Short In-frame Deletions Research Articles

Rice Yellow Mottle Virus resistance by genome editing of the Oryza sativa L. ssp. japonica nucleoporin gene OsCPR5.1 but not OsCPR5.2.

Rice yellow mottle virus (RYMV) causes one of the most devastating rice diseases in Africa. Management of RYMV is challenging. Genetic resistance provides the most effective and environment-friendly control. The recessive resistance locus rymv2 (OsCPR5.1) had been identified in African rice (Oryza glaberrima), however, introgression into Oryza sativa ssp. japonica and indica remains challenging due to crossing barriers. Here, we evaluated whether CRISPR/Cas9 genome editing of the two rice nucleoporin paralogs OsCPR5.1 (RYMV2) and OsCPR5.2 can be used to introduce RYMV resistance into the japonica variety Kitaake. Both paralogs had been shown to complement the defects of the Arabidopsis atcpr5 mutant, indicating partial redundancy. Despite striking sequence and structural similarities between the two paralogs, only oscpr5.1 loss-of-function mutants were fully resistant, while loss-of-function oscpr5.2 mutants remained susceptible, intimating that OsCPR5.1 plays a specific role inRYMVsusceptibility. Notably, edited lines with short in-frame deletions or replacements in the N-terminal domain (predicted to be unstructured) of OsCPR5.1 were hypersusceptible to RYMV. In contrast to mutations in the single Arabidopsis AtCPR5 gene, which caused severely dwarfed plants, oscpr5.1 and oscpr5.2 single and double knockout mutants showed neither substantial growth defects nor symptoms indicative lesion mimic phenotypes, possibly reflecting functional differentiation. The specific editing of OsCPR5.1, while maintaining OsCPR5.2 activity, provides a promising strategy for generating RYMV-resistance in elite Oryza sativa lines as well as for effective stacking with other RYMV resistance genes or other traits.

Whole exome sequencing analysis of canine urothelial carcinomas without BRAF V595E mutation: Short in-frame deletions in BRAF and MAP2K1 suggest alternative mechanisms for MAPK pathway disruption.

Molecular profiling studies have shown that 85% of canine urothelial carcinomas (UC) harbor an activating BRAF V595E mutation, which is orthologous to the V600E variant found in several human cancer subtypes. In dogs, this mutation provides both a powerful diagnostic marker and a potential therapeutic target; however, due to their relative infrequency, the remaining 15% of cases remain understudied at the molecular level. We performed whole exome sequencing analysis of 28 canine urine sediments exhibiting the characteristic DNA copy number signatures of canine UC, in which the BRAF V595E mutation was undetected (UDV595E specimens). Among these we identified 13 specimens (46%) harboring short in-frame deletions within either BRAF exon 12 (7/28 cases) or MAP2K1 exons 2 or 3 (6/28 cases). Orthologous variants occur in several human cancer subtypes and confer structural changes to the protein product that are predictive of response to different classes of small molecule MAPK pathway inhibitors. DNA damage response and repair genes, and chromatin modifiers were also recurrently mutated in UDV595E specimens, as were genes that are positive predictors of immunotherapy response in human cancers. Our findings suggest that short in-frame deletions within BRAF exon 12 and MAP2K1 exons 2 and 3 in UDV595E cases are alternative MAPK-pathway activating events that may have significant therapeutic implications for selecting first-line treatment for canine UC. We developed a simple, cost-effective capillary electrophoresis genotyping assay for detection of these deletions in parallel with the BRAF V595E mutation. The identification of these deletion events in dogs offers a compelling cross-species platform in which to study the relationship between somatic alteration, protein conformation, and therapeutic sensitivity.

SHINE: protein language model-based pathogenicity prediction for short inframe insertion and deletion variants.

Accurate variant pathogenicity predictions are important in genetic studies of human diseases. Inframe insertion and deletion variants (indels) alter protein sequence and length, but not as deleterious as frameshift indels. Inframe indel Interpretation is challenging due to limitations in the available number of known pathogenic variants for training. Existing prediction methods largely use manually encoded features including conservation, protein structure and function, and allele frequency to infer variant pathogenicity. Recent advances in deep learning modeling of protein sequences and structures provide an opportunity to improve the representation of salient features based on large numbers of protein sequences. We developed a new pathogenicity predictor for SHort Inframe iNsertion and dEletion (SHINE). SHINE uses pretrained protein language models to construct a latent representation of an indel and its protein context from protein sequences and multiple protein sequence alignments, and feeds the latent representation into supervised machine learning models for pathogenicity prediction. We curated training data from ClinVar and gnomAD, and created two test datasets from different sources. SHINE achieved better prediction performance than existing methods for both deletion and insertion variants in these two test datasets. Our work suggests that unsupervised protein language models can provide valuable information about proteins, and new methods based on these models can improve variant interpretation in genetic analyses.

Macrocyclization of Quinazoline-Based EGFR Inhibitors Leads to Exclusive Mutant Selectivity for EGFR L858R and Del19.

Activating mutations in the epidermal growth factor receptor (EGFR) are frequent oncogenic drivers of non-small-cell lung cancer (NSCLC). The most frequent alterations in EGFR are short in-frame deletions in exon 19 (Del19) and the missense mutation L858R, which both lead to increased activity and sensitization of NSCLC to EGFR inhibition. The first approved EGFR inhibitors used for first-line treatment of NSCLC, gefitinib and erlotinib, are quinazoline-based. However, both inhibitors have several known off-targets, and they also potently inhibit wild-type (WT) EGFR, resulting in side effects. Here, we applied a macrocyclic strategy on a quinazoline-based scaffold as a proof-of-concept study with the goal of increasing kinome-wide selectivity of this privileged inhibitor scaffold. Kinome-wide screens and SAR studies yielded 3f, a potent inhibitor for the most common EGFR mutation (EGFR Del19: 119 nM) with selectivity against the WT receptor (EGFR: >10 μM) and the kinome.

High-Resolution CRISPR Functional Genomics Reveals Critical Vulnerabilities of the NuRD Complex for Fetal Hemoglobin Control

Hemoglobinopathies resulting from mutations of the adult beta-globin gene affect millions worldwide. Persistently elevated levels of fetal hemoglobin (HbF) are without consequence in healthy individuals but convey a major clinical benefit in patients with sickle cell disease and beta-thalassemia. Fetal globin silencing relies on interactions between DNA binding factors and chromatin readers, writers, and erasers acting within multiprotein nuclear complexes. The nucleosome remodeling and deacetylase (NuRD) complex is an epigenetic chromatin modifier, including ATP-dependent nucleosome remodeling and histone deacetylase activities, that has been implicated in HbF control. BCL11A and ZBTB7A, two key transcription factors required for HbF repression in adult erythroid cells, physically interact with NuRD complex members. In order to identify potential target sites within the NuRD complex with maximal HbF de-repression and minimal cellular toxicity potential we set out to dissect the function of the NuRD complex in adult erythroid precursors by dense mutagenesis. We performed CRISPR-Cas9 mediated saturating mutagenesis of all coding regions of human NuRD complex members in human umbilical cord blood-derived erythroid progenitor (HUDEP-2) adult erythroid cells with HbF expression as the primary readout and cell fitness as a counter-screen. This approach distinguished four classes of NuRD complex members: those required for HbF repression but dispensable for cellular fitness (such as MTA2, MBD2, HDAC2, and GATAD2A), those required for both HbF repression and cell fitness (such as CHD4), those only required for cell fitness (such as RBBP4), and many not essential for either HbF repression or cell fitness. The NuRD paralogs essential for HbF repression correlated with the abundance of peptides retrieved by MTA2 immunoprecipitation followed by mass spectrometry, suggesting a NuRD sub-complex that silences fetal globin. We demonstrated that this tiling CRISPR screen approach identifies residues at which short in-frame deletions result in loss of function, including at catalytic residues of CHD4 and HDAC2, additional NuRD domains and conserved regions of unknown significance. By mapping functional data onto existing structures we detected sequences essential for HbF repression within the ELM2 and BAH domains of MTA2 that mediate key protein-protein interactions with HDAC1/2 and with chromatin. At CHD4, we found numerous critical positions where in-frame alleles resulted in robust HbF elevation to a greater magnitude than disruption of any other NuRD member. However nearly all of these HbF-inducing perturbations at CHD4 were inversely correlated with cell fitness. In contrast, we observed that in-frame deletions within the CHDCT2 domain of CHD4 resulted in profound elevation of HbF level but spared the negative impact on cell growth. Overall, these results demonstrate a sub-complex of NuRD essential for HbF silencing and identify key interfaces including at the potent HbF repressor CHD4 which could serve as selective small molecule development targets for therapeutic HbF re-induction. DisclosuresOrkin:Epizyme Inc.: Consultancy; Bioverativ: Consultancy.

The prognostic value of cytoskeletal regulation and vesicle trafficking signaling pathway mutation in breast cancer.

e13068 Background: Breast cancer (BC) is the most commonly diagnosed cancer in 2020, with an estimated 2.3 million new cases and the leading cause of cancer death in women. The cytoskeleton is a dynamic system of protein filaments that is regulated by dozens of proteins which are, in turn, regulated by phosphoinositides. Cytoskeletal regulation and vesicle trafficking (CRVT) signaling pathway is associated with many changes in the structure and movements of cells. Few studies addressing the association between genetic mutation of the CRVT pathway and the disease prognosis have been reported up to now. Here we investigate the genetic status of CRVT pathway as a biomarker for predicting overall survival (OS) and relapse-free survival (RFS) for breast cancer patients. Methods: We used cBioPortal platform to analyze a cohort of 10646 breast cancer samples. Mutations in 7 genes ( APC, BAP1, KEAP1, PAK1, PTEN, SRC, STAT3 ) are the most common indication in the CRVT signaling pathway. We define overall survival (OS) as the time between the procedure date when the tumor specimen was collected and the date of death or last follow-up visit. And we define relapse-free survival (RFS) as the time from surgical resection to relapse of disease. We estimated and compared the survival curves of these two groups using Kaplan-Meier method and log-rank tests. The statistical significance level was set to 5% ( p < 0.05). Results: Genomic alterations of the CRVT-indicator genes were found in 2582 (n = 2582/10646, 24.25%) samples with sorts of alterations identified including variants of non-synonymous mutations, splicing mutations, short in-frame insertion and deletion, short frame shift insertion and deletion, and copy number gain and loss. PTEN was most commonly altered (n = 2329), followed by PAK1 (n = 863), APC (n = 718), STAT3 (n = 341), KEAP1 (n = 317), BAP1(n = 292), and SRC (n = 278). Based on the result, patients were divided into the CRVT and non-CRVT group. The median OS of the CRVT group was 141.57 moth, which was significantly shorter than the OS of the non-CRVT group (log-rank test p= 0.000662). The median RFS of the CRVT group was 167.76 moth, which was also significantly shorter than the RFS of the non-CRVT group (log-rank test p= 0.0169). Conclusions: To the best of our knowledge, this study is the first investigation to evaluate the prognostic value of CRVT pathway mutation in patients with breast cancer. Our investigation shows that CRVT pathway mutation could significantly predict poor prognosis in breast cancer patients. The result of our analysis should be better confirmed with additional relevant research in the future using updated analyses.

Ratpost: a searchable database of protein-inactivating sequence variations in 40 sequenced rat-inbred strains.

Rat-inbred strains are essential as scientific tools. We have analyzed the publicly available genome sequences of 40 rat-inbred strains and provide an overview of sequence variations leading to amino acid changes in protein-coding genes, premature STOP codons or loss of STOP codons, and short in-frame insertions and deletions of all protein-coding genes across all these inbred lines. We provide an overview of the predicted impact on protein function of all these affected proteins in the database, by comparing their sequence with the sequences of the rat reference strain BN/SsNHsdMcwi. We also investigate the flaws of the protein-coding sequences of this reference strain itself, by comparing them with a consensus genome. These data can be retrieved via a searchable website (Ratpost.be) and allow a global, better interpretation of genetic background effects and a source of naturally defective alleles in these 40 sequenced classical and high-priority rat-inbred strains.

Targeting female flight for genetic control of mosquitoes.

Aedes aegypti Act4 is a paralog of the Drosophila melanogaster indirect flight muscle actin gene Act88F. Act88F has been shown to be haploinsufficient for flight in both males and females (amorphic mutants are dominant). Whereas Act88F is expressed in indirect flight muscles of both males and females, expression of Act4 is substantially female-specific. We therefore used CRISPR/Cas9 and homology directed repair to examine the phenotype of Act4 mutants in two Culicine mosquitoes, Aedes aegypti and Culex quinquefasciatus. A screen for dominant female-flightless mutants in Cx. quinquefasciatus identified one such mutant associated with a six base pair deletion in the CxAct4 coding region. A similar screen in Ae. aegypti identified no dominant mutants. Disruption of the AeAct4 gene by homology-dependent insertion of a fluorescent protein marker cassette gave a recessive female-flightless phenotype in Ae. aegypti. Reproducing the six-base deletion from Cx. quinquefasciatus in Ae. aegypti using oligo-directed mutagenesis generated dominant female-flightless mutants and identified additional dominant female-flightless mutants with other in-frame insertions or deletions. Our data indicate that loss of function mutations in the AeAct4 gene are recessive but that short in-frame deletions produce dominant-negative versions of the AeAct4 protein that interfere with flight muscle function. This makes Act4 an interesting candidate for genetic control methods, particularly population-suppression gene drives targeting female viability/fertility.

Folliculin variants linked to Birt-Hogg-Dubé syndrome are targeted for proteasomal degradation.

Germline mutations in the folliculin (FLCN) tumor suppressor gene are linked to Birt-Hogg-Dubé (BHD) syndrome, a dominantly inherited genetic disease characterized by predisposition to fibrofolliculomas, lung cysts, and renal cancer. Most BHD-linked FLCN variants include large deletions and splice site aberrations predicted to cause loss of function. The mechanisms by which missense variants and short in-frame deletions in FLCN trigger disease are unknown. Here, we present an integrated computational and experimental study that reveals that the majority of such disease-causing FLCN variants cause loss of function due to proteasomal degradation of the encoded FLCN protein, rather than directly ablating FLCN function. Accordingly, several different single-site FLCN variants are present at strongly reduced levels in cells. In line with our finding that FLCN variants are protein quality control targets, several are also highly insoluble and fail to associate with the FLCN-binding partners FNIP1 and FNIP2. The lack of FLCN binding leads to rapid proteasomal degradation of FNIP1 and FNIP2. Half of the tested FLCN variants are mislocalized in cells, and one variant (ΔE510) forms perinuclear protein aggregates. A yeast-based stability screen revealed that the deubiquitylating enzyme Ubp15/USP7 and molecular chaperones regulate the turnover of the FLCN variants. Lowering the temperature led to a stabilization of two FLCN missense proteins, and for one (R362C), function was re-established at low temperature. In conclusion, we propose that most BHD-linked FLCN missense variants and small in-frame deletions operate by causing misfolding and degradation of the FLCN protein, and that stabilization and resulting restoration of function may hold therapeutic potential of certain disease-linked variants. Our computational saturation scan encompassing both missense variants and single site deletions in FLCN may allow classification of rare FLCN variants of uncertain clinical significance.

New Drosophila Circadian Clock Mutants Affecting Temperature Compensation Induced by Targeted Mutagenesis of Timeless.

Drosophila melanogaster has served as an excellent genetic model to decipher the molecular basis of the circadian clock. Two key proteins, PERIOD (PER) and TIMELESS (TIM), are particularly well explored and a number of various arrhythmic, slow, and fast clock mutants have been identified in classical genetic screens. Interestingly, the free running period (tau, τ) is influenced by temperature in some of these mutants, whereas τ is temperature-independent in other mutant lines as in wild-type flies. This, so-called “temperature compensation” ability is compromised in the mutant timeless allele “ritsu” (timrit), and, as we show here, also in the timblind allele, mapping to the same region of TIM. To test if this region of TIM is indeed important for temperature compensation, we generated a collection of new mutants and mapped functional protein domains involved in the regulation of τ and in general clock function. We developed a protocol for targeted mutagenesis of specific gene regions utilizing the CRISPR/Cas9 technology, followed by behavioral screening. In this pilot study, we identified 20 new timeless mutant alleles with various impairments of temperature compensation. Molecular characterization revealed that the mutations included short in-frame insertions, deletions, or substitutions of a few amino acids resulting from the non-homologous end joining repair process. Our protocol is a fast and cost-efficient systematic approach for functional analysis of protein-coding genes and promoter analysis in vivo. Interestingly, several mutations with a strong temperature compensation defect map to one specific region of TIM. Although the exact mechanism of how these mutations affect TIM function is as yet unknown, our in silico analysis suggests they affect a putative nuclear export signal (NES) and phosphorylation sites of TIM. Immunostaining for PER was performed on two TIM mutants that display longer τ at 25°C and complete arrhythmicity at 28°C. Consistently with the behavioral phenotype, PER immunoreactivity was reduced in circadian clock neurons of flies exposed to elevated temperatures.

High-Resolution Sequencing of Viral Populations during Early Simian Immunodeficiency Virus Infection Reveals Evolutionary Strategies for Rapid Escape from Emerging Env-Specific Antibody Responses.

Primate lentiviruses, including the human and simian immunodeficiency viruses (HIV and SIV), produce infections marked by persistent, ongoing viral replication. This occurs despite the presence of virus-specific adaptive immune responses, including antibodies targeting the viral envelope glycoprotein (Env), and evolution of antibody-escape variants is a well-documented feature of lentiviral infection. Here, we examined the evolutionary dynamics of the SIV env gene during early infection (≤29 weeks postinfection) in a cohort of four SIVmac251-infected rhesus macaques. We tracked env evolution during acute and early infection using frequent sampling and ultradeep sequencing of viral populations, capturing a transmission bottleneck and the subsequent reestablishment of Env diversity. A majority of changes in the gp120 subunit mapped to two short clusters, one in the first variable region (V1) and one in V4, while most changes in the gp41 subunit appeared in the cytoplasmic domain. Variation in V1 was dominated by short duplications and deletions of repetitive sequence, while variation in V4 was marked by short in-frame deletions and closely overlapping substitutions. The most common substitutions in both patches did not alter viral replicative fitness when tested using a highly sensitive, deep-sequencing-based competition assay. Our results, together with the observation that very similar or identical patterns of sequence evolution also occur in different macaque species infected with related but divergent strains of SIV, suggest that resistance to early, strain-specific anti-Env antibodies is the result of temporally and mutationally predictable pathways of escape that occur during the early stages of infection.IMPORTANCE The envelope glycoprotein (Env) of primate lentiviruses mediates entry by binding to host cell receptors followed by fusion of the viral membrane with the cell membrane. The exposure of Env complexes on the surface of the virion results in targeting by antibodies, leading to selection for virus escape mutations. We used the SIV/rhesus macaque model to track in vivo evolution of variation in Env during acute/early infection in animals with and without antibody responses to Env, uncovering remarkable variation in animals with antibody responses within weeks of infection. Using a deep-sequencing-based fitness assay, we found substitutions associated with antibody escape had little to no effect on inherent replicative capacity. The ability to readily propagate advantageous changes that incur little to no replicative fitness costs may be a mechanism to maintain continuous replication under constant immune selection, allowing the virus to persist for months to years in the infected host.

The role of the cytopathologist in subtyping and epidermal growth factor receptor testing in non-small cell lung cancer: An institutional experience.

Approximately 10% of non-small-cell lung cancer (NSCLC) remains unclassifiable as NSCLC-not otherwise specified (NOS), after using a panel of immunomarkers. The present study was undertaken to assess sensitivity and specificity of immunomarkers in sub-typing NSCLC on fine needle aspiration cytology (FNAC). Epidermal growth factor receptor (EGFR) mutations were also detected in these samples. Sixty cases of NSCLC including 15 squamous cell carcinoma (SCC), 15 adenocarcinomas (ADC) and 30 NSCLC-NOS reported on FNAC were included in the study. A panel of CK7, CK5/6, TTF-1 and p63 was applied in these cases. DNA was extracted from 54 cases including 14 effusion samples, and EGFR mutations were detected. Classic ADC cases (n=15) showed 73.3% TTF-1 positivity and 100% CK7 positivity. Two cases of ADC showed aberrant expression of p63 and 2 cases showed CK5/6 positivity. 80% of classic SCC cases (n=15) showed strong nuclear p63 positivity and 86.6% were positive for CK5/6. TTF-1 was seen exclusively in ADC cases. Immunohistochemistry (IHC) using two immunomarkers (TTF-1 and p63) helped in subtyping 24/30(80%) cases of NSCLC. EGFR mutations were detected in 9/54 (16.7%) cases, and the most common mutation was short in-frame deletion in Exon 19. More than 90% of NSCLC can be sub-typed on cytology samples with the help of immunochemistry. The sensitivity of TTF-1, p63, CK5/6 and CK7 was 73.3%, 80%, 100% and 100%, respectively. The specificity of TTF-1, p63, CK5/6 and CK7 was 100%, 86.6%, 86.6% and 93.3%, respectively. TTF-1 is highly specific, and almost 80% of NSCLC-NOS cases can be sub-typed using TTF-1 and p63. EGFR mutations can be detected in cytology samples, and 16.7% samples were positive for EGFR mutations.

P3.02b-070 NSCLC with Detectable EGFR Mutation: Institutional Experience: Topic: EGFR Clinical

EGFR is one of the most commonly mutated proto-oncogenes in lung cancer. The frequency of such mutations varies from approximately 10% of lung adenocarcinomas in North American and European populations to as high as 50% in Asia. The leucine to arginine substitution at position 858 (L858R) in exon 21 and short in-frame deletions in exon 19 are the most common sensitizing mutations, comprising approximately 90% of cases. Approximately 50% of EGFR TKI resistance is due to a second site mutation, the T790M mutation occurring within exon 20. We describe our experience in patients with lung cancer with detectable EGFR mutation. Describe the epidemiological characteristics and the incidence of mutations in patients diagnosed with advanced NSCLC in our institution. Analyze toxicities and adherence to treatment. Evaluate treatments performed and the problem of the analysis of the biopsy. We analyzed patients from June 2012 to date. We found 61/326 (18.7%) patients with detectable EGFR mutation, 47 women (77%) and 39% smokers, 47 patients had advanced or unresectable disease. The most common sites of metastases were bone and lymph nodes. 27 of the 61 patients had mutation of exon 19, 27 patients with mutation of exon 21, others in 18 and 20. Five of them with detectable T790M mutation confirmed by repeat biopsy after progressing to ITK. The most common side effects were diarrhea and rash, 12 patients had toxicities which had to reduce dose or discontinue treatment. The most common side effects were diarrhea and rash, 12 patients had toxicities which had to reduce dose or discontinue treatment. We had a low incidence of invalid or not evaluable biopsies. although not all patients with detectable mutation were treated with ITK, who received such treatment had good adhesion and a low percentage of patients had to discontinue treatment.

Systematic analysis of somatic mutations driving cancer: Uncovering functional protein regions in disease development

Recent advances in sequencing technologies enable the large-scale identification of genes that are affected by various genetic alterations in cancer. However, understanding tumor development requires insights into how these changes cause altered protein function and impaired network regulation in general and/or in specific cancer types. In this work we present a novel method called iSiMPRe that identifies regions that are significantly enriched in somatic mutations and short in-frame insertions or deletions (indels). Applying this unbiased method to the complete human proteome, by using data enriched through various cancer genome projects, we identified around 500 protein regions which could be linked to one or more of 27 distinct cancer types. These regions covered the majority of known cancer genes, surprisingly even tumor suppressors. Additionally, iSiMPRe also identified novel genes and regions that have not yet been associated with cancer. While local somatic mutations correspond to only a subset of genetic variations that can lead to cancer, our systematic analyses revealed that they represent an accompanying feature of most cancer driver genes regardless of the primary mechanism by which they are perturbed during tumorigenesis. These results indicate that the accumulation of local somatic mutations can be used to pinpoint genes responsible for cancer formation and can also help to understand the effect of cancer mutations at the level of functional modules in a broad range of cancer driver genes. This article was reviewed by Sandor Pongor, Michael Gromiha and Zoltan Gaspari.

Mutational Consequences of Ciprofloxacin in Escherichia coli.

We examined the mutagenic specificity of the widely used antibiotic ciprofloxacin (CPR), which displays weak to moderate mutagenic activity in several bacteria and generates short in-frame deletions in rpoB in Staphylococcus aureus To determine the spectrum of mutations in a system where any gene knockout would result in a recovered mutant, including frameshifts and both short and long deletions, we examined CPR-induced mutations in the thymidylate synthase-encoding thyA gene. Here, any mutation resulting in loss of thymidylate synthase activity generates trimethoprim (Trm) resistance. We found that deletions and insertions in all three reading frames predominated in the spectrum. They tend to be short deletions and cluster in two regions, one being a GC-rich region with potential extensive secondary structures. We also exploited the well-characterized rpoB-Rif(r) system in Escherichia coli to determine that cells grown in the presence of sublethal doses of CPR not only induced short in-frame deletions in rpoB, but also generated base substitution mutations resulting from induction of the SOS system. Some of the specific point mutations prominent in the spectrum of a strain that overproduces the dinB-encoded Pol IV were also present after growth in CPR. However, these mutations disappeared in CPR-treated dinB mutants, whereas the deletions remained. Moreover, CPR-induced deletions also occurred in a strain lacking all three SOS-induced polymerases. We discuss the implications of these findings for the consequences of overuse of CPR and other antibiotics.

Systematic analysis of somatic mutations driving cancer: uncovering functional protein regions in disease development.

BackgroundRecent advances in sequencing technologies enable the large-scale identification of genes that are affected by various genetic alterations in cancer. However, understanding tumor development requires insights into how these changes cause altered protein function and impaired network regulation in general and/or in specific cancer types.ResultsIn this work we present a novel method called iSiMPRe that identifies regions that are significantly enriched in somatic mutations and short in-frame insertions or deletions (indels). Applying this unbiased method to the complete human proteome, by using data enriched through various cancer genome projects, we identified around 500 protein regions which could be linked to one or more of 27 distinct cancer types. These regions covered the majority of known cancer genes, surprisingly even tumor suppressors. Additionally, iSiMPRe also identified novel genes and regions that have not yet been associated with cancer.ConclusionsWhile local somatic mutations correspond to only a subset of genetic variations that can lead to cancer, our systematic analyses revealed that they represent an accompanying feature of most cancer driver genes regardless of the primary mechanism by which they are perturbed during tumorigenesis. These results indicate that the accumulation of local somatic mutations can be used to pinpoint genes responsible for cancer formation and can also help to understand the effect of cancer mutations at the level of functional modules in a broad range of cancer driver genes.ReviewersThis article was reviewed by Sándor Pongor, Michael Gromiha and Zoltán Gáspári.Electronic supplementary materialThe online version of this article (doi:10.1186/s13062-016-0125-6) contains supplementary material, which is available to authorized users.

117P Classification of EGFR mutations in Pakistani lung adenocarcinoma for predicting response to targeted therapy

Lung cancer is the 4th leading cause of death in all cancers. Adenocarcinomas of lung constitute up to 50% of NSCLC. In Pakistan like rest of the world lung cancer prevalence is high (15%) and primary lung adenocarcinoma constitute about 40–45% of primary lung cancer. Mutations in EGFR tyrosine kinase domain have been reported in adenocarcinoma. EGFR mutation screening has become imperative for the selection of metastatic NSCLC patients eligible for targeted treatment. This report presents distribution of EGFR mutations in 315 NSCLC patients. EGFR mutation in tumor samples was screened by multiplex real time PCR (Roche Diagnostics(R), USA) according to the manufacturer's instructions. Briefly, DNA from FFPE tissue, obtained from Histopathology sections, was extracted and amplified with primers and probes specific to 43 different EGFR mutations in Cobas z 480 instrument. The assay can detect 43 mutations in four exons (18–21) of EGFR gene, including several point mutations, deletions and insertions. Out of 315 patients, 208 were male and 107 were females; male to female ratio was 2:1. The mean age of the patients was 62 years and age distribution was 23 and 85 years. On the basis of immuno histopathological finding tumors were categorized into two groups; well to poorly differentiated adenocarcinoma 231(73%) and metastatic adenocarcinoma 84 (27%). EGFR mutation Del 19 was detected in 38 patients, its short in-frame deletion in exon 19, clustered around the amino acid resideus747–750(most common variant delL746-A750, delL747-T751insS, and delL747-P753insS). Whereas L858R point mutation (substitution of amino acid leucine to arginine) was found in 27 patients. In two patients compound mutation, two point mutations together [S768I and G719X] and another one insertion (amino acid residues inserted) on Exon 20 was observed. L858R (exon 21) and deletion 19 exon were most frequent mutations in primary lung adenocarcinoma patients of Pakistani origin, in published literature female preponderance was significant. Our study showed Del 19 and L858R were the most frequent mutations in Pakistani lung cancer patients. In additions, 25% of the patients were found eligible for targeted therapy.

Multilevel biological characterization of exomic variants at the protein level significantly improves the identification of their deleterious effects.

There are now many predictors capable of identifying the likely phenotypic effects of single nucleotide variants (SNVs) or short in-frame Insertions or Deletions (INDELs) on the increasing amount of genome sequence data. Most of these predictors focus on SNVs and use a combination of features related to sequence conservation, biophysical, and/or structural properties to link the observed variant to either neutral or disease phenotype. Despite notable successes, the mapping between genetic variants and their phenotypic effects is riddled with levels of complexity that are not yet fully understood and that are often not taken into account in the predictions, despite their promise of significantly improving the prediction of deleterious mutants. We present DEOGEN, a novel variant effect predictor that can handle both missense SNVs and in-frame INDELs. By integrating information from different biological scales and mimicking the complex mixture of effects that lead from the variant to the phenotype, we obtain significant improvements in the variant-effect prediction results. Next to the typical variant-oriented features based on the evolutionary conservation of the mutated positions, we added a collection of protein-oriented features that are based on functional aspects of the gene affected. We cross-validated DEOGEN on 36 825 polymorphisms, 20 821 deleterious SNVs, and 1038 INDELs from SwissProt. The multilevel contextualization of each (variant, protein) pair in DEOGEN provides a 10% improvement of MCC with respect to current state-of-the-art tools. The software and the data presented here is publicly available at http://ibsquare.be/deogen : wvranken@vub.ac.be Supplementary data are available at Bioinformatics online.

CT Gray-Level Texture Analysis as a Quantitative Imaging Biomarker of Epidermal Growth Factor Receptor Mutation Status in Adenocarcinoma of the Lung.

The purpose of this study was to investigate the radiogenomic correlation between CT gray-level texture features and epidermal growth factor receptor (EGFR) mutation status in adenocarcinoma of the lung. This retrospective study included 25 patients with exon 19 short inframe deletion (exon 19) and 21 patients with exon 21 L858R point (exon 21) EGFR mutations among 125 patients with EGFR mutant adenocarcinoma of the lung. The randomly formed control group consisted of 20 patients selected from 126 patients with EGFR mutation-negative (wild-type) adenocarcinomas. Five gray-level texture features (contrast, correlation, inverse difference moment, angular second moment, and entropy) were analyzed. Contrast differentiated both exon 19 (p = 0.00027) and exon 21 (p = 0.00001) mutants from the wild type. Wild-type adenocarcinomas had high scores for contrast (mean, 1598.547) compared with EGFR mutants (mean, 679.463). Correlation differentiated both exon 19 (p = 0.017) and exon 21 (p = 0.0015) mutants from wild-type adenocarcinomas. Inverse difference moment differentiated exon 19 mutants from exon 21 mutants (p = 0.019) and both exon 19 (p = 0.044) and exon 21 (p = 0.00001) mutants from wild-type adenocarcinomas. Angular second moment and entropy were not associated with statistically significant differences between mutation statuses. Contrast, correlation, and inverse difference moment texture features correlate with EGFR mutation status in adenocarcinoma of the lung. Further investigation with larger prospective studies is needed to validate the role of CT gray-level texture analysis as a quantitative imaging biomarker.

Muscular Dystrophy Mutations Impair the Nuclear Envelope Emerin Self-assembly Properties.

More than 100 genetic mutations causing X-linked Emery-Dreifuss muscular dystrophy have been identified in the gene encoding the integral inner nuclear membrane protein emerin. Most mutations are nonsense or frameshift mutations that lead to the absence of emerin in cells. Only very few cases are due to missense or short in-frame deletions. Molecular mechanisms explaining the corresponding emerin variants' loss of function are particularly difficult to identify because of the mostly intrinsically disordered state of the emerin nucleoplasmic region. We now demonstrate that this EmN region can be produced as a disordered monomer, as revealed by nuclear magnetic resonance, but rapidly self-assembles in vitro. Increases in concentration and temperature favor the formation of long curvilinear filaments with diameters of approximately 10 nm, as observed by electron microscopy. Assembly of these filaments can be followed by fluorescence through Thioflavin-T binding and by Fourier-transform Infrared spectrometry through formation of β-structures. Analysis of the assembly properties of five EmN variants reveals that del95-99 and Q133H impact filament assembly capacities. In cells, these variants are located at the nuclear envelope, but the corresponding quantities of emerin-emerin and emerin-lamin proximities are decreased compared to wild-type protein. Furthermore, variant P183H favors EmN aggregation in vitro, and variant P183T provokes emerin accumulation in cytoplasmic foci in cells. Substitution of residue Pro183 might systematically favor oligomerization, leading to emerin aggregation and mislocalization in cells. Our results suggest that emerin self-assembly is necessary for its proper function and that a loss of either the protein itself or its ability to self-assemble causes muscular dystrophy.