fall within this size range. For example, one complete IS6110 (insertion-like sequence element) insertion in M. tuberculosis is 1358 bp [17]. At the other end of this range, multi-drug resistant strains of methicillin-resistant S. aureus (MRSA) have many insertions and deletions in the range 47 bp—643 bp that affect their pathogenicity [18]. To detect the smallest indels, which fall below the minimum detectable Δl, we turn to the exquisite sequence specificity of digestion by restriction enzymes, which can identify sequence polymorphisms down to a single nucleotide variation.

Using these design principles, we present here two alternative modes of detection that illustrate the wide range of genomic variations that may be detected using a single sensor. For large insertions or deletions (Fig 1: Mode I, left panel), a nanopore may be used to discriminate the raw change in DNA length caused by the presence or absence of this sequence according to the duration of translocation events. For short indels, mutations, or single nucleotide variations (SNVs) (Fig 1: Mode II, right panel), which are more difficult to identify solely by length as discussed above, we utilize a restriction enzyme. The sample is only cut in the presence (or absence) of the critical sequence, and subsequent detection in a nanopore reveals either one or two fragments in the nanopore according to the observed durations and blockage levels of translocation events.

Event Diagram Discrimination of Sample Length and Composition

We first experimentally illustrate the practical length resolution of the nanopore platform for identifying sample length and composition. We analyzed samples containing mixtures of DNA fragments composed of one or two well-defined lengths. The resulting event diagrams create unique fingerprints that can be used to distinguish different lengths of DNA (Mode I) or whether or not a fragment of DNA has been cut (Mode II). Fig 2A–2E show event diagrams for 100 bp, 200 bp, 900 bp, 1000 bp, and 100+900 bp DNA in a single nanopore (diameter 4.8 nm, effective height 7 nm) at +300 mV bias (for additional examples, see Figs B-E in S1 File). Here, each translocation event is represented by its corresponding ion current event amplitude (ΔIB) and dwell time (tD). From comparison of Fig 2A and 2D, it is evident that insertions and deletions Δl several times larger than the base length (here: Δl:l0 = 9:1) are indeed easily distinguishable (Fig C in S1 File). Comparison of Fig 2A and 2B illustrates that Δl = 100 bp results in reasonably distinct event diagrams for l0 = 100 bp, which may be distinguished to >95% confidence with just a few events each, taking both dwell time and current amplitude into consideration (Fig D in S1 File). However, at l0 = 900 bp a minimum of several hundred events are required to confidently (>95%) differentiate l0 (Fig 2C) from l0 + Δl (1000 bp, Fig 2D), since their event diagrams overlap significantly (Fig E in S1 File). Returning to Eq 1, for Δl = 100 bp, we expect Δlog(tD) = 0.415 for l0 = 100 bp, and Δlog(tD) = 0.063 for l0 = 900 bp. For the data shown in Fig 2F, Δlog(tD) = 0.1 for l0 = 100 bp, and Δlog(tD) = 0.03 for l0 = 900 bp. The inability to easily and quickly discriminate the 900 bp DNA from the 1000 bp DNA demonstrates the practical limits set on Mode I sample identification according to the size of the insertion or deletion that must be detected.

Fig 2.  Translocation Event Diagrams Uniquely Identify DNA Fragment Lengths in a Single Nanopore.

http://journals.plos.org/plosone/article/figure/image?size=large&id=info:doi/10.1371/journal.pone.0142944.g002

Fig 2. Translocation Event Diagrams Uniquely Identify DNA Fragment Lengths in a Single Nanopore.

(a) 100 bp at 1 nM. (b) 200 bp at 1 nM. (c) 900 bp at 1 nM. (d) 1000 bp at 1 nM. (e) 1:1 combination of 100 bp and 900 bp, total concentration 2 nM. (f) Semilog(x) distributions of translocation dwell times for all samples (a)-(e). Translocations for all samples were collected in a single nanopore (4.8 nm diameter, effective thickness ~7 nm) with a +300 mV bias relative to trans (open pore current: 13 nA). To facilitate visualization of population density, a random white noise offset below the acquisition rate of this data (-2 μs < Δt < +2 μs, acquisition rate 250 kHz) has been added to each tD.    http://dx.doi.org:/10.1371/journal.pone.0142944.g002

Fig 2E illustrates how Mode II may overcome these limitations by digesting DNA into fragments: here, a highly asymmetric ratio of lengths in a mixed sample (100+900 bp) clearly facilitates sample identification as compared to the full length 1000 bp DNA (Fig 2D). However, Mode II also presents a more challenging case for quantitative discrimination between an uncut and a cut sample. Whereas single-length samples can be identified using either their tD or Idistribution (as shown in Fig 2F), the longer fragment in a cut sample may share significant overlap with the uncut sample. This is particularly true in the case of a highly asymmetric cut site.

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http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0142944.g003

Fig 3. Gaussian Mixture Models for Mode II Classification of 1000 bp vs. 900+100 bp DNA Fragments.

(a) 2-D GMM for 1000 bp DNA fragment translocations. (b) 2-D GMM for 900+100 bp DNA fragment translocations. (c) Bayesian posterior estimates p(A|Θ) of correctly identifying a data set Θ as Case A, calculated for each increment of N points in Θ, repeated 1000 times (first 50 shown in gray) and averaged (blue), each using M = 1500 points in the model data set. (d) Bayesian posterior estimates p(B|Θ) of correctly identifying a data set Θ as Case B, calculated for each increment of N points in Θ, repeated 1000 times (first 50 shown in gray) and averaged (red), all using M = 1500 points in the model data set. (e) Bayesian posterior estimates p(A|Θ) for test data sets ofN points given a model based on data set size M. Each point represents the average of 1000 separate bootstrap simulations. (f) Bayesian posterior estimates p(A|Θ) for test data sets of N points given a model based on data set size M. Each point represents the average of 1000 separate bootstrap simulations. Insets: range of N for which p(A|Θ) reaches 0.95. See Methods and S1 File for complete numerical simulation details.    http://dx.doi.org:/10.1371/journal.pone.0142944.g003

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http://journals.plos.org/plosone/article/figure/image?size=inline&id=info:doi/10.1371/journal.pone.0142944.g004

Fig 4. Gaussian Mixture Models of DNA Fragments for Actual Mode II Pathogen Typing at the SNV Level.

(a) Diagram of the main steps in sample preparation, detection, and classification: PCR fragments from isolated pathogens are subjected to a restriction digest, which recognizes and cuts only one genomic variant. Nanopore translocations are used to classify the pathogen according to the combination of fragment lengths detected. (b) ThemazG gene of the avirulent M. tuberculosis strain H37Ra is not cut by NaeI (942 bp), while the same gene in the closely related virulent strain H37Rv, which differs by only a single A-to-C mutation, is cut by NaeI (621bp + 321 bp). (c) Gaussian mixture model (one component) fit to translocations of mazG fragments from H37Ra. (d) Gaussian mixture model (two components) fit to translocations of mazG fragments from H37Rv. (e) Posterior probabilities for correctly identifying the H37Ra and H37Rv strains as a function of number of translocation events collected from an unknown sample, simulated using bootstrap sampling from nanopore translocation data. (f) The parC gene of the multi-drug-resistant MRSA strain FPR3757 is not cut by BseRI (886 bp) due to a single C-to-A mutation, while the closely related and less resistant strain HOU-MR is cut by BseRI (640bp + 245 bp). (g) Gaussian mixture model (one component) fit to translocations of parC fragments from FPR3757. (h) Gaussian mixture model (two components) fit to translocations of parC fragments from HOU-MR. (i) Posterior probabilities for correctly identifying the FPR3757 and HOU-MR strains as a function of number of translocation events collected from an unknown sample, simulated using bootstrap sampling from nanopore translocation data.    http://dx.doi.org:/10.1371/journal.pone.0142944.g004

Conclusion

Solid-state nanopore based biosensing is a rapidly growing field due to its practical and conceptual simplicity, portability and versatility. To date, few reports have demonstrated the utility of the method towards clinical diagnostic applications. Yet as we have shown here, nanopores are well-suited to make statistically robust diagnostic classifications among different DNA lengths with real single-molecule data, even in cases where the distributions significantly overlap. Utilizing a Bayesian statistical model, we have demonstrated that nanopore sensing can be used to discriminate among pathogens based on well-known genomic variations. Both large indels (Mode I) or short indels and single nucleotide variations (Mode II) can be targeted using proper sequence-specific digestion with off-the-shelf restriction enzymes. Furthermore, the Bayesian classifiers indicate the statistical confidence of each classification as a function of the number of nanopore events obtained in each measurement. Even at this preliminary stage of development we find that only a few tens of events (obtained in just a few minutes using a single pore) are sufficient to produce a statistically reliable result with well-defined and small error margins.

Our method is general and can be adapted to address many different “multiple-choice” clinical questions using a nanopore biosensor or other single molecule approaches. Future extensions of this work may seek to design and implement large panels of critical sites that represent the minimum sets necessary to characterize genomic variation for various applications in healthcare and research, and to develop additional sensing modalities. Although the primary design challenge currently remains linked to the location and availability of restriction digestion sites, we expect that the ongoing development of designer restriction enzymes, for example systems based on modular zinc fingers [27], TALENs [28], or CRISPR-like proteins will provide additional design flexibility for this technique.

The nanopore fingerprinting approach presented here addresses clear needs in clinical molecular diagnostics for a rapid and simple sensor that can identify a wide range of genomic variation in pathogens to inform treatment options. We have shown here discrimination of both large and small scale genomic variations between pathogen strains, down to single SNVs. The large, flexible sample design space for lengths, cut sites, and enzyme selection at each critical locus ensures that the technique is highly customizable for different genomic variation panels that could profile pathogenicity, antibiotic resistance, or even sequence type. The inherent scalability, minimal sample requirements, speed, and simple readout of the nanopore platform would all facilitate on-site and perhaps even automated use: As successive events are recorded, an increasingly clear fingerprint of translocation times and blockage levels will permit online software to “call” the sample as soon as enough events have been accumulated. Our technique is highly portable and customizable, and the binary data would be readily transferrable among different labs.

 

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Reporter: Aviva Lev-Ari, PhD, RN

 

PCR is an organisation dedicated to education and information in the field of cardiovascular therapies, most notably for cardiolovascular intervention and interventional medicine.
Its activities cover a large spectrum, from the organisation of annual courses in Europe, Asia and the Middle East to editing a scientific journal, publishing textbooks as well as providing training seminars on thematic subjects.

[92] TUESDAY 21 MAY

Abstract & Case Corner
Complex and unusual interventions for structural heart disease 12:30 – 13:30
Congenital disease treatment in children and adults 13:30 – 15:00
Challenges during percutaneous balloon mitral valvuloplasty 15:00 – 16:30
Percutaneous treatment of mitral regurgitation 16:45 – 18:15
Interactive Case Corner
Interactive case corner #1 13:15 – 14:45
Interactive case corner #2 15:00 – 16:30
Interactive case corner #3 16:45 – 18:15
Main arena
Opening 10:00 – 13:00
2013 Great Debate: The burning issues – Bioresorbable scaffolds and dual antiplatelet therapy 
With an unrestricted educational grant from MEDTRONIC
13:00 – 14:30
Presentation of the 2013 Ethica award by Jean Fajadet & William Wijns 14:30 – 15:00
From late breaking trial to clinical practice 15:00 – 16:45
Moderated Poster Area
Moderated posters 1 16:45 – 18:15
PCR Sharing Centre
Understand what you see with the iPad Atlas of OCT – Interactive OCT image interpretation 14:00 – 15:30
Do you want to become comfortable with health economics? Practical example: is TAVI cost effective? 15:40 – 16:40
Peripheral Abstract & Case Corner
Renal artery stenting: what you cannot leave behind 12:30 – 14:00
Subclavian artery angioplasty: rare but real 14:00 – 15:30
In vascular disease, think global! 15:30 – 16:30
Room 241
Embolic stroke and cardiovascular interventions 13:30 – 15:00
RSICA@EuroPCR – Combined structural heart disease interventions 
With the collaboration of the Russian Scientific Society of Interventional Cardioangiology
15:00 – 16:30
Percutanous haemodynamic support in high-risk PCI and cardiogenic shock: your safety net in the cathlab 
With an unrestricted educational grant from ABIOMED
16:45 – 18:15
Room 242AB
How to decide between antegrade versus retrograde recanalisation of coronary chronic total occlusions? 12:30 – 13:30
Techniques for antegrade revascularisation of coronary chronic total occlusion 13:30 – 14:30
Techniques for retrograde coronary chronic total occlusion recanalisation 14:30 – 15:30
Coronary chronic total occlusion: from procedural success to long-term outcome 15:30 – 16:30
Coronary chronic total occlusion: set up your strategy to achieve success while keeping it simple 
With an unrestricted educational grant from ABBOTT VASCULAR
16:45 – 18:15
Room 243
A decade of experience with DES: insights from large registries and randomised clinical trials 12:30 – 14:00
DES: updated evidence from randomised clinical trials 14:00 – 15:00
Coronary perforation and interventional devices 15:00 – 16:30
Coronary dissection: management of rare and common cases 16:45 – 18:15
Room 251
Managing challenges during TAVI 12:30 – 14:00
Current and future technologies in the cathlab 14:00 – 15:30
TAVI update 15:30 – 16:30
Room 252AB
Renal denervation for resistant hypertension: procedural aspects, clinical effects and off-target indications 12:30 – 14:00
Selecting the right patient for catheter-based renal sympathetic denervation: a case-based discussion 14:00 – 15:30
Emerging technologies for transcatheter aortic valve therapies – Part I 
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15:30 – 16:30
Catheter-based renal sympathetic denervation: long-term Symplicity clinical evidence, new data and future perspectives 
With an unrestricted educational grant from MEDTRONIC
16:45 – 18:15
Room 253
Interventional strategies for thrombus management in STEMI 12:30 – 14:00
Stent for Life and 2012 ESC STEMI guidelines implementation 14:00 – 15:30
Primary PCI for STEMI: prevention of thrombus embolism 15:30 – 16:30
Clot, too much clot, new clots: primary PCI for STEMI 16:45 – 18:15
Room 341
Outcome in contemporary coronary intervention 12:30 – 14:00
Cardiovascular Innovation Pipeline – New stents, scaffolds and drug-eluting balloons 14:00 – 15:30
Procedural factors determining outcome in high-risk patients 15:30 – 16:30
Novelties in peripheral interventions 16:45 – 17:45
Room 342A
Is there consensus in approach to coronary chronic total occlusion management? 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Cardiovascular Society of India
12:30 – 14:00
Patients in whom PCI is preferred over CABG 
Under the auspices of the Working Group on Interventional Cardiology of the Croatian Cardiac Society, the Working Group on Interventional Cardiology of the Cyprus Society of Cardiology, the South African Society of Cardiovascular Interventions (SASCI) and the Working Group on Interventional Cardiology of the Serbian Society of Cardiology
14:00 – 16:30
Mechanical device support during PCI: when, to whom and which device? 
With an unrestricted educational grant from MAQUET Cardiovascular GETINGE GROUP
16:45 – 18:15
Room 342B
Use of intravascular imaging during PCI 12:30 – 13:30
Impact of IVUS in a real-world practice 13:30 – 14:30
Use of adjunctive imaging during PCI in ACS 14:30 – 15:30
Use of adjunctive imaging during PCI 15:30 – 16:30
Unsettled issues with oral antiplatelet therapy: which one? How much? How long? 16:45 – 18:15
Room 343
Risk scores to aid decision making between CABG and PCI – Role of SYNTAX Score II 12:30 – 14:00
Intra-coronary haemodynamic parameters for evaluation of coronary lesion severity during cardiac catheterisation: how should we use them for clinical decision making? 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Working Group on Interventional Cardiology of the Netherlands Society of Cardiology (WIC)
14:00 – 15:30
Percutaneous interventions for congenital disease 15:30 – 16:30
Strategies in percutaneous management of left main stem stenosis 16:45 – 18:15
Room 351
TAVI results from worldwide registries 12:30 – 14:00
Overcoming TAVI challenges 14:00 – 15:30
Managing difficulties during TAVI 15:30 – 16:30
Transapical TAVI and other surgical transcatheter techniques 
With an unrestricted educational grant from EDWARDS LIFESCIENCES, JENAVALVE, MEDTRONIC and SYMETIS S.A.
16:45 – 18:15
Room 352A
You are facing a patient who needs a PCI: how to build your strategy and select your material? 14:00 – 15:30
Clinical impact of stent design – What’s new in 2013? 15:30 – 16:30
How to prevent distal embolisation during PCI of diseased saphenous vein graft 16:45 – 18:15
Room 352B
Various imaging techniques for TAVI procedures 12:30 – 14:00
TAVI nightmares 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Working Group on Interventional Cardiology (AGIK) of the German Society of Cardiology (DGK)
14:00 – 15:30
Percutaneous valve implantation for rare causes 15:30 – 16:30
TAVI: predictors of clinical outcomes 16:45 – 18:15
Room 353
Non-aortic transcatheter valvular interventions 12:30 – 13:30
All you need to know about interventions for mitral regurgitation 13:30 – 15:00
Percutaneous treatment options for degenerative mitral regurgitation 15:00 – 16:30
Atrial septal defect and left atrial appendage closure 16:45 – 18:15
Room Cordis
Training Village: Radial approach for coronary diagnostic and interventions – hands-on with the experts 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
13:00 – 15:00
Training Village: Catheter-based renal sympathetic denervation: introduction to an irrigated technology 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
15:30 – 16:30
Training Village: Catheter-based renal sympathetic denervation: introduction to an irrigated technology 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
16:30 – 17:30
Training Village: Femoral artery access and haemostasis 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
17:30 – 18:30
Room Maillot
Trials and innovations for peripheral interventions 13:00 – 14:00
Revascularisation strategies in patients with lower limb disease 14:00 – 16:30
Titanium-nitride-oxide active coated stents in renal applications: the true indications of renal stenting after ASTRAL and after the introduction of denervation 
With an unrestricted educational grant from HEXACATH
16:45 – 18:15
Room Medtronic Academia
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
13:00 – 14:30
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
14:45 – 16:15
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
16:30 – 18:00
Room St Jude Medical
Training Village: PCI optimisation – Focus on FFR 
With an unrestricted educational grant from ST. JUDE MEDICAL
14:00 – 15:00
Training Village: PCI optimisation – Focus on FFR 
With an unrestricted educational grant from ST. JUDE MEDICAL
15:15 – 16:15
Training Village: Left atrial appendage 
With an unrestricted educational grant from ST. JUDE MEDICAL
15:45 – 17:15
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
16:30 – 17:30
Theatre Bleu
NIC@EuroPCR – Interventional procedures complicated with fatal outcome 
With the collaboration of the National Intervention Council of India
14:00 – 16:30
Left main PCI using transradial approach 
With an unrestricted educational grant from TERUMO
16:45 – 18:45
Theatre Bordeaux
Expanding the indication for TAVI: who, why and when? 14:30 – 16:30
Tips and tricks on the four key steps of left atrial appendage closure: selection, planning, imaging, and guidance 
PHILIPS and ST JUDE MEDICAL
16:45 – 18:45
Theatre Havane
Learning bifurcations – How to successfully perform PCI in your patient presenting complex bifurcation lesions requiring two stents 14:00 – 15:30
Interactive case-based discussion on complex bifurcations 15:40 – 16:30
Incorporating bioresorbable vascular scaffolds in daily clinical practice: the time has come 
With an unrestricted educational grant from ABBOTT VASCULAR

 

[173] WEDNESDAY 22 MAY

Abstract & Case Corner
Left main treatment: dedicated stents, complex strategies and post-CABG situation 08:00 – 09:30
Left main PCI for left main disease intervention: outcome in 2013 09:45 – 10:45
Treatment of left main stem stenosis in high-risk patients 10:45 – 11:45
Fistula and haematoma during PCI 12:00 – 13:00
PCI challenges: just another day in the cathlab? 13:00 – 14:00
Retrieval techniques of lost ‘bits and pieces’ during PCI 14:10 – 15:40
Unusual causes of ACS 15:40 – 16:40
Stent deformation during PCI 16:45 – 18:15
Interactive Case Corner
Interactive case corner #4 08:00 – 09:30
Interactive case corner #5 09:45 – 11:15
Interactive case corner #6 12:00 – 13:30
Interactive case corner #7 14:10 – 15:40
Interactive case corner #8 16:45 – 18:15
Main arena
Complex cardiovascular interventions and new techniques – Master LIVE demonstrations by Jean Fajadet & Talib Majwal and expert panel discussion 08:00 – 11:45
Complex cardiovascular interventions and new techniques – Master LIVE demonstrations by Karl Heinz Kuck & Talib Majwal and expert panel discussion 14:10 – 16:45
Moderated Poster Area
Moderated posters 2 12:00 – 14:00
Moderated posters 3 16:45 – 18:15
Nurses and Technicians Corner
Moderated posters 12:00 – 13:00
PCR Sharing Centre
Do you want to become comfortable with pathophysiology? Practical example: hypertensive patients 08:00 – 09:00
Understand what you see with the iPad Atlas of OCT – Interactive OCT image interpretation 09:45 – 11:15
Do you want to become comfortable with health economics? Practical example: is renal denervation cost effective? 14:10 – 15:10
Do you want to become comfortable with data analysis? 15:40 – 16:40
Peripheral Abstract & Case Corner
Thoraco-abdominal aneurysm treatment 08:00 – 09:30
Endovascular aortic aneurysm repair: an evergrowing story 09:45 – 10:45
Aortic aneurysms: fundamentals to innovation 10:45 – 11:45
Renal artery stenting: challenging but rewarding cases 12:00 – 13:00
How to manage aorto-renal rupture and dissection 13:00 – 14:00
Aneurysm and false aneurysm management for superficial femoral artery and popliteal artery 14:10 – 15:40
Multilevel vascular interventions 15:40 – 16:40
Complications and great saves on carotid interventions 16:45 – 17:45
Room 241
Percutaneous mitral valve repair with the MitraClip system: determinants of outcome 08:00 – 09:30
Technical and approach issues in renal artery stenting 
Under the auspices of the Working Group on Interventional Cardiology of the Bulgarian Society of Cardiology, the Working Group on Interventional Cardiology of the Macedonian Society of Cardiology and the Working Group on Interventional Cardiology of the Romanian Society of Cardiology
09:45 – 11:45
Chronic total occlusion and multivessel disease: can novel imaging help to reduce risks? 
With an unrestricted educational grant from PHILIPS and INFRAREDX
12:00 – 13:00
Cardioprotective strategies to reduce ischaemic injury during PCI 
With an unrestricted educational grant from MENARINI
13:05 – 14:05
How to avoid patient-prosthesis mismatch and aortic regurgitation after aortic valve interventions 14:10 – 15:40
Hot Line – First-in-man in valvular heart disease 15:40 – 16:40
New frontiers – Exploring reduced contrast volume and fluoroscopy time with the GPSCath balloon dilatation catheter for complex percutaneous transluminal angioplasty procedures 
With an unrestricted educational grant from TELEFLEX
16:45 – 18:15
Room 242AB
Innovative stents and scaffolds 08:00 – 09:40
Emerging technologies for transcatheter aortic valve therapies – Part II 09:45 – 11:45
Real-world considerations for selecting antiplatelet therapy in high-risk ACS patients: putting evidence into clinical practice 
This educational programme is accredited by EBAC for one hour of External CME credit – Programme supported by an unrestricted educational grant from ASTRAZENECA
12:00 – 13:30
Hot Line – Trial updates and registries 14:10 – 15:10
Managing patients with unprotected left main coronary artery disease 
With the collaboration of China Interventional Therapeutics (CIT)
15:10 – 16:40
Catheter-based renal sympathetic denervation – Building momentum with the next generation Vessix system 
With an unrestricted educational grant from BOSTON SCIENTIFIC
16:45 – 18:15
Room 243
Challenging coronary artery intervention in ACS 
Under the auspices of the Iranian Society of Interventional Cardiology (ISOIC) and the Russian Society of Interventional Cardioangiology (RSICA)
08:00 – 09:30
Hot Line – First-in-man & novel DES and scaffolds 09:45 – 11:45
Management of complex coronary disease in Asia Pacific 
With an unrestricted educational grant from MEDTRONIC
12:00 – 13:30
Complex cardiovascular intervention in patients primarily reported as ACS 
Under the auspices of the Working Group on Interventional Cardiology of the Czech Society of Cardiology and the Working Group on Interventional Cardiology of the Slovak Society of Cardiology
14:10 – 15:40
How to improve the STEMI treatment in large territories like Russia? 15:40 – 16:40
Impact of thrombus aspiration device on the results of primary PCI 16:45 – 18:15
Room 251
Primary PCI in complex STEMI with cardiogenic shock 08:00 – 09:30
Learning FFR – Assisting for FFR measurement in the cathlab 09:45 – 11:15
Synchronising polymer absorption and drug elution with the Synergy stent. Implications for healing and dual antiplatelet therapy duration 
With an unrestricted educational grant from BOSTON SCIENTIFIC
12:00 – 13:00
The Direct Flow valve: innovation for improving outcomes in TAVI 
With an unrestricted educational grant from DIRECT FLOW MEDICAL
13:05 – 14:05
Assisting for PCI through radial approach 14:10 – 15:40
Pre-procedure risk assessment to prevent complications after/during PCI 15:40 – 16:40
Clinical value of anti-restenosis and pro-healing Combo stent 
With an unrestricted educational grant from ORBUSNEICH
16:45 – 18:15
Room 252AB
How to treat a patient with complex multivessel disease and/or left main disease 
Under the auspices of the Argentine College of Interventional Cardioangiologist (CACI) and the Atheroma Coronary and Interventional Cardiology Group (GACI)
08:00 – 09:30
GRCI@EuroPCR – Challenging cases in the catheterisation laboratory: international viewpoint Gestion de cas complexes en salle de cathétérisme: approche internationale 
Bilingual session in collaboration with the GRCI (Groupe de Réflexion sur la Cardiologie Interventionnelle) Session bilingue en collaboration avec le GRCI (Groupe de Réflexion sur la Cardiologie Interventionnelle)
09:45 – 11:15
Advancing innovations in catheter-based renal sympathetic denervation 
CORDIS, JOHNSON & JOHNSON
12:00 – 13:30
Device-based interventions in heart failure: targeting deleterious mechanisms of heart failure progression 14:10 – 15:40
Novel devices for acute or chronic heart failure 15:40 – 16:40
DES and dual antiplatelet therapy: customising treatment duration to your patient 
With an unrestricted educational grant from ABBOTT VASCULAR
16:45 – 18:15
Room 253
Transradial approach for complex coronary interventions in patients with ACS 
Under the auspices of the Working Group on Interventional Cardiology of the Hungarian Society of Cardiology and the Working Group on Interventional Cardiology of the Macedonian Society of Cardiology
08:00 – 09:30
How to write a scientific manuscript and get it published! 09:45 – 10:45
From bench to cathlab: clinical implication of stent design 10:45 – 11:45
Conduction disturbances after TAVI 12:00 – 13:00
Overcoming TAVI challenges 13:00 – 14:00
Planning is the key to avoiding TAVI complications 
Under the auspices of the Association of Cardiovascular Interventions (ACVI) of the Polish Cardiac Society and the South African Society of Cardiovascular Intervention (SASCI)
14:10 – 15:40
How to write a scientific abstract and get it accepted! 15:40 – 16:40
Use of DES in specific subsets of patients/lesions 16:45 – 18:15
Room 341
Tough calls in primary PCI: STEMI and multivessel disease 
Under the auspices of the Working Group on Interventional Cardiology of the Israeli Heart Society and the Working Group on Interventional Cardiology of the Slovenian Society of Cardiology
08:00 – 09:30
Antegrade or retrograde strategy for coronary chronic total occlusion recanalisation? 09:45 – 10:45
Complicated coronary chronic total occlusion recanalisation 10:45 – 11:45
Resistant hypertension and its treatment across the world 
With an unrestricted educational grant from TERUMO
12:00 – 13:00
Coronary intervention in the elderly population 13:00 – 14:00
PCI in the elderly: when to stop, when to intervene 
Under the auspices of the Working Group on Interventional Cardiology of the Dutch Society of Cardiology and the Working Group on Interventional Cardiology (GTCI) of the Tunisian Society of Cardiology and Cardiovascular Surgery
14:10 – 15:40
Percutaneous revascularisation from coronary chronic total occlusion: results from registries 15:40 – 16:40
Single-guide catheter techniques for retrograde recanalisations for coronary chronic total occlusions 16:45 – 18:15
Room 342A
Intravascular diagnostics – Does it really change our treatment strategy? 
Under the auspices of the Working Group on Interventional Cardiology of the Danish Society of Cardiology and the Working Group on Interventional Cardiology of the Norwegian Society of Cardiology
08:00 – 09:30
Multivessel disease: “a tale of two cities” 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Working Group on Interventional Cardiology of the Cyprus Society of Cardiology
09:45 – 11:15
Self-expanding stents: a NEW solution for patients presenting with atypical coronary anatomy 
With an unrestricted educational grant from STENTYS
12:00 – 13:30
Complex primary PCI in high-risk STEMI patients 14:10 – 15:40
Non-left main bifurcation stenting: tips and tricks 15:40 – 16:40
Ischaemia-driven revascularisation: the evolution of FFR in daily practice 
With an unrestricted educational grant from ST. JUDE MEDICAL
16:45 – 18:15
Room 342B
Unusual causes of STEMI in young women 08:00 – 09:30
Different approaches for thrombus removal during primary PCI 09:45 – 10:45
Primary PCI for STEMI when stent is not the solution 10:45 – 11:45
Revascularisation strategies for multivessel disease patients: stents, bypasses or both? 12:00 – 13:00
Complex PCI in patients with multivessel disease 13:00 – 14:00
Challenging cases from Turkey 
With the collaboration of the Turkish Society of Cardiology’s Association of Percutaneous Cardiovascular Interventions
14:10 – 15:10
Individualised antiplatelet therapy based on testing or genotyping: idea from the past or solution for the future 15:40 – 16:40
Real life use of bioabsorbable vascular scaffold in coronary disease 16:45 – 18:15
Room 343
Patent foramen ovale closure in patients with cryptogenic stroke – Timed out or role respected? 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Working Group on Interventional Cardiology of the Danish Society of Cardiology
08:00 – 09:30
Multislice computed tomography: emerging indication in interventional cardiology 09:45 – 10:45
The role of non-invasive imaging to guide percutaneous coronary revascularisation procedures 10:45 – 11:45
FFR in the real world 12:00 – 13:00
FFR are we working with the best threshold? 13:00 – 14:00
STEMI and multivessel disease 
Under the auspices of the Working Group on Interventional Cardiology of the Georgian Society of Cardiology and the Working Group on Interventional Cardiology of the Kazakhstanese Society of Cardiology
14:10 – 15:40
Coronary aneurysms and ACS 15:40 – 16:40
Left ventricular assistance devices in acute ischaemic heart failure 16:45 – 18:15
Room 351
All you need to know about TAVI 08:00 – 09:30
New devices for TAVI 09:45 – 10:45
Percutaneous valve implantation: new valves and new indications 10:45 – 11:45
Complex patients today and tomorrow: Medtronic DES solutions from Resolute Integrity to bioresorbable stents 
With an unrestricted educational grant from MEDTRONIC
12:00 – 13:30
All you need to know about OCT 14:10 – 15:40
Use of OCT during PCI 15:40 – 16:40
The Medtronic transcatheter valve programmes – Recapturability, transapical technology and mitral solutions 
With an unrestricted educational grant from MEDTRONIC
16:45 – 18:15
Room 352A
You are facing an elderly patient presenting with high risk NSTE-ACS: how do you successfully perform PCI? 08:00 – 09:30
Radial approach – Fundamental rules 09:45 – 10:40
Radial approach – Navigation from radial to brachial 10:50 – 11:45
Radial access: anything new? 12:00 – 13:00
When there is no access site, remember that the arteries lead to the heart 13:00 – 14:00
You are facing a patient presenting with an acute STEMI: how do you successfully perform PCI? 14:10 – 15:40
Radial approach – Navigation from brachial artery to ascending aorta 15:45 – 16:40
Radial access: a gold standard worldwide? 16:45 – 18:15
Room 352B
TAVI: typical and atypical complications 
Under the auspices of the Association of Cardiovascular Interventions (ACVI) of Polish Cardiac Society and the Saudi Arabia Cardiology Interventional Group (SACIG) of the Saudia Heart Association
08:00 – 09:30
AICT@EuroPCR – How Asia performs PCI of coronary chronic total occlusion 
With the collaboration of the Asian Interventional Cardiovascular Therapeutics (AICT)
09:45 – 11:15
Titanium-nitride-oxide bioactive stent: the evidence-based choice in STEMI and NSTEMI patients 
With an unrestricted educational grant from HEXACATH
12:00 – 13:30
TAVI and coronary artery disease: what is the best treatment strategy? 
Under the auspices of the Working Group on Interventional Cardiology of the Latvian Society of Cardiology and the Russian Society of Interventional Cardioangiology
14:10 – 15:40
TAVI and coronary artery disease 15:40 – 16:40
A new combination of factor Xa inhibition and standard antiplatelet therapy to prevent more recurrent cardiovascular events in ACS 
With an unrestricted educational grant from BAYER HEALTHCARE PHARMACEUTICALS
16:45 – 18:15
Room 353
Czech Republic shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Czech Society of Cardiology
08:00 – 08:45
India shares its most educational cases 
Under the auspices of the Cardiovascular Society of India
08:45 – 09:30
Hungary shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Hungarian Society of Cardiology
09:45 – 10:30
Saudi Arabia shares its most educational cases 
Under the auspices of the Saudi Arabia Cardiology Interventional Society (SACIS) of the Saudia Heart Association
10:30 – 11:15
South Africa shares its most educational cases 
Under the auspices of the South African Society of Cardiovascular Intervention (SASCI)
11:15 – 12:00
Diabetes and coronary artery disease: a bad association! 12:00 – 13:00
Renal function and clinical outcome after PCI 13:00 – 14:00
Switzerland shares its most educational cases 
Under the auspices of the Working Goup on Interventional Cardiology and ACS of the Swiss Society of Cardiology
14:10 – 14:55
United Kingdom shares its most educational cases 
Under the auspices of the British Cardiovascular Intervention Society (BCIS)
14:55 – 15:40
Serbia shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Serbian Society of Cardiology
15:40 – 16:25
Iran shares its most educational cases 
Under the auspices of the Iranian Society of Interventional Cardiology (ISOIC)
16:45 – 17:30
Germany shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology (AGIK) of the German Society of Cardiology (DGK)
17:30 – 18:15
Room Cordis
Training Village: Radial approach for coronary diagnostic and interventions – hands-on with the experts 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
09:00 – 11:00
Training Village: Catheter-based renal sympathetic denervation: introduction to an irrigated technology 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
14:00 – 15:00
Training Village: Advanced tips and tricks: vessel preparation and post dilation 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
15:30 – 16:30
Room Maillot
Carotid LIVE session: the “state-of-the-art” of stroke prevention 08:00 – 09:55
Access is key for carotid artery stenting in complex aortic arches 10:00 – 10:50
Embolic protection devices for carotid artery stenting 10:50 – 11:45
Access is critical 
With an unrestricted educational grant from COOK MEDICAL
12:00 – 13:30
Visceral and renal artery interventions 14:10 – 15:40
Guest lectures: how I survived the peripheral endovascular battle? 15:40 – 16:40
The evolving evidence of IN.PACT drug-eluting balloon in claudication and critical limb ischaemia 
With an unrestricted educational grant from MEDTRONIC
16:45 – 18:15
Room Medtronic Academia
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
09:00 – 10:30
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
10:30 – 12:00
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
12:30 – 14:00
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
14:15 – 15:45
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
16:00 – 17:30
Room St Jude Medical
Training Village: Left atrial appendage 
With an unrestricted educational grant from ST. JUDE MEDICAL
09:00 – 10:30
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
10:15 – 11:15
Training Village: Left atrial appendage 
With an unrestricted educational grant from ST. JUDE MEDICAL
10:45 – 12:15
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
11:30 – 12:30
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
14:00 – 15:00
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
15:15 – 16:15
Training Village: Left atrial appendage 
With an unrestricted educational grant from ST. JUDE MEDICAL
15:45 – 17:15
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
16:30 – 17:30
Talk ‘LIVE’ Corner
Talk ‘LIVE’ 17:00 – 18:30
Theatre Bleu
Structured care pathways for NSTE-ACS: best practice examples 08:00 – 09:30
Complex bifurcation stenting: LIVE demonstration of emerging techniques 09:45 – 11:45
Do we really need dedicated stents to treat bifurcation lesions? 
With an unrestricted educational grant from BIOSENSORS INTERNATIONAL
12:00 – 14:00
Bioresorbable coronary scaffolds in practice 14:10 – 16:10
Acurate positioning of transapical and transfemoral aortic valves with self-seating and self-sealing design 
With an unrestricted educational grant from SYMETIS S.A.
16:45 – 18:45
Theatre Bordeaux
The best way to diagnose ischaemia in my patient? Convince me! Personal views from interventional cardiologists 08:00 – 09:30
Can left atrial appendage or patent foramen ovale closure prevent embolic stroke? 09:45 – 11:45
Optimising PCI outcomes using OCT and FFR in patients with stable and acute coronary artery disease 
With an unrestricted educational grant from ST. JUDE MEDICAL
12:00 – 14:00
What to do with coronary artery disease in TAVI candidates? 14:10 – 16:10
The next frontier for catheter-based renal sympathetic denervation for patients with resistant hypertension 
With an unrestricted educational grant from COVIDIEN
16:45 – 18:45
Theatre Havane
Learning access for TAVI – Access options for TAVI 08:00 – 09:30
Learning transseptal puncture and mitral balloon valvuloplasty – Transseptal puncture and mitral balloon valvuloplasty made easy 10:15 – 11:45
An in-depth look into the BIOFLOW trials: a modern limus-eluting stent with bioabsorbable polymer 
With an unrestricted educational grant from BIOTRONIK
12:00 – 13:30
Learning atrial closure procedures – Patent foramen ovale and left atrial appendage closure made easy 14:10 – 15:40
Interactive case-based discussion – complications on atrial closure procedures 15:45 – 16:40
Interventional management of high-risk ACS and STEMI: don’t just do it… do it right! 
With an unrestricted educational grant from TERUMO and THE MEDICINES COMPANY
16:45 – 18:15

[172] THURSDAY 23 MAY

Abstract & Case Corner
FFR or IVUS to guide coronary revascularisation? Do you believe in morphology or function? 08:00 – 09:30
Role of imaging in in-stent restenosis 09:45 – 10:45
Diagnostics and management of stent fracture 10:45 – 11:45
PCI of totally occluded saphenous vein graft 12:00 – 13:00
Interventional management of unusual causes of angina 13:00 – 14:00
The role of drug-eluting balloons in contemporary coronary intervention 14:10 – 15:40
Management of late in-stent restenosis 15:40 – 16:40
Coronary perforation management 16:45 – 17:45
Interactive Case Corner
Interactive case corner #9 08:00 – 09:30
Interactive case corner #10 09:45 – 11:15
Interactive case corner #11 12:00 – 13:30
Interactive case corner #12 14:10 – 15:40
Interactive case corner #13 16:45 – 18:15
Main arena
Complex cardiovascular interventions and new techniques – Master LIVE demonstrations by Corrado Tamburino, Martyn Thomas & Simon Redwood and expert panel discussion 08:00 – 11:45
Complex cardiovascular interventions and new techniques – Master LIVE demonstrations by Christian Hamm & Corrado Tamburino and expert panel discussion 14:10 – 16:45
Moderated Poster Area
Moderated posters 4 12:00 – 14:00
Moderated posters 5 16:45 – 18:15
PCR Sharing Centre
Understand what you see with the iPad Atlas of OCT – Interactive OCT image interpretation 08:00 – 09:30
Do you want to be more confident when developing and delivering PowerPoint presentations? 14:10 – 15:10
Do you want to become comfortable with data analysis? 15:40 – 16:40
Peripheral Abstract & Case Corner
Tips and tricks in carotid artery stenting 08:00 – 09:30
Carotid artery stenting: clinical outcome 09:45 – 10:45
Acute procedural events in carotid artery stenting 10:45 – 11:45
Carotid artery stenting: novelties in risk assessment 12:00 – 13:00
Carotid artery stenting: challenging scenarios 13:00 – 14:00
Aorto-iliac angioplasty: what is new in 2013 14:10 – 15:40
Iliac angioplasty 15:40 – 16:40
Complex aortic interventions 
With the collaboration of the International Society of Endovascular Specialists
16:45 – 18:15
Room 241
How I treat complications after peripheral endovascular intervention 
Under the auspices of the Italian Society for Vascular and Endovascular Surgery (SICVE) and the Vascular Surgery Society of Southern Africa (VASSA)
08:00 – 09:30
Cardiovascular Innovation Pipeline – New valves and devices 09:45 – 10:45
Radiation safety during PCI 10:45 – 11:45
Innovating vascular restoration: paving the way for the DESolve scaffold platform 
With an unrestricted educational grant from ELIXIR MEDICAL
12:00 – 13:30
How to prevent and treat ilio-femoral complications of TAVI? 14:10 – 15:40
Emerging technologies for transcatheter mitral valve therapies 2013 – Part I: transcatheter mitral valve repair devices 15:40 – 16:40
Tryton growing clinical experience and data displacing provisional stenting? 
With an unrestricted educational grant from TRYTON MEDICAL
16:45 – 18:15
Room 242AB
Challenges in complex percutaneous valve treatment: the combination of aortic stenosis and significant functional mitral regurgitation 08:00 – 09:30
Preclinical studies of upcoming bioresorbable scaffolds 09:45 – 11:45
The Embolic Protection Stent – Beyond current techniques: a more effective solution in STEMI primary PCI 
With an unrestricted educational grant from INSPIRE MD
12:00 – 13:30
Effect of catheter-based renal sympathetic denervation: is there a role beyond resistant hypertension? 14:10 – 15:40
Contribution of renal denervation to the treatment of resistant hypertension: a health technology assessment perspective 15:40 – 16:40
Edwards TAVI: a predictable procedure with sustained clinical results 
With an unrestricted educational grant from EDWARDS LIFESCIENCES
16:45 – 18:15
Room 243
PCI of bifurcation lesions: results from registries and new dedicated stents 08:00 – 09:30
Non-left main bifurcation stenting: tips and tricks 09:45 – 10:45
Non-left main bifurcation lesions: tips and tricks 10:45 – 11:45
Stent thrombosis: management challenges 12:00 – 13:00
Very late stent thrombosis 13:00 – 14:00
Innovations in Cardiovascular Interventions@EuroPCR 2013 
With the collaboration of Innovations in Cardiovascular Interventions (ICI)
14:10 – 15:40
Intervention for prevention of stroke 15:40 – 16:40
Stent thrombosis: new evidence from clinical trials and registries 16:45 – 18:15
Room 251
Best clinical abstract presentations 08:00 – 09:30
Best nurse research abstract session 09:45 – 11:15
Nurses and Technicians best presentation award and closing ceremony 11:15 – 11:45
The Portico TAVI system – How new design translates into clinical results 
With an unrestricted educational grant from ST. JUDE MEDICAL
12:00 – 13:00
Emerging clinical use of drug-eluting balloons in challenging atherosclerotic lesions 
With an unrestricted educational grant from BIOTRONIK
13:05 – 14:05
Challenging cases from Taiwan 
With the collaboration of the Taiwan Society of Cardiovascular Interventions
14:10 – 15:40
Left main dissection during PCI 15:40 – 16:40
Cre8: welcome back confidence in short dual antiplatelet therapy with effective DES 
With an unrestricted educational grant from CID
16:45 – 18:15
Room 252AB
All you need to know about catheter-based renal sympathetic denervation 08:00 – 09:30
Antiplatelet and antithrombotic therapy in PCI: a balancing act 09:45 – 11:15
Clinical update on EnligHTN, the original multi-electrode catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from ST. JUDE MEDICAL
12:00 – 13:30
Up-to-date primary PCI technique 14:10 – 15:40
GPIIbIII inhibitors : still useful in 2013? 15:40 – 16:40
What do YOU think? A case-based discussion on biodegradable versus durable polymer DES in complex patients 
With an unrestricted educational grant from BIOSENSORS INTERNATIONAL
16:45 – 18:15
Room 253
Restenosis after failure of CABG and PCI 
Under the auspices of the Working Group on Interventional Cardiology of the Danish Society of Cardiology and the Working Group on Interventional Cardiology of the Finnish Society of Cardiology
08:00 – 09:30
How to write a scientific manuscript and get it published! 09:45 – 10:45
ABC for biotechnology innovators@EuroPCR 
With the collaboration of Innovations in Cardiovascular Interventions (ICI)
10:45 – 11:45
Tools and techniques for PCI of coronary chronic total occlusion 12:00 – 13:00
How to treat coronary chronic total occlusion with limited resources and material? 13:00 – 14:00
New challenges for high-risk primary PCI in 2013 
Under the auspices of the Association of Cardiovascular Interventions (ACVI) of the Polish Cardiac Society and the Working Group of Acute Cardiology of the Slovenian Society of Cardiology
14:10 – 15:40
The unusual coronary chronic total occlusion: recanalisation in bypass patients, acute myocardial infarction and anomalous coronaries 15:40 – 16:40
New generation DES: comparison with older DES 16:45 – 18:15
Room 341
Prevention and management of complications after TAVI 
Under the auspices of the Portuguese Association for Interventional Cardiology (APIC) and the Working Group on Interventional Cardiology of the Spanish Society of Cardiology
08:00 – 09:30
Incidence and prevention of cerebrovascular events after TAVI 09:45 – 10:45
Challenges before, during and after TAVI 10:45 – 11:45
TAVI and bleeding complication 12:00 – 13:00
TAVI and kidney injury 13:00 – 14:00
TAVI with coronary artery disease 
Under the auspices of the Working Goup on Interventional Cardiology (EWGIC) of the Egyptian Society of Cardiology and the Working Group on Interventional Cardiology of the Lebanese Society of Cardiology
14:10 – 15:40
TAVI in unique clinical scenarios 15:40 – 16:40
TAVI technical issues 16:45 – 18:15
Room 342A
How to treat a patient with significant paravalvular leak after TAVI 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Atheroma Coronary and Interventional Cardiology Group (GACI)
08:00 – 09:30
Interventional treatment of acute ischaemic stroke: which role for STEMI networks? 09:45 – 11:45
Self-expanding stents: a NEW solution to optimise primary PCI beyond the open artery 
With an unrestricted educational grant from STENTYS
12:00 – 13:30
Cardiovascular Innovation Pipeline – Treatment of resistant hypertension 14:10 – 15:40
Hot Line – Registries and first-in-man for structural heart disease 15:40 – 16:40
Patient with STEMI: learn the best from East and West 
With an unrestricted educational grant from TERUMO
16:45 – 18:15
Room 342B
Determinants of outcome in STEMI patients 08:00 – 09:30
Resuscitated cardiac arrest – Burning interventional questions 09:45 – 11:45
Updates on contrast-induced nephropathy 12:00 – 13:00
Updates on myocardial revascularisation in patients with chronic kidney disease and haemodialysis 13:00 – 14:00
Unusual causes of STEMI 14:10 – 15:40
You cannot miss this great session on Rotablator! 15:40 – 16:40
Rotational atherectomy in complex coronary cases 16:45 – 18:15
Room 343
Challenges in acute myocardial infarction 
Under the auspices of the Working Group on Interventional Cardiology of the Austrian Society of Cardiology and the Working Goup on Interventional Cardiology and ACS of the Swiss Society of Cardiology
08:00 – 09:30
New methods for physiological assessment of coronary stenosis? 09:45 – 10:45
Complex PCI: which role for self-expanding stents? 10:45 – 11:45
Clinical value of IVUS during ACS: when you lose your way 12:00 – 13:00
Clinical value of IVUS: what others don’t tell 13:00 – 14:00
Challenging prosthetic mitral valve malfunction 
Under the auspices of the Working Group on Interventional Cardiology (AGIK) of the German Cardiac Society (DGK) and the Working Group on Invasive Cardiology of the Italian Society of Invasive Cardiology (SICI-GISE)
14:10 – 15:40
Clinical value of IVUS during coronary chronic total occlusion PCI: with a little help from your friend 15:40 – 16:40
Room 351
All you need to know about bioresorbable scaffolds 08:00 – 09:30
Hot Line – Evolving procedural strategies 09:45 – 11:45
Treating complex lesions and patients with bioresorbable vascular scaffolds 
With an unrestricted educational grant from ABBOTT VASCULAR
12:00 – 13:30
Bioresorbable vascular scaffolds in chronic total occlusions and calcified lesions 14:10 – 15:40
Bioresorbable scaffolds: clinical results 15:40 – 16:40
Complex cases of mitral regurgitation: how far can you go with MitraClip? 
With an unrestricted educational grant from ABBOTT VASCULAR
16:45 – 18:15
Room 352A
You are a practitioner who wishes to successfully start a peripheral percutaneous transluminal angioplasty (PTA) programme 08:00 – 09:30
Radial approach – Cannulation of the targeted vessels ostia 09:45 – 10:40
Forum on radial approach 10:50 – 11:45
Difficult diagnosis and management of ACS 12:00 – 13:00
Acute heart failure due to ACS 13:00 – 14:00
Cardiovascular Innovation Pipeline – Novel interventional approaches for heart failure 15:40 – 16:40
Radial access: problem or solution? 16:45 – 18:15
Room 352B
All you need to know about treatment of coronary chronic total occlusion 08:00 – 09:30
Renal denervation: novel approaches and first-in-man results 09:45 – 10:45
Management of intra-coronary thrombus during primary PCI 10:45 – 11:45
Provisional treatment approach of a distal left main and true bifurcation lesion: combination of a dedicated stent in the main branch and drug-eluting balloon in the side branch 
With an unrestricted educational grant from MINVASYS
12:00 – 13:30
EuroIntervention / European Heart Journal@EuroPCR 14:10 – 15:40
Unusual presentation of coronary aneurysms 15:40 – 16:40
How to treat aorto-ostial coronary dissection 16:45 – 18:15
Room 353
Tunisia shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology (GTCI) of the Tunisian Society of Cardiology and Cardiovascular Surgery
08:00 – 08:45
Italy shares its most educational cases 
Under the auspices of the Working Group on Invasive Cardiology of the Italian Society of Invasive Cardiology (SICI-GISE)
08:45 – 09:30
Egypt shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology (EWGIC) of the Egyptian Society of Cardiology
09:45 – 10:30
Kazakhstan shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Association of Cardiologists of Kazakhstan
11:15 – 12:00
Stent dislodgement during PCI 12:00 – 13:00
Aortic damage during percutaneous intervention 13:00 – 14:00
Spain shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Spanish Society of Cardiology
14:10 – 14:55
Sweden shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Swedish Society of Cardiology
14:55 – 15:40
Argentina shares its most educational cases 
Under the auspices of the Argentine College of Interventional Cardioangiologist (CACI)
15:40 – 16:25
Portugal shares its most educational cases 
Under the auspices of the Portuguese Association for Interventional Cardiology (APIC)
16:45 – 17:30
Greece shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Hellenic Cardiological Society
17:30 – 18:15
Room Cordis
Training Village: Endovascular complication management: renal access 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
09:00 – 10:00
Training Village: Catheter-based renal sympathetic denervation: introduction to an irrigated technology 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
10:30 – 11:30
Training Village: Radial approach for coronary diagnostic and interventions – hands-on with the experts 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
13:00 – 15:00
Training Village: Advanced Exoseal: achieving haemostasis and managing access site complications 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
15:30 – 16:30
Training Village: Importance of vessel pre- and post- dilatation for better patient outcomes 
With an unrestricted educational grant from CORDIS CARDIAC & VASCULAR INSTITUTE
16:45 – 18:00
Room Maillot
Solutions for complex abdominal aortic aneurysm 08:00 – 09:55
Therapeutic embolisation – Part I: tools and techniques for coronary and peripheral arteries 10:00 – 11:45
Left atrial appendage closure for stroke prevention: what every interventional cardiologist should know 
With an unrestricted educational grant from BOSTON SCIENTIFIC
12:00 – 13:00
New hopes for critical limb ischaemia 
With an unrestricted educational grant from TERUMO
13:05 – 14:05
Solutions for complex thoracic aortic disease 14:10 – 15:40
Therapeutic embolisation – Part II: clinical applications for coronary and peripheral arteries 15:40 – 16:40
Titanium-nitride-oxide active stent in ACS patients with or without bleeding risks 
With an unrestricted educational grant from HEXACATH
16:45 – 18:15
Room Medtronic Academia
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
09:00 – 10:30
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
10:30 – 12:00
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
12:30 – 14:00
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
14:15 – 15:45
Training Village: Hands-on introduction to the new Symplicity Spyral catheter-based renal sympathetic denervation system 
With an unrestricted educational grant from MEDTRONIC ACADEMIA
16:00 – 17:30
Room St Jude Medical
Training Village: PCI optimisation – Focus on OCT 
With an unrestricted educational grant from ST. JUDE MEDICAL
09:00 – 10:00
Training Village: PCI optimisation – Focus on OCT 
With an unrestricted educational grant from ST. JUDE MEDICAL
10:15 – 11:15
Training Village: Left atrial appendage 
With an unrestricted educational grant from ST. JUDE MEDICAL
10:45 – 12:15
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
11:30 – 12:30
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
14:00 – 15:00
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
15:15 – 16:15
Training Village: Left atrial appendage 
With an unrestricted educational grant from ST. JUDE MEDICAL
15:45 – 17:15
Training Village: TAVI 
With an unrestricted educational grant from ST. JUDE MEDICAL
16:30 – 17:30
Talk ‘LIVE’ Corner
Talk ‘LIVE’ 17:00 – 18:30
Theatre Bleu
Revascularisation in a patient with ischaemic heart failure and reduced left ventricular function 08:00 – 09:30
Treatment of coronary chronic total occlusion: Japan meets Europe 
With the collaboration of Complex Cardiovascular Therapeutics (CCT)
09:45 – 11:45
Left main and complex bifurcation stenting 
With an unrestricted educational grant from TERUMO
12:00 – 14:00
Am I treating the right lesion? Angiography versus ischaemia-based coronary revascularisation in stable coronary artery disease patients 14:10 – 16:10
Catheter-based renal sympathetic denervation: introducing the new Symplicity Spyral and Flex systems 
With an unrestricted educational grant from MEDTRONIC
16:45 – 18:45
Theatre Bordeaux
Enabling technologies for TAVI 08:00 – 09:30
Percutaneous treatment options for functional mitral regurgitation 09:45 – 11:45
Optimising TAVI procedures and patients outcomes: Medtronic’s new technologies and valve-in-valve procedure with Evolut 
With an unrestricted educational grant from MEDTRONIC
12:00 – 14:00
Valve-in-valve 14:10 – 16:10
Physiological stenosis assessment with FFR and instant wave-free ratio: we need both! 
With an unrestricted educational grant from VOLCANO
16:45 – 18:45
Theatre Havane
Optimal management of your NSTE-ACS patient with complex multivessel disease 08:00 – 09:30
Learning renal denervation – Critical appraisal on device-based therapies targeting the sympathetic system 09:45 – 11:45
Contemporary ACS antithrombotic therapy 
With an unrestricted educational grant from THE MEDICINES COMPANY
12:00 – 13:30
Learning ostial PCI – How to successfully perform PCI in a patient presenting ostial left main and ostial right coronary artery 14:10 – 15:40
Interactive case-based discussion – complications on ostial PCI 15:45 – 16:40
Contemporary coronary chronic total occlusion PCI: integrating the hybrid approach to your practice 
With an unrestricted educational grant from BOSTON SCIENTIFIC
16:45 – 18:15

 

 

 

[59] FRIDAY 24 MAY

Abstract & Case Corner
How to close paravalvular leak 09:00 – 10:30
Cases you have never seen 10:45 – 11:45
Unusual cases in the cathlab: diagnostic challenges 11:45 – 12:45
Interactive Case Corner
Interactive case corner #14 09:00 – 10:30
Interactive case corner #15 10:45 – 12:15
Main arena
Complex cardiovascular interventions and new techniques – Master LIVE demonstrations by Farrel Hellig, Martyn Thomas & Simon Redwood and expert panel discussion 09:00 – 13:00
PCR Sharing Centre
Do you want to be more confident when developing and delivering PowerPoint presentations? 09:00 – 10:00
Peripheral Abstract & Case Corner
Femoro-popliteal angioplasty : could new devices improve mid-term follow-up? 09:00 – 10:30
Chronic total occlusion revascularisation for superficial femoral artery 10:45 – 12:15
Room 241
Bioresorbable versus durable polymer coatings for DES 09:00 – 10:30
All you need to know about drug-coated balloons in coronary and peripheral vascular disease 10:45 – 12:15
Room 242A
Fully-absorbable jacket, in-stent restenosis and bypasses: new avenues for bioabsorbable vascular scaffolds? 09:00 – 10:30
Bioresorbable scaffolds: lessons learned from intracoronary imaging 10:45 – 11:45
Managing difficult stent cases 11:45 – 12:45
Room 242B
Challenging cases of saphenous vein graft interventions 09:00 – 10:30
Overcoming challenges during PCI 10:45 – 11:45
Helpful techniques during “extreme” PCI 11:45 – 12:45
Room 243
Predictors of in-stent restenosis and stent thrombosis after DES implantation 09:00 – 10:30
Stent thrombosis: overcoming challenging scenarios 10:45 – 12:15
Room 251
Emerging technologies for transcatheter mitral valve therapies 2013 – Part II: transcatheter replacement technologies 09:00 – 11:00
Novel catheter-based therapies of mitral regurgitation 11:00 – 12:00
Room 252A
Developments in percutaneous closure of the left atrial appendage 09:00 – 10:30
Percutaneous treatment of complex coronary aneurysms 10:45 – 11:45
Percutaneous management of complex coronary aneurysms 11:45 – 12:45
Room 252B
Primary PCI when the left main is the culprit 09:00 – 10:30
Primary PCI when the left main is the culprit 10:45 – 11:45
Left main dissection during PCI 11:45 – 12:45
Room 253
Below-the-knee angioplasty: risk stratification and DES benefits 09:00 – 10:00
Severe aortic stenosis combined with coronary artery disease in high-risk patient 
Under the auspices of the Working Group on Interventional Cardiology of the Hellenic Cardiological Society and the Working Group on Interventional Cardiology of the Israeli Heart Society
10:45 – 12:15
Room 341
Slow flow and no flow in PCI, not only in ACS: how to prevent and how to treat it? 
Under the auspices of the Working Group on Interventional Cardiology of the Danish Society of Cardiology and the Working Group on Interventional Cardiology of the Swedish Society of Cardiology
09:00 – 10:30
Renal denervation for resistant hypertension 10:45 – 12:15
Room 342A
Cardiogenic shock and intra-aortic balloon pump 
Under the auspices of the Luxembourg Society of Cardiology and the Working Group on Interventional Cardiology of the Norwegian Society of Cardiology
09:00 – 10:30
PCI of bifurcation lesions: impact of procedural techniques on clinical outcome 10:45 – 11:45
Bifurcation lesion: problems and solutions 11:45 – 12:45
Room 342B
TAVI or not TAVI: that is the question 
Under the auspices of the British Cardiovascular Intervention Society (BCIS) and the Working Group on Interventional Cardiology of the Spanish Society of Cardiology
09:00 – 10:30
Unfrequent indications for TAVI 10:45 – 11:45
TAVI in patients with previous cardiac valve operations 11:45 – 12:45
Room 343
Insights from OCT 09:00 – 10:30
Importance of OCT during PCI today 10:45 – 12:15
Room 351
Primary PCI for STEMI 
Under the auspices of the Working Group on Interventional Cardiology (BWGIC) of the Belgium Society of Cardiology and the Working Group on Interventional Cardiology of the Scottish Cardiac Society
09:00 – 10:30
All you need to know about radial approach for PCI 10:45 – 12:15
Room 352A
Learning rotablator – How to easily and successfully use rotational atherectomy 09:00 – 10:30
Novel techniques using rotational atherectomy 10:45 – 12:15
Room 352B
All you need to know about antiplatelet and antithrombotic pharmacology for PCI: NSTEMI and STEMI 09:00 – 10:30
Management of acute coronary artery occlusion during PCI 10:45 – 11:45
Retrieval of ‘things’ left behind during PCI 11:45 – 12:45
Room 353
Israel shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Israeli Heart Society
09:00 – 09:45
Macedonia shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Macedonian Society of Cardiology
09:45 – 10:30
Cyprus shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Cyprus Society of Cardiology
10:45 – 11:30
Austria shares its most educational cases 
Under the auspices of the Working Group on Interventional Cardiology of the Austrian Society of Cardiology
11:30 – 12:15
Room Maillot
Hybrid angio suite 09:00 – 10:30
Large size percutaneous access for endoaortic procedures 10:45 – 12:45
Theatre Bleu
Coronary perforation: management and implications 09:00 – 10:30
Device-based left ventricular cavity reduction in heart failure 10:45 – 12:15
Theatre Bordeaux
SOLACI@EuroPCR 
With the collaboration of the Sociedad Latino Americana de Cardiologia Intervencionista (SOLACI)
09:00 – 10:30
Tips and tricks for a successful catheter-based renal sympathetic denervation in difficult anatomies 10:45 – 12:45
Theatre Havane
Optimal management of your patient with coronary chronic total occlusion 09:00 – 10:30
The ‘undefeatable’ coronary chronic total occlusion: warriors at work 10:45 – 11:45
Challenging retrograde recanalisations of coronary chronic total occlusion 11:45 – 12:45
 SOURCE:

 

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Zinc-Finger Nucleases (ZFNs) and Transcription Activator–Like Effector Nucleases (TALENs)

Reporter: Larry H Bernstein, MD, FCAP

 

TALENs and ZFNs are associated with different mutation signatures

Y Kim,  J Kweon  & Jin-Soo Kim

Zinc-finger nucleases (ZFNs) and transcription activator–like effector nucleases (TALENs) are of great interest for genome engineering in higher eukaryotic cells and organisms. These enzymes

  1. contain the same FokI nuclease domain and
  2. induce site-specific DNA cleavage.

http://www.nature.com/nmeth/journal/v10/n3/extref/nmeth.2364-S1.pdf

http://www.nature.com/nmeth/journal/v10/n3/full/nmeth.2364.html?WT.ec_id=NMETH-201303

English: Bacterial multi-drug resistance syste...

English: Bacterial multi-drug resistance system: complex of dimeric transcription-activator protein BmrR with bound TPP, untwisting the DNA to position the two promoter sites (top) for transcription. Coordinates from PDB file 1R8E, Brennan lab; displayed in KiNG. (Photo credit: Wikipedia)

                                   

English: Diagram of a typical rAAV vector

English: Diagram of a typical rAAV vector (Photo credit: Wikipedia)

Splicing activation

Splicing activation (Photo credit: Allen Gathman)

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Reporter: Aviva Lev-Ari, PhD, RN

J Cardiovasc Transl Res. 2012 Sep 7. [Epub ahead of print]

Next Generation Diagnostics in Inherited Arrhythmia Syndromes : A Comparison of Two Approaches.

Ware JSJohn SRoberts AMBuchan RGong SPeters NSRobinson DOLucassen ABehr ERCook SA.

Source

MRC Clinical Sciences Centre, Imperial College London, London, UK, j.ware@imperial.ac.uk.

Abstract

Next-generation sequencing (NGS) provides an unprecedented opportunity to assess genetic variation underlying human disease. Here, we compared two NGS approaches for diagnostic sequencing in inherited arrhythmia syndromes. We compared PCR-based target enrichment and long-read sequencing (PCR-LR) with in-solution hybridization-based enrichment and short-read sequencing (Hyb-SR). The PCR-LR assay comprehensively assessed five long-QT genes routinely sequenced in diagnostic laboratories and “hot spots” in RYR2. The Hyb-SR assay targeted 49 genes, including those in the PCR-LR assay. The sensitivity for detection of control variants did not differ between approaches. In both assays, the major limitation was upstream target capture, particular in regions of extreme GC content. These initial experiences with NGS cardiovascular diagnostics achieved up to 89 % sensitivity at a fraction of current costs. In the next iteration of these assays we anticipate sensitivity above 97 % for all LQT genes. NGS assays will soon replace conventional sequencing for LQT diagnostics and molecular pathology.

PMID: 22956155 [PubMed]
Source: 
http://www.ncbi.nlm.nih.gov/pubmed/22956155

Researchers in the UK have compared a PCR-based and a capture hybridization-based assay for sequencing panels of inherited cardiovascular disease genes and have found both to be suitable for diagnostics in principle, though their sensitivity needs to be optimized.

According to James Ware, a clinical lecturer at Imperial College London, the purpose of the study, published online this month in the Journal of Cardiovascular Translational Research, was to evaluate different approaches for sequencing cardiovascular disease genes, both for molecular diagnosis and for large-scale resequencing research studies.

His group, in the National Institute for Health Research Royal Brompton Cardiovascular Biomedical Research Unit, is interested in a range of inherited heart disease types, including cardiomyopathies and inherited arrhythmia syndromes such as long QT syndrome.

For their study, they compared two next-gen sequencing assays: a PCR-based approach that uses Fluidigm’s Access Array to amplify 96 amplicons in five LQT genes and one other gene, followed by sequencing on the 454 GS Junior; and an in-solution hybridization approach that uses Agilent’s SureSelect to target 49 inherited arrhythmia genes and sequences them on Life Technologies’ SOLiD 4.

The study focused on the sensitivity of the assays, or how well they were able to capture their intended targets, rather than their specificity, or their ability to avoid false positives.

Ware said that at the time of the study, PCR and in-solution capture were the two main target selection methods available. The researchers are still using both approaches but are now employing “a wide range of sequencers” from various providers for both types of assays, including Illumina instruments and Life Tech’s Ion Torrent.

For their comparison, they analyzed 48 samples, of which they sequenced 33 with both approaches and 15 using either one or the other.

The samples included 19 known variants in three disease genes, of which the hybridization-SOLiD method detected 17 and the PCR-454 method 14. Undetected variants were generally in areas that were not well covered, either due to a failure in enrichment, sequencing, or because the alignment was not unique. One variant that was missed by both approaches fell in a very GC-rich region.

Consumables costs for both assays were considerably lower than with Sanger sequencing: While sequencing five genes by Sanger costs more than $700 in consumables, the five-gene PCR/454 assay cost about $55 and the 49-gene hybridization/SOLiD assay cost about $200, according to the study.

Turnaround time is the shortest for Sanger sequencing, which, according to the study, can be done in one day for five genes and 17 samples, not including sample prep. The PCR/454 assay takes about two days for target enrichment and sequencing 48 samples, and the hybridization/SOLiD assay takes about two weeks for sequencing alone, they wrote.

Overall, Ware said, both sequencing approaches performed “reasonably well” and are significantly cheaper than Sanger sequencing. He said that in the UK, molecular diagnosis for inherited cardiovascular disease has traditionally been performed by Sanger, at a cost of approximately £500 to £1,000 ($800 to $1,600) for several genes involved in a clinical condition. However, for cost reasons, not all relevant genes are usually sequenced.

Target selection was the performance-limiting step for both approaches, a result the researchers expected. “It sounds obvious, but not all genes are equally easy to target,” Ware said. For example, in the hybridization assay, the overall target coverage was about 98 percent, but for some genes, it was only 80 percent or 90 percent. The two most important genes in long QT syndrome, KCNQ1 and KCNH2, “proved to be the hardest to sequence.”

Thus, for diagnostic use of NGS gene panels, “it’s important to know not just how the system performs overall but really how it’s performing for the specific genes you’re interested in,” he said.

To use either approach in diagnostics, the target selection step would need to be optimized. Ware’s team has already improved both assays and is now trying them in a number of fully Sanger-sequenced samples to study both sensitivity and specificity.

Longer term, the sensitivity of next-gen sequencing could approach that of Sanger sequencing, he said. And even if it does not reach 100 percent, because NGS approaches can target so many more genes, “maybe you can afford a very slight tradeoff in the per-gene sensitivity if the overall diagnostic sensitivity of the panel goes up,” he said. “At the moment, because we don’t have that much experience in sequencing the less-common genes, we don’t exactly know where that tradeoff lies.” In addition, any gaps could be filled by Sanger sequencing, while the test would probably still be cost effective.

Each approach also has some features that make it more suitable for certain applications. The PCR-based method has a fast turnaround and an “extremely user-friendly workflow,” Ware said, but it can only accommodate a small number of genes at the moment. His team also found it to be easier to optimize and improve. Thus, in the short term, PCR and sequencing “is probably closer to providing a diagnostic solution,” he said, especially for conditions where only a few genes are causative.

The hybridization-based approach, on the other hand, has much greater capacity, and there are advantages in “having a single assay that covers everything,” he said. It might also be possible to detect copy number variants using this approach, but not the more limited PCR method, he added.

Ware and his colleagues are currently using the hybridization approach to study a large panel of genes in 2,000 well-phenotyped volunteers, both healthy individuals and heart disease patients.

They have also started to use the hybridization method to sequence the TTN gene, truncating mutations in which were recently found to be a common cause of dilated cardiomyopathy. They are running the TTN test routinely for patients consented for research diagnostic testing that is not available anywhere else. Because this gene is so large, it is “completely impractical to be sequenced by conventional Sanger,” Ware said.

Julia Karow tracks trends in next-generation sequencing for research and clinical applications for GenomeWeb’s In Sequenceand Clinical Sequencing News. E-mail her here or follow her GenomeWeb Twitter accounts at @InSequence and@ClinSeqNews.

 

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Molecular pathogen identification comes to the bedside

Reporter:  Larry H Bernstein, MD, FCAP

The developments in molecular diagnostics have been proceeding at a rapid pace.  Naturally it is not surprising that it would reach into clinical microbiology early.  Microbiology and virology have many methods for validation of type of pathogen, and the identification of new pathogens can require delay because of use of a State laboratory.  This will be less an issue with the consolidation of regional facilities and associated laboratories.

I present an example of point-of-care technology from the University of California, Davis developed by Gerald Kost and colleagues with UC Lawrence Livermore National Point-of-Care Technologies Center .

Tran NK, Wisner DH, Albertson TE, Cohen S, et al.  Multiplex polymerase chain reaction pathogen detection in patients with suspected septicemia after trauma, emergency, and burn surgery. Surgery 2012 Mar;151(3):456-63. Epub 2011 Oct 5.  nktran@ucdavis.edu

The goal of the study:  to determine the clinical value of multiplex polymerase chain reaction (PCR) study for enhancing pathogen detection in patients with suspected septicemia after trauma, emergency, and burn surgery.

Finding: PCR-based pathogen detection quickly reveals occult bloodstream infections in these high-risk patients and may accelerate the initiation of targeted antimicrobial therapy.

Type study: a prospective observational study

Population:  30 trauma and emergency surgery patients compared to 20 burn patients.

Method:  Whole- routine blood cultures (BCs) were tested using a new multiplex, PCR-based, pathogen detection system. PCR results were compared to culture data.

Arbitrated Case Review

Arbitrated case review was performed by a medical intensivist, 3 trauma surgeons, 3 burn surgeons, 1 microbiologist, and an infectious disease physician to determine antimicrobial adequacy based on paired PCR/BC results. The arbitrated case review process is adapted from a previous study. Physicians were first presented cases with only BC results. Cases were then represented with PCR results included.

Results:

  • PCR detected rapidly more pathogens than culture methods.
  • Acute Physiology and Chronic Health Evaluation II (APACHE II), Sequential Organ Failure Assessment (SOFA), and Multiple Organ Dysfunction (MODS) scores were greater in PCR-positive versus PCR-negative trauma and emergency surgery patients (P ≤ .033).
  • Negative PCR results (odds ratio, 0.194; 95% confidence interval, 0.045-0.840; P = .028) acted as an independent predictor of survival for the combined surgical patient population.

CONCLUSION:

  • PCR results were reported faster than blood culture results.
  • Severity scores were significantly greater in PCR-positive trauma and emergency surgery patients.
  • The lack of pathogen DNA as determined by PCR served as a significant predictor of survival in the combined patient population.
  • PCR testing independent of traditional prompts for culturing may have clinical value in burn patients.

NK Tran, et al.  Multiplex Polymerase Chain Reaction Pathogen Detection in Trauma, Emergency, and Burn Surgery Patients with Suspected Septicemia.  Surgery. 2012 March; 151(3): 456–463. PMID: 21975287 [PubMed – indexed for MEDLINE] PMCID: PMC3304499 On-line 2011 October 5.
doi:  10.1016/j.surg.2011.07.030
PMCID: PMC3304499.  NIHMSID: NIHMS288960

Plymerase chain reaction, PCR

Plymerase chain reaction, PCR (Photo credit: Wikipedia)

 

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Harvard Group Using Bio-Rad Digital PCR System as Part of NHGRI-Funded Study of Multi-Allelic CNV

 

Reporter: Aviva Lev-Ari, PhD, RN

August 23, 2012

Researchers in the Department of Genetics at the Harvard University Medical School have been awarded $500,000 by the National Institutes of Health for the first year of a four-year project to study multi-allelic copy number variation in the human genome.

As part of the research, the Harvard team is using a Bio-Rad QX100 Droplet Digital PCR system as one of two methods to analyze multi-allelic CNVs in human cohorts. The researchers are also using a computational method that compares available whole-genome sequencing data.

Steven McCarroll, a professor of genetics at Harvard Med and director of genetics at the Stanley Center for Psychiatric Research at the Broad Institute, is principal investigator on the grant, which is being administered by NIH’s National Human Genome Research Institute.

According to a recently published grant abstract, McCarroll and colleagues seek to analyze multi-allelic CNVs, which involve genes and other functional elements for which three or more segregating alleles give rise to a wide range of copy numbers — between two and 10 — per diploid human genome.

These multi-allelic CNVs have been “refractory to widely used analysis methods and are not assessed in the genome-scale molecular or statistical approaches used to study genetically complex phenotypes in humans,” the researchers wrote.

The project builds on research that McCarroll’s group previously conducted on characterizing multi-allelic duplication CNVs of a megabase-long inversion polymorphism in a particular locus of chromosome 17 called 17q21.31, which contains markers previously associated with female fertility, female meiotic recombination, and neurological disease.

As part of that research, published in the August 2012 issue of Nature Genetics, the group analyzed read depth in the locus by applying an algorithm called Genome Structure in Populations, or Genome STRiP, to whole-genome sequencing data from 946 unrelated individuals sampled as part of the 1000 Genomes Project; and used droplet-based digital PCR to analyze 120 parent-offspring trios from HapMap.

http://www.nature.com/ng/journal/v44/n8/full/ng.2334.html

They found that their measurements of integer copy number varied from two to eight, and were 99.1 percent concordant across 234 genotypes in overlapping samples, thus validating both the computational and digital PCR methods.

More specifically, for the digital PCR assay, the group designed a pair of PCR primers and a dual-labeled fluorescence-FRET oligonucleotide probe to both the CNV locus and a two-copy control locus. Then they used a droplet generator from QuantaLife to compartmentalize the PCR reaction into uniform 1-nanoliter emulsion-based droplets containing zero, one, or very few template molecules for each locus; and a droplet reader from QuantaLife to count the number of positive and negative droplets, comparing the droplet counts of the CNV locus to the control locus to determine absolute copy number.

QuantaLife originally developed the droplet-based digital PCR system, but was acquired in October by Bio-Rad, which rebranded the platform as the QX100 Droplet Digital PCR system (PCR Insider, 10/6/2011).

Annette Tumolo, director of the digital biology center at Bio-Rad, told PCR Insider this week that McCarroll has access to two such platforms, one of which is in use at Harvard and was obtained from QuantaLife, and one of which Bio-Rad sold to the Broad Institute.

Tumolo said that Bio-Rad maintains “an active and positive relationship” with the McCarroll lab. “They’ve gotten great results [with the QX100], and were able to rapidly publish the Nature Genetics paper,” Tumolo said.

Under the new NHGRI grant, McCarroll and colleagues plan to “accurately analyze mCNVs in reference populations” using both the computational and digital PCR approach, the researchers wrote in their grant abstract.

“By analyzing these data in a statistical framework that incorporates information about genotypes, allele frequencies, inheritance, and haplotypes, we will place mCNV alleles onto the haplotype maps created by HapMap and 1000 Genomes, and render mCNVs accessible to genotype imputation to the fullest extent possible,” the grant abstract states.

In addition, McCarroll’s group hopes to “deeply characterize mCNVs at 10 biomedically important loci, to understand these polymorphisms at the levels of population genetics, mutational rates and histories, and relationships to clinical phenotypes. Finally, we will pilot inexpensive in silico genome-wide association studies for mCNVs based on statistical imputation into existing GWAS data sets.”

The end goal of the project is to discover relationships between disease risk and gene dosage, which will help reveal the molecular etiology of human disease, the researchers wrote.

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Ben Butkus is senior editor of GenomeWeb’s premium content and the editor of PCR Insider. He covers technologies and trends in PCR, qPCR, nucleic acid amplification, and sample prep. E-mail him here or follow his GenomeWeb Twitter account at@PCRInsider.

 

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