scholarly journals Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations

2016 ◽  
Author(s):  
Jonathan S. Brammeld ◽  
Mia Petljak ◽  
Inigo Martincorena ◽  
Steven P. Williams ◽  
Luz Garcia Alonso ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Point Mutations ◽  
Gene Mutations ◽  
Resistance Mechanisms ◽  
Chemical Mutagenesis ◽  
Specific Gene ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Genome Wide

AbstractDrug resistance is an almost inevitable consequence of cancer therapy and ultimately proves fatal for the majority of patients. In many cases this is the consequence of specific gene mutations that have the potential to be targeted to re-sensitize the tumor. The ability to uniformly saturate the genome with point mutations without chromosome or nucleotide sequence context bias would open the door to identify all putative drug resistance mutations in cancer models. Here we describe such a method for elucidating drug resistance mechanisms using genome-wide chemical mutagenesis allied to next-generation sequencing. We show that chemically mutagenizing the genome of cancer cells dramatically increases the number of drug-resistant clones and allows the detection of both known and novel drug resistance mutations. We have developed an efficient computational process that allows for the rapid identification of involved pathways and druggable targets. Such a priori knowledge would greatly empower serial monitoring strategies for drug resistance in the clinic as well as the development of trials for drug resistant patients.

scholarly journals Genome-wide chemical mutagenesis screens allow unbiased saturation of the cancer genome and identification of drug resistance mutations

2017 ◽  
Vol 27 (4) ◽  
pp. 613-625 ◽  
Author(s):  
Jonathan S. Brammeld ◽  
Mia Petljak ◽  
Inigo Martincorena ◽  
Steven P. Williams ◽  
Luz Garcia Alonso ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Cancer Genome ◽  
Chemical Mutagenesis ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Genome Wide

scholarly journals Coupling chemical mutagenesis to next generation sequencing for the identification of drug resistance mutations in Leishmania

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Arijit Bhattacharya ◽  
Philippe Leprohon ◽  
Sophia Bigot ◽  
Prasad Kottayil Padmanabhan ◽  
Angana Mukherjee ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Next Generation Sequencing ◽  
Chemical Mutagenesis ◽  
Next Generation ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Paromomycin Resistance ◽  
Allelic Replacement ◽  
Genome Wide ◽  
Generation Sequencing

AbstractCurrent genome-wide screens allow system-wide study of drug resistance but detecting small nucleotide variants (SNVs) is challenging. Here, we use chemical mutagenesis, drug selection and next generation sequencing to characterize miltefosine and paromomycin resistant clones of the parasite Leishmania. We highlight several genes involved in drug resistance by sequencing the genomes of 41 resistant clones and by concentrating on recurrent SNVs. We associate genes linked to lipid metabolism or to ribosome/translation functions with miltefosine or paromomycin resistance, respectively. We prove by allelic replacement and CRISPR-Cas9 gene-editing that the essential protein kinase CDPK1 is crucial for paromomycin resistance. We have linked CDPK1 in translation by functional interactome analysis, and provide evidence that CDPK1 contributes to antimonial resistance in the parasite. This screen is powerful in exploring networks of drug resistance in an organism with diploid to mosaic aneuploid genome, hence widening the scope of its applicability.

scholarly journals Phenotyping of Cytomegalovirus Drug Resistance Mutations by Using Recombinant Viruses Incorporating a Reporter Gene

2005 ◽  
Vol 49 (7) ◽  
pp. 2710-2715 ◽  
Author(s):  
Sunwen Chou ◽  
Laura C. Van Wechel ◽  
Heather M. Lichy ◽  
Gail I. Marousek
Keyword(s):  
Drug Resistance ◽  
Reporter Gene ◽  
Gene Mutations ◽  
Resistance Mutations ◽  
Recombinant Viruses ◽  
Drug Resistance Mutations ◽  
Drug Concentrations ◽  
Unique Restriction ◽  
Seap Activity ◽  
Ganciclovir Resistance

ABSTRACT A new recombinant phenotyping method was developed for the analysis of drug resistance mutations in human cytomegalovirus (CMV). CMV strain T2211 was derived from strain AD169 by inserting unique restriction sites and a secreted alkaline phosphatase (SEAP) reporter gene for rapid viral quantitation. Specific viral UL97 and pol gene mutations were transferred by recombination into T2211, and their drug resistance phenotypes (for ganciclovir, foscarnet, or cidofovir) were determined by the drug concentrations required to reduce supernatant SEAP activity by 50% (IC50). Changes in the IC50 conferred by the mutations tested (UL97 M460V, C592G, A594V, and L595S and pol del981-2) were similar to those previously reported in marker transfer and conventional plaque reduction assays. The combination of UL97 C592G and pol del981-2 conferred much higher ganciclovir resistance than either mutation alone. The UL97 polymorphism D605E had no measurable effect on ganciclovir susceptibility, alone or in combination with common UL97 resistance mutations. Transfer into strain T2211 facilitates the phenotyping of newly observed mutations, combinations of mutations, and clinical CMV sequences without an accompanying viral isolate.

scholarly journals Low-Frequency Drug Resistance in HIV-Infected Ugandans on Antiretroviral Treatment Is Associated with Regimen Failure

2016 ◽  
Vol 60 (6) ◽  
pp. 3380-3397 ◽  
Author(s):  
Fred Kyeyune ◽  
Richard M. Gibson ◽  
Immaculate Nankya ◽  
Colin Venner ◽  
Samar Metha ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Treatment Failure ◽  
Sanger Sequencing ◽  
Antiretroviral Treatment ◽  
Low Frequency ◽  
Drug Resistant ◽  
Viral Quasispecies ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Hiv 1

Most patients failing antiretroviral treatment in Uganda continue to fail their treatment regimen even if a dominant drug-resistant HIV-1 genotype is not detected. In a recent retrospective study, we observed that approximately 30% of HIV-infected individuals in the Joint Clinical Research Centre (Kampala, Uganda) experienced virologic failure with a susceptible HIV-1 genotype based on standard Sanger sequencing. Selection of minority drug-resistant HIV-1 variants (not detectable by Sanger sequencing) under antiretroviral therapy pressure can lead to a shift in the viral quasispecies distribution, becoming dominant members of the virus population and eventually causing treatment failure. Here, we used a novel HIV-1 genotyping assay based on deep sequencing (DeepGen) to quantify low-level drug-resistant HIV-1 variants in 33 patients failing a first-line antiretroviral treatment regimen in the absence of drug-resistant mutations, as screened by standard population-based Sanger sequencing. Using this sensitive assay, we observed that 64% (21/33) of these individuals had low-frequency (or minority) drug-resistant variants in the intrapatient HIV-1 population, which correlated with treatment failure. Moreover, the presence of these minority HIV-1 variants was associated with higher intrapatient HIV-1 diversity, suggesting a dynamic selection or fading of drug-resistant HIV-1 variants from the viral quasispecies in the presence or absence of drug pressure, respectively. This study identified low-frequency HIV drug resistance mutations by deep sequencing in Ugandan patients failing antiretroviral treatment but lacking dominant drug resistance mutations as determined by Sanger sequencing methods. We showed that these low-abundance drug-resistant viruses could have significant consequences for clinical outcomes, especially if treatment is not modified based on a susceptible HIV-1 genotype by Sanger sequencing. Therefore, we propose to make clinical decisions using more sensitive methods to detect minority HIV-1 variants.

scholarly journals Evolution of Fluconazole-ResistantCandida albicansStrains by Drug-Induced Mating Competence and Parasexual Recombination

mBio ◽  
2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Christina Popp ◽  
Bernardo Ramírez-Zavala ◽  
Sonja Schwanfelder ◽  
Ines Krüger ◽  
Joachim Morschhäuser
Keyword(s):  
Drug Resistance ◽  
Mating Type ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Type Locus ◽  
Content Type ◽  
Mating Type Locus ◽  
Resistant Progeny

ABSTRACTThe clonal population structure ofCandida albicanssuggests that (para)sexual recombination does not play an important role in the lifestyle of this opportunistic fungal pathogen, an assumption that is strengthened by the fact that mostC. albicansstrains are heterozygous at the mating type locus (MTL) and therefore mating-incompetent. On the other hand, mating might occur within clonal populations and allow the combination of advantageous traits that were acquired by individual cells to adapt to adverse conditions. We have investigated if parasexual recombination may be involved in the evolution of highly drug-resistant strains exhibiting multiple resistance mechanisms against fluconazole, an antifungal drug that is commonly used to treat infections byC. albicans. Growth of strains that were heterozygous forMTLand different fluconazole resistance mutations in the presence of the drug resulted in the emergence of derivatives that had become homozygous for the mutated allele and the mating type locus and exhibited increased drug resistance. WhenMTLa/aandMTLα/α cells of these strains were mixed in all possible combinations, we could isolate mating products containing the genetic material from both parents. The initial mating products did not exhibit higher drug resistance than their parental strains, but further propagation under selective pressure resulted in the loss of the wild-type alleles and increased fluconazole resistance. Therefore, fluconazole treatment not only selects for resistance mutations but also promotes genomic alterations that confer mating competence, which allows cells in an originally clonal population to exchange individually acquired resistance mechanisms and generate highly drug-resistant progeny.IMPORTANCESexual reproduction is an important mechanism in the evolution of species, since it allows the combination of advantageous traits of individual members in a population. The pathogenic yeastCandida albicansis a diploid organism that normally propagates in a clonal fashion, because heterozygosity at the mating type locus (MTL) inhibits mating between cells. Here we show thatC. albicanscells that have acquired drug resistance mutations during treatment with the commonly used antifungal agent fluconazole rapidly develop further increased resistance by genome rearrangements that result in simultaneous loss of heterozygosity for the mutated allele and the mating type locus. This enables the drug-resistant cells of a population to switch to the mating-competent opaque morphology and mate with each other to combine different individually acquired resistance mechanisms. The tetraploid mating products reassort their merged genomes and, under selective pressure by the drug, generate highly resistant progeny that have retained the advantageous mutated alleles. Parasexual propagation, promoted by stress-induced genome rearrangements that result in the acquisition of mating competence in cells with adaptive mutations, may therefore be an important mechanism in the evolution ofC. albicanspopulations.

scholarly journals Effect of Mutation and Genetic Background on Drug Resistance in Mycobacterium tuberculosis

2012 ◽  
Vol 56 (6) ◽  
pp. 3047-3053 ◽  
Author(s):  
Lukas Fenner ◽  
Matthias Egger ◽  
Thomas Bodmer ◽  
Ekkehardt Altpeter ◽  
Marcel Zwahlen ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Mycobacterium Tuberculosis ◽  
Hot Spot ◽  
Multidrug Resistant ◽  
Beijing Genotype ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Content Type ◽  
Drug Resistance Mutations ◽  
Resistant Strains

ABSTRACTBacterial factors may contribute to the global emergence and spread of drug-resistant tuberculosis (TB). Only a few studies have reported on the interactions between different bacterial factors. We studied drug-resistantMycobacterium tuberculosisisolates from a nationwide study conducted from 2000 to 2008 in Switzerland. We determined quantitative drug resistance levels of first-line drugs by using Bactec MGIT-960 and drug resistance genotypes by sequencing the hot-spot regions of the relevant genes. We determined recent transmission by molecular methods and collected clinical data. Overall, we analyzed 158 isolates that were resistant to isoniazid, rifampin, or ethambutol, 48 (30.4%) of which were multidrug resistant. Among 154 isoniazid-resistant strains,katGmutations were associated with high-level andinhApromoter mutations with low-level drug resistance. OnlykatG(S315T) (65.6% of all isoniazid-resistant strains) andinhApromoter −15C/T (22.7%) were found in molecular clusters.M. tuberculosislineage 2 (includes Beijing genotype) was associated with any drug resistance (adjusted odds ratio [OR], 3.0; 95% confidence interval [CI], 1.7 to 5.6;P< 0.0001). Lineage 1 was associated withinhApromoter −15C/T mutations (OR, 6.4; 95% CI, 2.0 to 20.7;P= 0.002). We found that the genetic strain background influences the level of isoniazid resistance conveyed by particular mutations (interaction tests of drug resistance mutations across all lineages;P< 0.0001). In conclusion,M. tuberculosisdrug resistance mutations were associated with various levels of drug resistance and transmission, andM. tuberculosislineages were associated with particular drug resistance-conferring mutations and phenotypic drug resistance. Our study also supports a role for epistatic interactions between different drug resistance mutations and strain genetic backgrounds inM. tuberculosisdrug resistance.

scholarly journals Combination of Drugs and Drug-Resistant Reverse Transcriptase Results in a Multiplicative Increase of Human Immunodeficiency Virus Type 1 Mutant Frequencies

2002 ◽  
Vol 76 (18) ◽  
pp. 9253-9259 ◽  
Author(s):  
Louis M. Mansky ◽  
Dennis K. Pearl ◽  
Lisa C. Gajary
Keyword(s):  
Human Immunodeficiency Virus ◽  
Drug Resistance ◽  
Reverse Transcriptase ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Immunodeficiency Virus ◽  
Hiv 1 ◽  
Mutant Frequency

ABSTRACT Replication of drug-resistant human immunodeficiency virus type 1 (HIV-1) in the presence of drug can lead to the failure of antiretroviral drug treatment. Drug failure is associated with the accumulation of drug resistance mutations. Previous studies have shown that 3′-azido-3′-deoxythymidine (AZT), (−)2′,3′-dideoxy-3′-thiacytidine (3TC), and AZT-resistant HIV-1 reverse transcriptase (RT) can increase the virus in vivo mutation rate. In this study, the combined effects of drug-resistant RT and antiretroviral drugs on the HIV-1 mutant frequency were determined. In most cases, a multiplicative effect was observed with AZT-resistant or AZT/3TC dually resistant RT and several drugs (i.e., AZT, 3TC, hydroxyurea, and thymidine) and led to increases in the odds of recovering virus mutants to over 20 times that of the HIV-1 mutant frequency in the absence of drug or drug-resistance mutations. This observation indicates that HIV-1 can mutate at a significantly higher rate when drug-resistant virus replicates in the presence of drug. These increased mutant frequencies could have important implications for HIV-1 population dynamics and drug therapy regimens.

scholarly journals Characterisation of HIV-1 transmission clusters and drug-resistant mutations in Denmark, 2004 to 2016

2018 ◽  
Vol 23 (44) ◽  
Author(s):  
Andreas Petersen ◽  
Susan A Cowan ◽  
Jens Nielsen ◽  
Thea K Fischer ◽  
Jannik Fonager
Keyword(s):  
Drug Resistance ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Transmission Networks ◽  
Late Presenters ◽  
Drug Resistance Mutations ◽  
Hiv Epidemic ◽  
Immunodeficiency Virus ◽  
Sex With Men ◽  
Hiv 1

This study describes the prevalence of human immunodeficiency virus (HIV) drug resistance mutations among 1,815 patients in Denmark from 2004 to 2016 and characterises transmission clusters. POL sequences were analysed for subtype, drug resistance mutations and phylogenetic relationship. The prevalence of surveillance drug resistance mutations (SDRM) was 6.7%, while the prevalence of drug resistance mutations (DRM) with a clinical impact was 12.3%. We identified 197 transmission clusters with 706 patients. Patients 40 years or older were less likely to be members of a transmission cluster and patients in transmission clusters were less likely to be infected abroad. The proportion of late presenters (LP) was lower in active compared with inactive clusters. Large active clusters consisted of more men who have sex with men (MSM), had members more frequently infected in Denmark and contained a significantly lower proportion of LP and significantly fewer patients with DRM than small active clusters. Subtyping demonstrated that the Danish HIV epidemic is gradually becoming more composed of non-B subtypes/circulating recombinant forms. This study shows that active HIV-1 transmission has become increasingly MSM-dominated and that the recent increase in SDRM and DRM prevalence is not associated with more sustained transmission within identified transmission networks or clusters.

scholarly journals The frequency of HIV-1 infection and surveillance drug-resistant mutations determination among Iranians with high-risk behaviors, during 2014 to 2020

2021 ◽  
Author(s):  
Saba Garshasbi ◽  
Arezoo Marjani ◽  
Ali Alipour ◽  
Khadijeh Khanaliha ◽  
Maryam Esghaei ◽  
...  
Keyword(s):  
Drug Resistance ◽  
High Risk ◽  
Reverse Transcriptase ◽  
Risk Behaviors ◽  
Resistance Mutation ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
High Risk Behaviors ◽  
Drug Resistance Mutations ◽  
Hiv 1

Background and Objectives: Human immunodeficiency virus (HIV) has various transmission routes. Instant antiretroviral therapy (ART) is the recommended treatment for HIV infection. Highly active antiretroviral therapy (HAART) significantly decreases the acquired immunodeficiency syndrome (AIDS) and AIDS-related co-morbidities. Notwithstanding the suit- ability of HAART, the antiretrovirals (ARVs) have adverse effects and antiretroviral drug resistance mutations are reported among those who receive ARVs. In this survey, the abundance of HIV-1 infection in Iranians with high-risk behaviors, and detection of the surveillance drug-resistant mutations (SDRMs) were evaluated. Materials and Methods: This cross-sectional study was conducted on 250 individuals with high-risk behaviors from Sep- tember 2014 to February 2020. HIV-1 Ag/Ab in plasma samples was detected using enzyme immunoassay (EIA) kits. The conserved region of HIV-1 was detected in the plasma samples by real-time polymerase chain reaction (PCR) assay. Further- more, in individuals with positive HIV-1 RNA, HIV-1 viral load testing was performed. After amplification and sequencing of the HIV-1 protease, reverse transcriptase, and integrase genes, surveillance drug resistance mutation (SDRM) and phylo- genetic analysis were determined. Results: Out of the 250 participants with high-risk behaviors, six (2.4%) were infected with HIV-1. According to the phy- logenetic analysis, the CRF35_AD (83.3% or 5/6) was the dominant subtype, followed by CRF01_AE (16.7% or 1/6). In this research, in none of the HIV-1 infected patients, SDRM for protease inhibitors (PIs), nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), and integrase inhibitors (INs) were observed. Nevertheless, in one of the patients, V179L mutation was detected which is a rare non-polymorphic mutation and is listed as a rilpivirine (RPV) -associated resistance mutation. Conclusion: The results of the current survey revealed that 2.4% of people with high-risk behaviors are infected with HIV and the level of drug resistance mutations (DRMs) in these people is very low.

Assessment of Transmitted HIV-1 Drug Resistance Mutations Using Ultra- Deep Pyrosequencing in a Turkish Cohort

2018 ◽  
Vol 16 (3) ◽  
pp. 216-221 ◽  
Author(s):  
Uluhan Sili ◽  
Burak Aksu ◽  
Aysun Tekin ◽  
Ufuk Hasdemir ◽  
Guner Soyletir ◽  
...  
Keyword(s):  
Drug Resistance ◽  
Virologic Failure ◽  
Treatment Success ◽  
Transmitted Drug Resistance ◽  
Drug Resistant ◽  
Resistance Mutations ◽  
Drug Resistance Mutations ◽  
Sensitivity Score ◽  
Minority Variants ◽  
Genotypic Sensitivity Score

Background: Antiretroviral treatment (ART) reduces morbidity and mortality caused by human immunodeficiency virus (HIV) infection; however, the emergence of drug-resistant strains poses an important obstacle to treatment success. Using conventional sequencing methods to determine antiretroviral resistance, mutations present in ≥20% of quasispecies can be identified, but drug-resistant minority variants can lead to virologic failure. Objective: We aimed to assess transmitted drug resistance mutations (TDRMs) within minority variants using ultra-deep pyrosequencing (UDPS). Method: Treatment-naive adult patients were included in this observational study. Surveillance TDRMs were classified as ≥20% or at minority variant level (≥2% – <20%). Genotypic sensitivity score calculated by using all pre-treatment drug resistance mutations (PDRMs) was also evaluated. Results: Thirty-six patients were analyzed. Any TDRM at ≥20% level was detected in 8.3% of the patients (n=3). This prevalence increased to 30.6% (n=11) with the inclusion of minority variants. All non-nucleoside reverse transcriptase inhibitor and protease inhibitor-related TDRMs were within minority variants. The genotypic sensitivity score of rilpivirine-based regimens was considerably diminished when minority variants were included in the PDRM analysis. Conclusion: UDPS was used for the first time to assess TDRM in a Turkish HIV cohort and uncovered several mutations hidden within minority variants. UDPS may be preferred to detect PDRMs for avoiding virologic failure with rilpivirine-based ART regimens.

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