OncoScape: Exploring the cancer aberration landscape by genomic data fusion

Although large-scale efforts for molecular profiling of cancer samples provide multiple data types for many samples, most approaches for finding candidate cancer genes rely on somatic mutations and DNA copy number only. We present a new method, OncoScape, which exploits five complementary data types across 11 cancer types to identify new candidate cancer genes. We find many rarely mutated genes that are strongly affected by other aberrations. We retrieve the majority of known cancer genes but also new candidates such as STK31 and MSRA with very high confidence. Several genes show a dual oncogene- and tumor suppressor-like behavior depending on the tumor type. Most notably, thewell-known tumor suppressor RB1 shows strong oncogene-like signal in colon cancer. We applied OncoScape to cell lines representing ten cancer types, providing the most comprehensive comparison of aberrations in cell lines and tumor samples to date. This revealed that glioblastoma, breast and colon cancer show strong similarity between cell lines and tumors, while head and neck squamous cell carcinoma and bladder cancer, exhibit very little similarity between cell lines and tumors. To facilitate exploration of the cancer aberration landscape, we created a web portal enabling interactive analysis of OncoScape results (http://ccb.nki.nl/software/oncoscape).Pattern Recognition and Bioinformatic

A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of Ca2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca 2+ Influx

Mutations in the CACNA1A gene, encoding the pore-forming Ca2.1 (P/Q-type) channel α subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α domain III (ΔF1502). Functional studies of ΔF1502 Ca2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba 2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α mutation, detected by whole-exome sequencing, and analyze its functional consequences on Ca2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca 2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca 2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca 2+ current density through ΔF1502 Ca2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of Ca2.1 within a wide range of voltage depolarization. ΔF1502 also slowed Ca2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on Ca2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca 2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function Ca2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A -associated phenotypic spectru

A Single Amino Acid Deletion (ΔF1502) in the S6 Segment of CaV2.1 Domain III Associated with Congenital Ataxia Increases Channel Activity and Promotes Ca2+ Influx

Mutations in the CACNA1A gene, encoding the pore-forming CaV2.1 (P/Q-type) channel α1A subunit, result in heterogeneous human neurological disorders, including familial and sporadic hemiplegic migraine along with episodic and progressive forms of ataxia. Hemiplegic Migraine (HM) mutations induce gain-of-channel function, mainly by shifting channel activation to lower voltages, whereas ataxia mutations mostly produce loss-of-channel function. However, some HM-linked gain-of-function mutations are also associated to congenital ataxia and/or cerebellar atrophy, including the deletion of a highly conserved phenylalanine located at the S6 pore region of α1A domain III (ΔF1502). Functional studies of ΔF1502 CaV2.1 channels, expressed in Xenopus oocytes, using the non-physiological Ba2+ as the charge carrier have only revealed discrete alterations in channel function of unclear pathophysiological relevance. Here, we report a second case of congenital ataxia linked to the ΔF1502 α1A mutation, detected by whole-exome sequencing, and analyze its functional consequences on CaV2.1 human channels heterologously expressed in mammalian tsA-201 HEK cells, using the physiological permeant ion Ca2+. ΔF1502 strongly decreases the voltage threshold for channel activation (by ~ 21 mV), allowing significantly higher Ca2+ current densities in a range of depolarized voltages with physiological relevance in neurons, even though maximal Ca2+ current density through ΔF1502 CaV2.1 channels is 60% lower than through wild-type channels. ΔF1502 accelerates activation kinetics and slows deactivation kinetics of CaV2.1 within a wide range of voltage depolarization. ΔF1502 also slowed CaV2.1 inactivation kinetic and shifted the inactivation curve to hyperpolarized potentials (by ~ 28 mV). ΔF1502 effects on CaV2.1 activation and deactivation properties seem to be of high physiological relevance. Thus, ΔF1502 strongly promotes Ca2+ influx in response to either single or trains of action potential-like waveforms of different durations. Our observations support a causative role of gain-of-function CaV2.1 mutations in congenital ataxia, a neurodevelopmental disorder at the severe-most end of CACNA1A-associated phenotypic spectrum.This work was supported by grants from the Spanish Ministry of Economy and Competitiveness (SAF2012-31089 to JMF-F; SEV-2012-0208 to Centre for Genomic Regulation, “Centro de Excelencia Severo Ochoa 2013-2017”; and MDM-2014-0370 through the “María de Maeztu” Programme for Units of Excellence in R&D to “Departament de Ciències Experimentals i de la Salut”), FEDER Funds, Fondo de Investigación Sanitaria, Instituto Carlos III, Spain (RIC RD12/0042/0014, Red HERACLES, and Grant PI12/1005 to AM). AM-G is a predoctoral fellow supported by Vall d’Hebron Institut de Recerca, Barcelona, Spain. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Brain MRI of the proband at the age of 14 months (A), 28 months (B), and 4 and a half years (C,D).

<p>After the initial normal findings (A), note the progressive cerebellar atrophy mainly involving the complete vermis (indicated by the arrows in B, C). The hemispheres, displaying prominence of the cerebellar folia, were eventually affected (D).</p

ΔF1502 effects on Ca²⁺ influx evoked by a 42 Hz train of 2 ms action potential-like waveforms (APWs).

<p>(A) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 10) and ΔF1502 (filled circles, n = 11) Ca_V2.1 channels expressed in tsA-201 HEK cells, before stimulation with a 42 Hz train of 2 ms APWs. In this series of experiments, maximal Ca²⁺ current density through Ca_V2.1 channels is still significantly reduced by ΔF1502 (left panel: from -94.26 ± 18.9 pA/pF (for WT, n = 10) to -47.76 ± 5.7 pA/pF (for ΔF1502, n = 11), P < 0.05, Student’s <i>t</i> test) and the significant left-shift induced by ΔF1502 on the Ca_V2.1 voltage-dependent activation is also noticed (right panel: WT V_{1/2 act} = 2.32 ± 1.18 mV (n = 10) <i>versus</i> ΔF1502 V_{1/2 act} = -17.74 ± 0.35 mV (n = 11), P < 0.0001, Student’s <i>t</i> test). (B) Representative Ca²⁺ current traces evoked by every 200^th pulse of a 42 Hz train of medium (2 ms) APWs (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146035#sec002" target="_blank">Materials and Methods</a> for details) obtained from two tsA-201 HEK cells expressing either WT (left) or ΔF1502 (right) Ca_V2.1 channels. Dotted lines stand for the zero current level. The corresponding current density-voltage relationships (left) and normalized I-V curves (right), obtained from these two cells before stimulation with a 42 Hz train of 2 ms APWs, are shown at the bottom (maximal Ca²⁺ current density through WT and ΔF1502 Ca_V2.1 channels are -115.28 pA/pF and -52.27 pA/pF, respectively; V_{1/2 act} values for WT and ΔF1502 Ca_V2.1 channels are 2.52 mV and -17.23 mV, respectively). (C) Average data for Ca²⁺ influx normalized by cell size (Q_Ca²⁺) in response to every 5^th pulse of a 42 Hz train of medium (2 ms) APWs, obtained from cells expressing WT (blue symbols, n = 10) or ΔF1502 (red symbols, n = 11) Ca_V2.1 channels.</p

<i>De novo</i> heterozygous <i>CACNA1A</i> deletion in congenital ataxia with cerebellar atrophy.

<p>(A) Pedigree of the affected individual carrying the <i>de novo</i> heterozygous ΔF1502 mutation. White symbols denote healthy individuals and grey, congenital ataxia. (B) Electropherograms showing the deleted nucleotides (bracket) (NM_001127221.1-transcript variant 3:c.4503-4505delCTT) leading to a F1052 deletion (NP_001120693.1). Note the double wild-type (WT) and mutant (ΔF1502) sequence in the patient’s electropherogram (heterozygous mutation carrier).</p

ΔF1502 induces a gain-of-function in the heterologously expressed Ca_V2.1 channel by affecting its activation and deactivation properties.

<p>(A) Representative current traces elicited by 20 ms depolarizing pulses from -80 mV to the indicated voltages (inset), illustrating the difference in voltage-dependence and activation kinetics between wild-type (WT) (left) and ΔF1502 (right) Ca_V2.1 channels. Dotted lines indicate the zero current level. (B) Representative current traces showing distinct deactivation kinetics of WT (left) and ΔF1502 (right) Ca_V2.1 channels, obtained by hyperpolarizing the cells during 30 ms at the indicated voltages (inset) following a 20 ms depolarizing pulse to +20 mV (for WT channels) or -5 mV (for ΔF1502 channels). The zero current level is indicated by dotted lines. (C) Average current density-voltage relationships (left) and normalized I-V curves (right) for WT (open circles, n = 27) and ΔF1502 (filled circles, n = 19) Ca_V2.1 channels expressed in tsA-201 HEK cells. Red box indicates the voltage range at which peak Ca²⁺ current densities through ΔF1502 channels exceed those produced by WT channels. Average V_{1/2 act}, k_act and V_rev values were (in mV): WT (open circles, n = 27) 3.8 ± 0.6, 3.5 ± 0.15 and 62.4 ± 1.4; ΔF1502 (filled circles, n = 19) -17.1 ± 0.9, 4.4 ± 0.19 and 51.6 ± 2.2, respectively. Average activation (D) and deactivation (E) kinetics of WT (open circles) and ΔF1502 Ca_V2.1 Ca²⁺ currents (filled circles) at the indicated voltages.</p

eDiVA-Classification and prioritization of pathogenic variants for clinical diagnostics

Mendelian diseases have shown to be an and efficient model for connecting genotypes to phenotypes and for elucidating the function of genes. Whole-exome sequencing (WES) accelerated the study of rare Mendelian diseases in families, allowing for directly pinpointing rare causal mutations in genic regions without the need for linkage analysis. However, the low diagnostic rates of 20-30% reported for multiple WES disease studies point to the need for improved variant pathogenicity classification and causal variant prioritization methods. Here, we present the exome Disease Variant Analysis (eDiVA; http://ediva.crg.eu), an automated computational framework for identification of causal genetic variants (coding/splicing single-nucleotide variants and small insertions and deletions) for rare diseases using WES of families or parent-child trios. eDiVA combines next-generation sequencing data analysis, comprehensive functional annotation, and causal variant prioritization optimized for familial genetic disease studies. eDiVA features a machine learning-based variant pathogenicity predictor combining various genomic and evolutionary signatures. Clinical information, such as disease phenotype or mode of inheritance, is incorporated to improve the precision of the prioritization algorithm. Benchmarking against state-of-the-art competitors demonstrates that eDiVA consistently performed as a good or better than existing approach in terms of detection rate and precision. Moreover, we applied eDiVA to several familial disease cases to demonstrate its clinical applicability.This project has received funding from the “la Caixa” Foundation, the CRG emergent translational research award and the European Union's H2020 Research and Innovation Programme under the grant agreement No 635290 (PanCanRisk)

eDiVA-Classification and prioritization of pathogenic variants for clinical diagnostics

Mendelian diseases have shown to be an and efficient model for connecting genotypes to phenotypes and for elucidating the function of genes. Whole-exome sequencing (WES) accelerated the study of rare Mendelian diseases in families, allowing for directly pinpointing rare causal mutations in genic regions without the need for linkage analysis. However, the low diagnostic rates of 20-30% reported for multiple WES disease studies point to the need for improved variant pathogenicity classification and causal variant prioritization methods. Here, we present the exome Disease Variant Analysis (eDiVA; http://ediva.crg.eu), an automated computational framework for identification of causal genetic variants (coding/splicing single-nucleotide variants and small insertions and deletions) for rare diseases using WES of families or parent-child trios. eDiVA combines next-generation sequencing data analysis, comprehensive functional annotation, and causal variant prioritization optimized for familial genetic disease studies. eDiVA features a machine learning-based variant pathogenicity predictor combining various genomic and evolutionary signatures. Clinical information, such as disease phenotype or mode of inheritance, is incorporated to improve the precision of the prioritization algorithm. Benchmarking against state-of-the-art competitors demonstrates that eDiVA consistently performed as a good or better than existing approach in terms of detection rate and precision. Moreover, we applied eDiVA to several familial disease cases to demonstrate its clinical applicability.This project has received funding from the “la Caixa” Foundation, the CRG emergent translational research award and the European Union's H2020 Research and Innovation Programme under the grant agreement No 635290 (PanCanRisk)

Evolutionary conservation of the F1502 residue and predicted location at the channel pore.

<p>(A) Sequence alignment of individual S6 segments at domains I to IV (DI-DIV) of human Ca_V2.x channel α₁ subunits (P/Q type Ca_V2.1; N-type Ca_V2.2; R-type Ca_V2.3), human Ca_V1.x (L-type) channel α₁ subunits, and the bacterial sodium channel Na_VAb (top); sequence alignment of S6-DIII of Ca_V2.1 channels from different species (as indicated). The three Phenylalanine’s group (in red) is conserved in the human Ca_V2.1 channel α_1A subunit, where F1502 is located at the third position. This particular amino acid residue is only conserved in S6-DIII of Ca_V2 type channels. The phenylalanine’s group is totally conserved in S6-DIII of Ca_V2.1 channels from different species. The alignments were performed with T-Coffee (T-Coffee). (B,C,D) Location of the F1502 homologous methionine residue (M209), using the Na_VAb structure as a model (PDB 4EKW). A methionine residue is also present at the F1502 position in L-type channels. The side view (B) show a red highlighted M209 residue in Na_VAb, which lines the inner pore vestibule of the channel. A view from the cytoplasm looking up through the channel pore show the arrangement of M209 residue in the four Na_VAb subunits (C), and a zoom of the pore region from the same view is shown in (D). Images were generated using UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311) [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0146035#pone.0146035.ref079" target="_blank">79</a>].</p