RESUMEN
In the TMC435-C101 study, 6 patients infected with hepatitis C virus genotype 1 were treated with the protease inhibitor TMC435 (200 mg once daily) as monotherapy for 5 days. Approximately 1.5 years later, 5 of these patients were re-treated with TMC435 (200 mg once daily) plus pegylated interferon alfa-2a and ribavirin (PegIFNα-2a and RBV) for 4 weeks, followed by PegIFNα-2a and RBV until week 48 (in the Optimal Protease inhibitor Enhancement of Response to therApy [OPERA-1] study). TMC435-resistant variants, which emerged in all 5 patients during the TMC435-C101 study, were no longer detected at the beginning of the OPERA-1 study based on virus population sequencing. During the OPERA-1 study, 3 patients had a sustained virologic response; deep sequencing indicated low-level persistence of resistant variants in the remaining 2 patients, which might have affected their response to re-treatment.
Asunto(s)
Hepacivirus/genética , Hepatitis C/tratamiento farmacológico , Compuestos Heterocíclicos con 3 Anillos/uso terapéutico , Inhibidores de Proteasas/uso terapéutico , ARN Viral/sangre , Sulfonamidas/uso terapéutico , Antivirales/uso terapéutico , Farmacorresistencia Viral , Quimioterapia Combinada , Genotipo , Hepatitis C/sangre , Hepatitis C/virología , Humanos , Interferón-alfa/uso terapéutico , Masculino , Mutación , Polietilenglicoles/uso terapéutico , ARN Viral/efectos de los fármacos , Proteínas Recombinantes/uso terapéutico , Ribavirina/uso terapéutico , Simeprevir , Carga ViralRESUMEN
BACKGROUND: The dynamics of raltegravir-resistant variants and their impact on virologic response in 23 HIV-1-infected patients, who started a salvage raltegravir-containing regimen, were investigated. METHODS: Integrase population sequencing and Ultra-Deep-454 Pyrosequencing (UDPS) were performed on plasma samples at baseline and at raltegravir failure. All integrase mutations detected at a frequency ≥1% were considered to be reliable for the UDPS analyses. Phylogenetic and phenotypic resistance analyses were also performed. RESULTS: At baseline, primary resistance mutations were not detected by both population and UDPS genotypic assays; few secondary mutations (T97A-V151I-G163R) were rarely detected and did not show any statistically association either with virologic response at 24-weeks or with the development of resistant variants at failure. At UDPS, not all resistant variants appearing early during treatment evolved as major populations during failure; only specific resistance pathways (Y143R-Q148H/R-N155H) associated with an increased rate of fitness and phenotypic resistance were selected. CONCLUSIONS: Resistance to raltegravir in integrase strand transfer inhibitor-naive patients remains today a rare event, which might be changed by future extensive use of such drugs. In our study, pathways of resistance at failure were not predicted by baseline mutations, suggesting that evolution plus stochastic selection plays a major role in the appearance of integrase-resistance mutations, whereas fitness and resistance are dominant factors acting for the late selection of resistant quasispecies.
Asunto(s)
Fármacos Anti-VIH/administración & dosificación , Farmacorresistencia Viral , Infecciones por VIH/virología , Integrasa de VIH/genética , VIH-1/genética , Mutación Missense , Pirrolidinonas/administración & dosificación , Adulto , Femenino , Genotipo , Infecciones por VIH/tratamiento farmacológico , VIH-1/clasificación , VIH-1/enzimología , VIH-1/aislamiento & purificación , Humanos , Masculino , Pruebas de Sensibilidad Microbiana , Datos de Secuencia Molecular , Fenotipo , Filogenia , Raltegravir Potásico , Terapia Recuperativa/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
OBJECTIVES: Cerebrospinal fluid (CSF) biomarkers of Alzheimer's disease (AD) are well-established in research settings, but their use in routine clinical practice remains a largely unexploited potential. Here, we examined the relationship between CSF biomarkers, measured by a fully automated immunoassay platform, and brain ß-amyloid (Aß) deposition status confirmed by amyloid positron emission tomography (PET). METHODS: One hundred ninety-nine CSF samples from clinically diagnosed AD patients enrolled in a clinical study and who underwent amyloid PET were used for the measurement of CSF biomarkers Aß 1-40 (Aß40), Aß 1-42 (Aß42), total tau (t-Tau), and phosphorylated tau-181 (p-Tau181) using the LUMIPULSE system. These biomarkers and their combinations were compared to amyloid PET classification (negative or positive) using visual read assessments. Several combinations were also analyzed with a multivariable logistic regression model. RESULTS: Aß42, t-Tau, and p-Tau181, and the ratios of Aß42 with other biomarkers had a good diagnostic agreement with amyloid PET imaging. The multivariable logistic regression analysis showed that amyloid PET status was associated with Aß40 and Aß42, but other factors, such as MMSE, sex, t-Tau, and p-Tau181, did not significantly add information to the model. CONCLUSIONS: CSF biomarkers measured with the LUMIPULSE system showed good agreement with amyloid PET imaging. The ratio of Aß42 with the other analyzed biomarkers showed a higher correlation with amyloid PET than Aß42 alone, suggesting that the combinations of biomarkers could be useful in the diagnostic assessment in clinical research and potentially in routine clinical practice.
Asunto(s)
Enfermedad de Alzheimer , Humanos , Enfermedad de Alzheimer/diagnóstico por imagen , Enfermedad de Alzheimer/líquido cefalorraquídeo , Amiloide/metabolismo , Biomarcadores/líquido cefalorraquídeo , Tomografía de Emisión de Positrones , Encéfalo/metabolismoRESUMEN
BACKGROUND: Determination of HIV-1 tropism is a pre-requisite to the use of CCR5 antagonists. This study evaluated the potential of population genotypic tropism tests (GTTs) in clinical practice, and the correlation with phenotypic tropism tests (PTTs) in patients accessing routine HIV care. METHODS: Forty-nine consecutive plasma samples for which an original Trofile(TM) assay was performed were obtained from triple-class-experienced patients in need of a therapy change. Viral tropism was defined as the consensus of three or more tropism calls obtained from the combination of two independent population PTT assays (Trofile Biosciences, San Francisco, CA, USA, and Virco, Beerse, Belgium), population GTTs and GTTs based on ultra-deep sequencing. If no consensus was reached, a clonal PTT was performed in order to finalize the tropism call. This two-step approach allowed the definition of a reference tropism call. RESULTS: According to the reference tropism result, 35/49 samples were CCR5 tropic (R5) (patients eligible for maraviroc treatment) and 14/49 were assigned as non-R5 tropic. The non-R5 samples [patients not eligible for maraviroc treatment according to the FDA/European Medicines Agency (EMEA) label] group included both the CXCR4 (X4) samples and the dual and mixed CCR5/CXCR4 (R5/X4) samples. Compared with Trofile(TM) population PTTs, population GTTs showed a higher sensitivity (97%) and a higher negative predictive value (91%), but almost equal specificity and an equal positive predictive value. CONCLUSIONS: In line with recent reports from clinical trial data, our data support the use of population genotypic tropism testing as a tool for tropism determination before the start of maraviroc.
Asunto(s)
Ciclohexanos/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , VIH-1/efectos de los fármacos , VIH-1/genética , Triazoles/uso terapéutico , Tropismo Viral , Genotipo , Infecciones por VIH/metabolismo , VIH-1/fisiología , Humanos , Maraviroc , Fenotipo , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismoRESUMEN
Resistance to hepatitis C virus (HCV) inhibitors targeting viral enzymes has been observed in in vitro replicon studies and during clinical trials. The factors determining the emergence of resistance and the changes in the viral quasispecies population under selective pressure are not fully understood. To assess the dynamics of variants emerging in vitro under various selective pressures with TMC380765, a potent macrocyclic HCV NS3/4A protease inhibitor, HCV genotype 1b replicon-containing cells were cultured in the presence of a low, high, or stepwise-increasing TMC380765 concentration(s). HCV replicon RNA from representative samples thus obtained was analyzed using (i) population, (ii) clonal, and (iii) 454 deep sequencing technologies. Depending on the concentration of TMC380765, distinct mutational patterns emerged. In particular, culturing with low concentrations resulted in the selection of low-level resistance mutations (F43S and A156G), whereas high concentrations resulted in the selection of high-level resistance mutations (A156V, D168V, and D168A). Clonal and 454 deep sequencing analysis of the replicon RNA allowed the identification of low-frequency preexisting mutations possibly contributing to the mutational pattern that emerged. Stepwise-increasing TMC380765 concentrations resulted in the emergence and disappearance of multiple replicon variants in response to the changing selection pressure. Moreover, two different codons for the wild-type amino acids were observed at certain NS3 positions within one population of replicons, which may contribute to the emerging mutational patterns. Deep sequencing technologies enabled the study of minority variants present in the HCV quasispecies population present at baseline and during antiviral drug pressure, giving new insights into the dynamics of resistance acquisition by HCV.
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Evolución Biológica , Resistencia a Medicamentos , Hepacivirus/genética , Mutación Missense , Inhibidores de Proteasas/farmacología , Selección Genética/efectos de los fármacos , Secuencia de Bases , Línea Celular , Análisis Mutacional de ADN , Hepacivirus/crecimiento & desarrollo , Humanos , ARN Viral/análisis , ARN Viral/genética , Replicón/genéticaRESUMEN
BACKGROUND: HIV-1 infected patients for whom standard gp160 phenotypic tropism testing failed are currently excluded from co-receptor antagonist treatment. To provide patients with maximal treatment options, massively parallel sequencing of the envelope V3 domain, in combination with tropism prediction tools, was evaluated as an alternative tropism determination strategy. Plasma samples from twelve HIV-1 infected individuals with failing phenotyping results were available. The samples were submitted to massive parallel sequencing and to confirmatory recombinant phenotyping using a fraction of the gp120 domain. RESULTS: A cut-off for sequence reads interpretation of 5 to10 times the sequencing error rate (0.2%) was implemented. On average, each sample contained 7 different V3 haplotypes. V3 haplotypes were submitted to tropism prediction algorithms, and 4/14 samples returned with presence of a dual/mixed (D/M) tropic virus, respectively at 3%, 10%, 11%, and 95% of the viral quasispecies. V3 tropism prediction was confirmed by gp120 phenotyping, except for two out of 4 D/M predicted viruses (with 3 and 95%) which were phenotypically R5-tropic. In the first case, the result was discordant due to the limit of detection for the phenotyping technology, while in the latter case the prediction algorithms were not computing the viral tropism correctly. CONCLUSIONS: Although only demonstrated on a limited set of samples, the potential of the combined use of "deep sequencing + prediction algorithms" in cases where routine gp160 phenotype testing cannot be employed was illustrated. While good concordance was observed between gp120 phenotyping and prediction of R5-tropic virus, the results suggest that accurate prediction of X4-tropic virus would require further algorithm development.
RESUMEN
Orally bioavailable CXCR4 and CCR5 coreceptor antagonists are being developed for the treatment of HIV-1 infection. A new tropism-testing platform, which offers various options depending on the needs, was established. Each option has specific characteristics in terms of sensitivity, information, throughput and cost. The platform consists of four assays, all based on a one-step RT-PCR of the main part of the HIV envelope glycoprotein gp120 (called 'NH(2)-V4'). Population-based sequencing of gp120's V3 loop is generally cheap and easy to run, and was chosen as the first test in the platform's cascade. Given its drawbacks such as limited sensitivity, additional tests were developed. A sensitive assay using NH(2)-V4 gp120 clonal sequencing and tropism prediction enabled us to demonstrate the quasispecies diversity present in 13 patient samples. For phenotyping, an eGFP-containing HIV backbone deleted for NH(2)-V4 was constructed and used for clonal and population tropism determination. As expected, clonal NH(2)-V4 gp120 phenotyping demonstrated significant correlation between prediction algorithms and phenotype-based classification. The absence of the N-linked glycosylation motif in V3 was associated with CXCR4 usage. Finally, population NH(2)-V4 gp120 phenotypic tropism determination appeared to be a promising tool for the detection of minority species present in the amplified envelope fragments.
Asunto(s)
Proteína gp120 de Envoltorio del VIH/genética , VIH-1/fisiología , Receptores CCR5/metabolismo , Receptores CXCR4/metabolismo , Algoritmos , Secuencia de Aminoácidos , Antagonistas de los Receptores CCR5 , Genotipo , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/aislamiento & purificación , Humanos , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Fenotipo , Filogenia , Receptores CXCR4/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Sensibilidad y Especificidad , Virología/métodosRESUMEN
Nonsense, missense, and even silent mutation-associated exon skipping is recognized in an increasing number of genes as a novel form of splicing mutation. The analysis of individual mutations of this kind can shed light on basic pre-mRNA splicing mechanisms. Using cDNA-based mutation detection analysis, we have identified one missense and six nonsense mutations that lead to different extents of exon-lacking transcripts in neurofibromatosis type 1 (NF1) patients. We confirmed mutation-associated exon skipping in a heterologous hybrid minigene context. There is evidence that the disruption of functional exonic splicing enhancer (ESE) sequences is frequently the mechanism underlying mutation-associated exon skipping. Therefore, we examined the wild-type and mutant NF1 sequences with two available ESE-prediction programs. Either or both programs predicted the disruption of ESE motifs in six out of the seven analyzed mutations. To ascertain the function of the predicted ESEs, we quantitatively measured their ability to rescue splicing of an enhancer-dependent exon, and found that all seven mutant ESEs had reduced splicing enhancement activity compared to the wild-type sequences. Our results suggest that the wild-type sequences function as ESE elements, whose disruption is responsible for the mutation-associated exon skipping observed in the NF1 patients. Further, this study illustrates the utility of ESE-prediction programs for delineating candidate sequences that may serve as ESE elements. However, until more refined prediction algorithms have been developed, experimental data, preferably from patient tissues, remain indispensable to assess the clinical significance, particularly of missense and silent mutations, and to understand the structure-function relationship in the corresponding protein.
Asunto(s)
Elementos de Facilitación Genéticos , Exones/genética , Mutación Missense , Neurofibromatosis 1/genética , Empalme del ARN/genética , Alelos , Simulación por Computador , Análisis Mutacional de ADN , ADN Complementario , Humanos , Transcripción GenéticaRESUMEN
BACKGROUND: Mutation analysis of the neurofibromatosis type 1 (NF1) gene has shown that about 30% of NF1 patients carry a splice mutation resulting in the production of one or several shortened transcripts. Some of these transcripts were also found in fresh lymphocytes of healthy individuals, albeit typically at a very low level. Starting from this initial observation, we were interested to gain further insight into the complex nature of NF1 mRNA processing. RESULTS: We have used a RT-PCR plasmid library based method to identify novel NF1 splice variants. Several transcripts were observed with specific insertions/deletions and a survey was made. This large group of variants detected in one single gene allows to perform a comparative analysis of the factors involved in splice regulation. Exons that are prone to skipping were systematically analysed for 5' and 3' splice site strength, branch point strength and secondary structure. CONCLUSION: Our study revealed a complex splicing pattern, generating a great diversity in NF1 transcripts. We found that, on average, exons that are spliced out in part of the mRNA have significantly weaker acceptor sites. Some variants identified in this study could have distinct roles and might expand our knowledge of neurofibromin.
RESUMEN
Previously, we have shown that the NF1 gene gives rise to multiple novel splice variants. In the present study, nine NF1 variants were quantified by real-time PCR in various human tissues. Some of these variants were expressed at low to moderate low levels and possible implications of these findings are discussed. Interestingly, two variants (NF1-DeltaE4b and NF1-DeltaE43) were shown to be highly expressed in specific tissues. NF1-DeltaE43 lacks a nuclear targeting sequence and might be functionally different from full-length NF1. These novel NF1 splice variants might expand our understanding of the role of neurofibromin.
Asunto(s)
Empalme Alternativo , Expresión Génica , Neurofibromatosis 1/genética , Neurofibromina 1/genética , ARN Mensajero , Animales , Células Cultivadas , Genes de Neurofibromatosis 1/fisiología , Humanos , Linfocitos/citología , Linfocitos/metabolismo , Ratones , Distribución TisularRESUMEN
Mutations in the neurofibromatosis type 1 (NF1) tumor suppressor gene predispose individuals to a variety of benign and malignant tumors. Many tumor suppressors 'shuttle' between the nucleus and the cytoplasm, thus regulating their function. By expressing different NF1 constructs in COS-7 cells (encompassing exons 28-49 and fused to the green fluorescent protein), we identified a functional nuclear localization signal (NLS) in exon 43. Mutation of the NLS completely abolishes the nuclear entry of the NF1-derivative fusion protein. A highly expressed splice variant that lacks this NLS controls the localization and hence the function of neurofibromin. The localization of neurofibromin in the nucleus may provide novel clues to unknown functions for NF1.
Asunto(s)
Transporte Activo de Núcleo Celular , Núcleo Celular/metabolismo , Genes Supresores de Tumor , Neurofibromina 1/metabolismo , Señales de Localización Nuclear/metabolismo , Empalme Alternativo , Animales , Células COS , Carcinoma Hepatocelular/genética , Chlorocebus aethiops , Citoplasma/metabolismo , Exones , Regulación Neoplásica de la Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Neoplasias Hepáticas/genética , Proteínas Luminiscentes , Mutagénesis Sitio-Dirigida , Neurofibromina 1/química , Neurofibromina 1/genética , Señales de Localización Nuclear/genética , Proteínas Recombinantes de Fusión/metabolismo , Fracciones Subcelulares , Transfección , Células Tumorales CultivadasRESUMEN
BACKGROUND: Expression of human Interleukin-5 receptor alpha (hIL-5Ralpha) is controlled by alternative splicing, which generates two different transcripts encoding a membrane-anchored and a soluble form of the receptor, respectively. Although the study of the expression and regulation of hIL-5Ralpha is of crucial importance in the field of immunological processing, methods and techniques until now described lack sufficient sensitivity for detection of small differences in the expression of these isoforms. The aim of this study was to develop a reliable and sensitive real-time quantitative PCR assay to analyse the expression level of each isoform. METHODS: For the quantitative real-time PCR assay, two standard curves specific for each splice variant were constructed. PCR amplifications were performed on CDNA from peripheral blood, eosinophilic chronic rhinosinusitis and normal nasal tissue using a common forward and two specific reverse primers, in combination with SYBR Green I as the detection format. RESULTS AND CONCLUSION: We have developed an accurate and reliable assay for quantification of interleukin-5 receptor alpha mRNA isoforms over a broad dynamic range of input molecules. Importantly, excess of one isoform did not influence accurate quantification of the other isoform. Quantification of hIL-5Ralpha variants in human samples demonstrated an overexpression of both membrane-anchored and soluble encoding variants in eosinophilic chronic rhinosinusitis tissue and peripheral blood in patients with eosinophilic chronic rhinosinusitis compared to healthy subjects. The implementation of this assay will allow a better understanding of the regulatory mechanisms of the hIL-5Ralpha gene and hence its role in the pathogenesis of chronic inflammatory diseases.
Asunto(s)
Reacción en Cadena de la Polimerasa/métodos , ARN Mensajero/análisis , Receptores de Interleucina/genética , Empalme Alternativo , Benzotiazoles , Diaminas , Eosinofilia/inmunología , Colorantes Fluorescentes , Humanos , Subunidad alfa del Receptor de Interleucina-5 , Datos de Secuencia Molecular , Compuestos Orgánicos , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Quinolinas , ARN Mensajero/metabolismo , Receptores de Interleucina/metabolismo , Receptores de Interleucina-5 , Reproducibilidad de los Resultados , Sinusitis/inmunología , Factores de TiempoRESUMEN
There are conflicting data on the impact of low frequency HIV-1 drug-resistant mutants on the response of first-line highly active antiretroviral therapy (HAART), more specifically containing a NNRTI. As population sequencing does not detect resistant viruses representing less than 15-25% of the viral population, more sensitive techniques have been developed but still need clinical validation. We evaluated ultra-deep sequencing (UDPS), recently more available and affordable, as a tool for the detection of HIV-1 minority species carrying drug resistant mutation (DRM) in a clinical setting. A retrospective analysis of the reverse transcriptase (RT) gene of plasma HIV-1 from 70 patients starting a NNRTI based regimen was performed. Minority populations were defined as representing > 1% and < 20% of the total viral population. Using UDPS, we could not confirm an association between the presence of low minority variants harbouring RT mutations at the start of therapy and primary or secondary therapeutic failure.
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Fármacos Anti-VIH/uso terapéutico , Infecciones por VIH/tratamiento farmacológico , Transcriptasa Inversa del VIH/genética , VIH-1/enzimología , VIH-1/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Inhibidores de la Transcriptasa Inversa/uso terapéutico , Adulto , Terapia Antirretroviral Altamente Activa , Farmacorresistencia Viral , Femenino , Infecciones por VIH/virología , Transcriptasa Inversa del VIH/antagonistas & inhibidores , VIH-1/clasificación , VIH-1/genética , Humanos , Masculino , Persona de Mediana Edad , Mutación , Estudios RetrospectivosRESUMEN
HIV-1 Protease (PR) and Reverse Transcriptase (RT) genotyping is well established for the management of antiretroviral (ARV) drug therapy, as it is able to detect gene mutations encoding resistance to ARV compounds or drug classes, that are associated with reduced drug susceptibility (i.e. phenotype). A correct phenotypic interpretation from the derived PR-RT genotype (i.e. virtual phenotype), requires a well characterized geno-phenotype correlative database and appropriate statistical predictive models. The applicability of the virtual phenotype for the patient, will, however, not only depend on the accuracy of the statistical models and the database they rely on, but also depend largely on the sequence information that is provided. Since HIV-1 evolves as a complex of closely related but non-identical viral genomes (i.e. quasispecies) it is crucial that the sequencing method used, is able to characterize most of the genetic mixtures that make up the different quasispecies within a single patient. US regulatory agencies require that developers of HIV-1 genotyping assays, determine and report the HIV-1 mixture detection level of their assay. Hence, the mixture scoring sensitivity of the population-based Sanger sequencing method, along with the defined mixture scoring rules, used to drive the virco(®)TYPE HIV-1 virtual phenotype, was investigated by comparing it to the 454 pyrosequencing technique, which is able to generate the complete viral population sequence. To this end the PR-RT coding sequence of 20 clinical isolates was determined by both sequencing methodologies. The genotyping assay which feeds the virco(®)TYPE HIV-1 virtual phenotype was able to call automatically 97.5% (i.e. 268 mixtures) and 95.3% (i.e. 326 mixtures) of the mixtures that were present between 25 and 75% and between 20 and 80% in the viral population, as detected by 454. From the not called mixtures, all but one did present a mixture sequence in the Sanger DNA chromatograms, however, with a peak surface area for the second peak that was below the threshold setting for automatic mixture calling in the basecaller software (i.e. 25%). Viral loads ranged from 470 to 629,000 copies/mL and exerted no effect on the mixture calling relationship between both sequencing methodologies (R(2)=0.92). In some occasions (i.e. 55 mixtures) the genotyping assay would detect automatically mixtures that were present below 20% in the viral population, when measured by 454. Hence, the mixture scoring sensitivity of the automated high throughput virco(®)TYPE HIV-1 genotyping assay is currently set at 97.5% and 95.3%, for mixtures present at 25 and 20% in the viral population and may identify occasionally mutations that are present at lower frequencies. These findings were not influenced by the viral load of the examined samples.
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VIH-1/genética , Análisis de Secuencia de ARN/métodos , Farmacorresistencia Viral , Genotipo , Infecciones por VIH/tratamiento farmacológico , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , Humanos , MutaciónRESUMEN
Ultra-deep sequencing (UDS) of amplicons is a major application for next-generation sequencing technologies, even more so for the 454 Genome Sequencer FLX. Especially for this application, errors that might be introduced during any of the sample processing or data analysis steps should be avoided or at least recognized, as they might lead to aberrant sequence variant calling. Since 454 pyrosequencing relies on PCR-driven target amplification, it is key to differentiate errors introduced during the amplification step from genuine minority variants. Thereto, optimal primer design is imperative because primer selection, primer dimer formation, and nonspecific binding may all affect the quality and outcome of amplicon-based deep sequencing. Also, other intrinsic PCR characteristics including amplification drift and the formation of secondary structures may influence sequencing data quality. We illustrate these phenomena using real life case studies and propose experimental and analytical evidence-based solutions for effective practice. Furthermore, because accuracy of the DNA polymerase is vital for reliable UDS results, a comparative analysis of error profiles from seven different DNA polymerases was performed and experimentally assessed in parallel by 454 sequencing. Finally, intra and interrun variability evaluation of the 454 sequencing protocol revealed highly reproducible results in amplicon-based UDS.
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Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodos , Secuencia de Bases , Humanos , Control de Calidad , Estándares de ReferenciaRESUMEN
Dual/mixed-tropic HIV-1 strains are predominant in a significative proportion of patients, though few information is available regarding the genetic characteristics, quasispecies composition, and susceptibility against CCR5-antagonists of the primary-isolates. For this reason, we investigated in deep details, both phenotypically and genotypically, the characteristics of 54 HIV-1 primary-isolates obtained from HIV-infected patients. Tropism was assessed by multiple-cycles phenotypic-assay on U87MG-CD4(+)-CCR5(+)-/CXCR4(+)-expressing cells. In vitro selection in PBMCs of X4-tropic viral strains following maraviroc-treatment was also performed. Phenotypic-assay reported pure R5-tropic viruses in 31 (57.4%) isolates, dual/mixed-tropic viruses in 22 (40.7%), and pure X4-tropic virus in only 1 (1.8%). Among dual/mixed-tropic isolates, 12 showed a remarkably higher replication-efficacy in CCR5-expressing cells (R5(+)/X4), and 2 in CXCR4-expressing cells (R5/X4(+)). Genotypic-tropism testing showed a correlation between PSSM-scores, geno2pheno false-positive-rate, and V3-net-charge with both CCR5-usage and syncytium-inducing ability. Moreover, specific gp120- and gp41-mutations were significantly associated with tropism and/or syncytium-inducing ability. Ultra-deep V3-pyrosequencing showed the presence of a swarm of genetically distinct species with a preference for CCR5-coreceptor not only in all pure R5-isolates, but also in 6/7 R5(+)/X4-tropic isolates. In both pure-X4 and R5/X4(+)-isolates, we observed extensive prevalence of X4-using species. In vitro selection-experiments with CCR5-inhibitor maraviroc (up to 2 months) showed no-emergence of X4-tropic variants for all R5- and R5(+)/X4-isolates tested (while X4-virus remained fully-resistant). In conclusion, our study shows that dual/mixed-tropic viruses are constituted by different species, whereby those with characteristics R5(+)/X4 are genotypically and phenotypically similar to the pure-R5 isolates; thus the use of CCR5-antagonists in patients with R5(+)/X4-tropic viruses may be a therapeutic-option that deserves further investigations.
Asunto(s)
Fármacos Anti-VIH/farmacología , Ciclohexanos/farmacología , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/fisiología , Triazoles/farmacología , Tropismo Viral , Replicación Viral/efectos de los fármacos , Línea Celular , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp41 de Envoltorio del VIH/genética , VIH-1/genética , VIH-1/crecimiento & desarrollo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Maraviroc , Datos de Secuencia Molecular , Receptores Virales/metabolismo , Acoplamiento ViralRESUMEN
OBJECTIVE: To compare the distribution of R5-like and X4-like HIV-1 envelope sequences in plasma and peripheral blood mononuclear cell (PBMC). METHODS: Clonal sequencing of the HIV-1 glycoprotein 120 region was performed on PBMC DNA and plasma RNA of 11 HIV-1 subtype B-infected patients with high probability of carrying X4 virus. Coreceptor use was predicted using the position-specific scoring matrix (PSSM). RESULTS: A total of 330 and 427 clonal envelope sequences were obtained from PBMC and plasma, respectively. PSSM interpretation revealed the presence of a mixture of predicted X4 and R5 sequences in 10 patients and pure R5 sequences in 1. The X4 sequences were significantly more represented in PBMC (with an average of 52.2% of the clonal proviral sequences scored X4) compared with plasma (19.7% X4 sequences) (P < 0.0001). At the single patient level, the higher representation of X4 sequences in PBMC reached statistical significance (P < 0.002) in 6 individuals. CONCLUSIONS: Mixtures of X4 and R5 sequences with highly divergent PSSM scores are present in both plasma and PBMC, but a shift toward a more abundant representation of X4-like PSSM scores in PBMC-derived DNA was apparent. Additional studies are needed to evaluate the clinical importance of these findings with regard to tropism prediction and the use of CCR5 antagonists.
Asunto(s)
Infecciones por VIH , VIH-1/clasificación , VIH-1/genética , Leucocitos Mononucleares/virología , Plasma/virología , Receptores CXCR4/metabolismo , Adulto , Clonación Molecular , ADN Viral/análisis , ADN Viral/sangre , ADN Viral/genética , Variación Genética , Proteína gp120 de Envoltorio del VIH/química , Proteína gp120 de Envoltorio del VIH/genética , Proteína gp120 de Envoltorio del VIH/metabolismo , Infecciones por VIH/virología , VIH-1/aislamiento & purificación , VIH-1/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , ARN Viral/análisis , ARN Viral/sangre , ARN Viral/genética , Análisis de Secuencia de ADNRESUMEN
The clinical trials of maraviroc showed that treatment failure was mostly associated with lack of X4 virus detection at baseline. The detection limit for X4 in tropism assays is ill defined around 10%. In the current study, quantification of X4-tropic minority populations was assessed on artificial mixed samples and 38 clinical isolates. These mixtures were subjected to tropism "clonal genotyping" or "population phenotyping". The detection of minority variants was dependant on the input of amplifiable copies. At VL > 4 log IU/ml, X4 quantification was deemed reliable. PCR founder effect and clonal resampling might result in misrepresentation of the minority species concentration at VL < 4 log. Fourteen of the clinical isolates contained dual/mixed X4-tropic virus, 5 of which were below 10% of the virus population. Currently, there is no indication what level of X4 would lead to treatment failure. Assays aiming for the detection of minority species should express results in function of VL.
RESUMEN
Patients with typical features of neurofibromatosis type 1 (NF1) limited to a specific body segment are usually referred to as having "segmental NF1", which is generally assumed to be the result of somatic mosaicism for a NF1 mutation. Mosaicism has also been demonstrated at the molecular level in some sporadic cases with phenotypically classic NF1. In the present report, we describe a patient with NF1 disease manifestations throughout the whole body, but leaving a few sharply delineated segments of the skin unaffected, suggestive of revertant mosaicism. A large intragenic deletion was found by mutation analysis using long-range RT-PCR. The intra-exonic breakpoints were characterized in exon 13 and exon 28, resulting in a deletion of 99,571 bp at the genomic level. The presence of two genetically distinct cell populations, confirming mosaicism for this NF1 mutation, was shown by analysis of several tissues. Revertant mosaicism was excluded by demonstrating heterozygosity for markers residing in the deletion region. The findings in this patient demonstrate two things: (1) although the entire body is affected, mosaicism can still be suspected at clinical examination and proven by DNA analysis and skin biopsies; (2) long-range RT-PCR is a feasible method for demonstrating large intragenic deletions in NF1.