RESUMEN
Human papillomavirus (HPV) genotyping is widely used, particularly in combination with high-risk (HR) HPV tests for cervical cancer screening. We developed a genotyping method using sequences of approximately 800 bp in the E6/E7 region obtained by PacBio single molecule real-time sequencing (SMRT) and evaluated its performance against MY09-11 L1 sequencing and after the APTIMA HPV genotyping assay. The levels of concordance of PacBio E6/E7 SMRT sequencing with MY09-11 L1 sequencing and APTIMA HPV genotyping were 100% and 90.8%, respectively. The sensitivity of PacBio E6/EA7 SMRT was slightly greater than that of L1 sequencing and, as expected, lower than that of HR-HPV tests. In the context of cervical cancer screening, PacBio E6/E7 SMRT is then best used after a positive HPV test. PacBio E6/E7 SMRT genotyping is an attractive alternative for HR and LR-HPV genotyping of clinical samples. PacBio SMRT sequencing provides unbiased genotyping and can detect multiple HPV infections and haplotypes within a genotype.
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Genotipo , Técnicas de Genotipaje , Papillomaviridae , Infecciones por Papillomavirus , Humanos , Infecciones por Papillomavirus/virología , Infecciones por Papillomavirus/diagnóstico , Femenino , Técnicas de Genotipaje/métodos , Papillomaviridae/genética , Papillomaviridae/clasificación , Papillomaviridae/aislamiento & purificación , Sensibilidad y Especificidad , Neoplasias del Cuello Uterino/virología , Neoplasias del Cuello Uterino/diagnóstico , Análisis de Secuencia de ADN/métodos , Detección Precoz del Cáncer/métodos , Proteínas Oncogénicas Virales/genética , ADN Viral/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
OBJECTIVES: To evaluate the routine use of the Sentosa ultra-deep sequencing (UDS) system for HIV-1 polymerase resistance genotyping in treatment-naïve individuals and to analyse the virological response (VR) to first-line antiretroviral treatment. METHODS: HIV drug resistance was determined on 237 consecutive samples from treatment-naïve individuals using the Sentosa UDS platform with two mutation detection thresholds (3% and 20%). VR was defined as a plasma HIV-1 virus load <50 copies/mL after 6 months of treatment. RESULTS: Resistance to at least one antiretroviral drug with a mutation threshold of 3% was identified in 29% and 16% of samples according to ANRS and Stanford algorithms, respectively. The ANRS algorithm also revealed reduced susceptibility to at least one protease inhibitor (PI) in 14.3% of samples, to one reverse transcriptase inhibitor in 12.7%, and to one integrase inhibitor (INSTI) in 5.1%. For a mutation threshold of 20%, resistance was identified in 24% and 13% of samples according to ANRS and Stanford algorithms, respectively. The 6 months VR was 87% and was similar in the 58% of patients given INSTI-based treatment, in the 16% given PI-based treatment and in the 9% given NNRTI-based treatment. Multivariate analysis indicated that the VR was correlated with the baseline HIV virus load and resistance to at least one PI at both 3% and 20% mutation detection thresholds (ANRS algorithm). CONCLUSIONS: The Vela UDS platform is appropriate for determining antiretroviral resistance in patients on a first-line antiretroviral treatment. Further studies are needed on the use of UDS for therapeutic management.
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Fármacos Anti-VIH , Infecciones por VIH , Inhibidores de Integrasa VIH , Seropositividad para VIH , VIH-1 , Humanos , VIH-1/genética , Genotipo , Infecciones por VIH/tratamiento farmacológico , Farmacorresistencia Viral/genética , Antirretrovirales/uso terapéutico , Seropositividad para VIH/tratamiento farmacológico , Inhibidores de Integrasa VIH/uso terapéutico , Secuenciación de Nucleótidos de Alto Rendimiento , Mutación , Fármacos Anti-VIH/farmacología , Fármacos Anti-VIH/uso terapéutico , Carga ViralRESUMEN
New variants and genetic mutations of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genome can only be identified using accurate sequencing methods. Single molecule real-time (SMRT) sequencing has been used to characterize Alpha and Delta variants, but not Omicron variants harboring numerous mutations in the SARS-CoV-2 genome. This study assesses the performance of a target capture SMRT sequencing protocol for whole genome sequencing (WGS) of SARS-CoV-2 Omicron variants and compared it to that of an amplicon SMRT sequencing protocol optimized for Omicron variants. The failure rate of the target capture protocol (6%) was lower than that of the amplicon protocol (34%, p < 0.001) on our data set, and the median genome coverage with the target capture protocol (98.6% [interquartile range (IQR): 86-99.4]) was greater than that with the amplicon protocol (76.6% [IQR: 66-89.6], [p < 0.001]). The percentages of samples with >95% whole genome coverage were 64% with the target capture protocol and 19% with the amplicon protocol (p < 0.05). The clades of 96 samples determined with both protocols were 93% concordant and the lineages of 59 samples were 100% concordant. Thus, target capture SMRT sequencing appears to be an efficient method for WGS, genotyping and detecting mutations of SARS-CoV-2 Omicron variants.
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COVID-19 , Humanos , SARS-CoV-2 , MutaciónRESUMEN
MOTIVATION: The circulating recombinant form of HIV-1 CRF02-AG is the most frequent non-B subtype in Europe. Anti-HIV therapy and pathophysiological studies on the impact of HIV-1 tropism require genotypic determination of HIV-1 tropism for non-B subtypes. But genotypic approaches based on analysis of the V3 envelope region perform poorly when used to determine the tropism of CRF02-AG. We, therefore, designed an algorithm based on information from the gp120 and gp41 ectodomain that better predicts the tropism of HIV-1 subtype CRF02-AG. RESULTS: We used a bio-statistical method to identify the genotypic determinants of CRF02-AG coreceptor use. Toulouse HIV Extended Tropism Algorithm (THETA), based on a Least Absolute Shrinkage and Selection Operator method, uses HIV envelope sequence from phenotypically characterized clones. Prediction of R5X4/X4 viruses was 86% sensitive and that of R5 viruses was 89% specific with our model. The overall accuracy of THETA was 88%, making it sufficiently reliable for predicting the tropism of subtype CRF02-AG sequences. AVAILABILITY AND IMPLEMENTATION: Binaries are freely available for download at https://github.com/viro-tls/THETA. It was implemented in Matlab and supported on MS Windows platform. The sequence data used in this work are available from GenBank under the accession numbers MK618182-MK618417.
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Infecciones por VIH , VIH-1 , Europa (Continente) , Genotipo , Proteína gp120 de Envoltorio del VIH , Receptores CCR5 , Receptores CXCR4 , Plata , Tropismo ViralRESUMEN
OBJECTIVES: To determine how the load of rilpivirine-resistant variants (mutational load) influences the virological response (VR) of HIV-1-infected patients to a rilpivirine-based first-line regimen. PATIENTS AND METHODS: Four hundred and eighty-nine patients infected with HIV-1 whose reverse transcriptase gene had been successfully resistance genotyped using next-generation sequencing were given a first-line regimen containing rilpivirine. Variables associated with the VR at 12 months were identified using a logistic model. The results were used to build a multivariate model for each mutational load threshold and the R2 variations were analysed to identify the mutational load threshold that best predicted the VR. RESULTS: The mutational load at baseline was the only variable linked to the VR at 12 months (Pââ<â0.01). The VR at 12 months decreased from 96.9% to 83.4% when the mutational load was >1700 copies/mL and to 50% when the mutational load was >â9000 copies/mL. The threshold of 9000 copies/mL was associated with the VR at 12 months with an OR of 36.7 (95% CI 4.7-285.1). The threshold of 1700 copies/mL was associated with the VR at 12 months with an OR of 7.2 (95% CI 1.4-36.8). CONCLUSIONS: There is quantifiable evidence that determining a mutational load threshold can be used to identify those patients on a first-line regimen containing rilpivirine who are at risk of virological failure. The clinical management of HIV-infected patients can be improved by evaluating the frequency of mutant variants at a threshold of <â20% together with the plasma HIV-1 viral load at the time of resistance genotyping.
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Farmacorresistencia Viral , Infecciones por VIH/tratamiento farmacológico , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , VIH-1/genética , Mutación , Rilpivirina/uso terapéutico , Terapia Antirretroviral Altamente Activa , Femenino , Genoma Viral , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Masculino , Rilpivirina/farmacología , Resultado del Tratamiento , Carga ViralRESUMEN
Hepatitis E virus genotype 3 (HEV-3) can lead to chronic infection in immunocompromised patients, and ribavirin is the treatment of choice. Recently, mutations in the polymerase gene have been associated with ribavirin failure but their frequency before treatment according to HEV-3 subtypes has not been studied on a large data set. We used single-molecule real-time sequencing technology to sequence 115 new complete genomes of HEV-3 infecting French patients. We analyzed phylogenetic relationships, the length of the polyproline region, and mutations in the HEV polymerase gene. Eighty-five (74%) were in the clade HEV-3efg, 28 (24%) in HEV-3chi clade, and 2 (2%) in HEV-3ra clade. Using automated partitioning of maximum likelihood phylogenetic trees, complete genomes were classified into subtypes. Polyproline region length differs within HEV-3 clades (from 189 to 315 nt). Investigating mutations in the polymerase gene, distinct polymorphisms between HEV-3 subtypes were found (G1634R in 95% of HEV-3e, G1634K in 56% of HEV-3ra, and V1479I in all HEV-3efg, clade HEV-3ra, and HEV-3k strains). Subtype-specific polymorphisms in the HEV-3 polymerase have been identified. Our study provides new complete genome sequences of HEV-3 that could be useful for comparing strains circulating in humans and the animal reservoir.
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Variación Genética , Genotipo , Virus de la Hepatitis E/clasificación , Virus de la Hepatitis E/genética , Hepatitis E/epidemiología , Hepatitis E/virología , Animales , Francia/epidemiología , Genoma Viral , Humanos , Mutación , Filogenia , Secuenciación Completa del GenomaRESUMEN
Objectives: To evaluate the diagnostic performance of the Vela next-generation sequencing (NGS) system in conjunction with the Sentosa SQ HIV Genotyping Assay for genotyping HIV-1. Methods: Plasma RNA was extracted and templates prepared with the Sentosa SX instrument before sequencing the HIV-1 polymerase on the Sentosa SQ301 Sequencer (PGM IonTorrent). The Vela NGS System was compared with direct sequencing and the 454 GS-FLX (Roche) and MiSeq (Illumina) systems for genotypic resistance testing on clinical samples. Results: The Vela NGS system detected majority resistance mutations in subtype B and CRF02-AG samples at 500 copies/mL and minority variants with a sensitivity of 5% at 100â¯000 copies/mL. The Vela NGS system and direct sequencing identified resistance mutations with 97% concordance in 46 clinical samples. Vela identified 1/20 of the 1%-5% mutations identified by 454, 5/12 of the 5%-20% mutations and 60/61 of the >20% mutations. Vela identified 3/14 of the 1%-5% mutations identified by MiSeq, 0/2 of the 5%-20% mutations and 47/47 of the >20% mutations. The resistance mutation quantifications by Vela and 454 were concordant (bias: 2.31%), as were those by Vela and MiSeq (bias: 1.06%). Conclusions: The Vela NGS system provides automated nucleic acid extraction, PCR reagent distribution, library preparation and bioinformatics analysis. The analytical performance was very good when compared with direct sequencing, but was less sensitive than two other NGS platforms for detecting minority variants.
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Automatización de Laboratorios/métodos , Farmacorresistencia Viral , Técnicas de Genotipaje/métodos , Infecciones por VIH/virología , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Pruebas de Sensibilidad Microbiana/métodos , Biología Computacional/métodos , Genes Virales , Genotipo , VIH-1/efectos de los fármacos , VIH-1/aislamiento & purificación , Mutación , ARN Viral/genética , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genéticaRESUMEN
The present study compares the performances of an in-house sequencing protocol developed on MiSeq, the Sanger method, and the 454 GS-FLX for detecting and quantifying drug-resistant mutations (DRMs) in the human immunodeficiency virus polymerase gene (reverse transcriptase [RT] and protease [PR]). MiSeq sequencing identified all the resistance mutations detected by bulk sequencing (n = 84). Both the MiSeq and 454 GS-FLX platforms identified 67 DRMs in the RT and PR regions, but a further 25 DRMs were identified by only one or other of them. Pearson's analysis showed good concordance between the percentage of drug-resistant variants determined by MiSeq and 454 GS-FLX sequencing (ρ = .77, P < .0001). The MiSeq platform is as accurate as the 454 GS-FLX Roche system for determining RT and PR DRMs and could be used for monitoring human immunodeficiency virus type 1 drug resistance.
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Variación Genética , Infecciones por VIH/virología , VIH-1/clasificación , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Mutación , Productos del Gen pol del Virus de la Inmunodeficiencia Humana/genética , Farmacorresistencia Viral , Proteasa del VIH/genética , Transcriptasa Inversa del VIH/genética , VIH-1/aislamiento & purificación , HumanosRESUMEN
Hepatitis E virus (HEV) can lead to chronic infection in solid-organ transplant patients. Ribavirin is efficient for treatment of chronically infected patients. Recently, the1634R mutation in the HEV polymerase has been associated with treatment failure. However, it is unclear if this mutation can be used as a prognostic marker of treatment outcome. We studied the prevalence of the 1634R mutation in the HEV polymerase of patients starting ribavirin therapy, the influence of the 1634R variants on the viral response, the frequency of the 1634R mutation in patients whose treatment failed, and its impact on ribavirin retreatment. We analyzed pretreatment samples from 63 solid-organ transplant patients with chronic hepatitis E using deep sequencing; 42 patients had a sustained virologic response (SVR), and 21 were non-SVR patients. We detected the 1634R variant by deep sequencing in 36.5% (23/63) of the patients (proportions, 1.3 to 100%). The 1634R variant was detected in 31.0% (13/42) of baseline plasma samples from patients with SVR and in 47.6% (10/21) in the other patients (P = 0.2). The presence of this mutation did not influence the initial decrease in viral RNA. Lastly, a second prolonged ribavirin treatment led to SVR in 70% of the patients who initially did not have SVR, despite the presence of the 1634R variant. We conclude that the presence of the 1634R variant at ribavirin initiation does not lead to absolute ribavirin resistance. Although its proportion increased in patients whose treatment failed, the presence of the 1634R variant did not compromise the response to a second ribavirin treatment.
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Antivirales/uso terapéutico , Virus de la Hepatitis E/efectos de los fármacos , Virus de la Hepatitis E/genética , Hepatitis E/tratamiento farmacológico , ARN Polimerasa Dependiente del ARN/genética , Ribavirina/uso terapéutico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Secuencia de Bases , Niño , Femenino , Marcadores Genéticos , Hepatitis E/virología , Humanos , Masculino , Persona de Mediana Edad , Mutación/genética , ARN Viral/genética , Análisis de Secuencia de ARN , Resultado del Tratamiento , Adulto JovenRESUMEN
We have recently demonstrated the remarkable efficiency of self-complementary (sc) AAV9 vectors for central nervous system (CNS) gene transfer following intravenous delivery in mice and larger animals. Here, we investigated whether gene delivery to motor neurons (MNs) could also be achieved via intramuscular (i.m.) scAAV9 injection and subsequent retrograde transport along the MNs axons. Unexpectedly, we found that a single injection of scAAV9 into the adult mouse gastrocnemius (GA) mediated widespread MN transduction along the whole spinal cord, without limitation to the MNs connected to the injected muscle. Spinal cord astrocytes and peripheral organs were also transduced, indicating vector spread from the injected muscle to both the CNS and the periphery through release into the blood circulation. Moreover, we showed that i.m. injection of scAAV9 vectors expressing "survival of motor neuron" (Smn) in spinal muscular atrophy (SMA) mice mediated high survival motor neuron (SMN) expression levels at both the CNS and the periphery, and increased the median lifespan from 12 days to 163 days. These findings represent to date the longest extent in survival obtained in SMA mice following i.m. viral vector gene delivery, and might generate a renewed interest in the use of i.m. adeno-associated viruses (AAV) delivery for the development of gene therapy strategies for MN diseases.
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Técnicas de Transferencia de Gen , Terapia Genética/métodos , Atrofia Muscular Espinal/terapia , Médula Espinal/patología , Animales , Sistema Nervioso Central/metabolismo , Sistema Nervioso Central/patología , Dependovirus/genética , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Vectores Genéticos , Inyecciones Intramusculares , Ratones , Ratones Transgénicos , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Músculo Esquelético/metabolismo , Músculo Esquelético/patología , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patología , Médula Espinal/metabolismo , TransgenesRESUMEN
BACKGROUND: HIV-1 resistance testing is recommended in clinical management and next-generation sequencing (NGS) methods are now available in many virology laboratories. OBJECTIVES: To evaluate the diagnostic performance of Long-Read Single Molecule Real-time (SMRT) sequencing (Sequel, PacBio) for HIV-1 polymerase genotyping. STUDY DESIGN: 111 prospective clinical samples (83 plasma and 28 leukocyte-enriched blood fraction) were analyzed for routine HIV-1 resistance genotyping using Sanger sequencing, Vela NGS, and SMRT sequencing. We developed a SMRT sequencing protocol and a bio-informatics pipeline to infer antiretroviral resistance on both haplotype and variant calling approaches. RESULTS: The polymerase was successfully sequenced by the three platforms in 98 % of plasma RNA samples for viral loads above 4 log copies/mL. The success rate decreased to 83 % using Sanger or Vela sequencing and to 67 % using SMRT sequencing for viral loads of 3 to 4 log copies/mL. Sensitivities of 50 %, 54 % and 61 % were obtained using SMRT, Vela, and Sanger sequencing, respectively, in cellular DNA from patients with prolonged undetectable plasma HIV-1 RNA. Ninety-eight percent of resistance-associated mutations (RAMs) identified with Sanger sequencing were detected using SMRT sequencing. Furthermore, 91 % of RAMs (> 5 % threshold) identified with Vela NGS were detected using SMRT sequencing. RAM quantification using Vela and SMRT sequencing was well correlated (Spearman correlation ρ = 0.82; P < 0.0001). CONCLUSIONS: SMRT sequencing of the full-length HIV-1 polymerase appeared performant for characterizing HIV-1 genotypic resistance on both RNA and DNA clinical samples. Long-read sequencing is a new tool for mutation haplotyping and resistance analysis.
RESUMEN
Spinal muscular atrophy (SMA) is the most common genetic disease leading to infant mortality. This neuromuscular disorder is caused by the loss or mutation of the telomeric copy of the 'survival of motor neuron' (Smn) gene, termed SMN1. Loss of SMN1 leads to reduced SMN protein levels, inducing degeneration of motor neurons (MN) and progressive muscle weakness and atrophy. To date, SMA remains incurable due to the lack of a method to deliver therapeutically active molecules to the spinal cord. Gene therapy, consisting of reintroducing SMN1 in MNs, is an attractive approach for SMA. Here we used postnatal day 1 systemic injection of self-complementary adeno-associated virus (scAAV9) vectors carrying a codon-optimized SMN1 sequence and a chimeric intron placed downstream of the strong phosphoglycerate kinase (PGK) promoter (SMNopti) to overexpress the human SMN protein in a mouse model of severe SMA. Survival analysis showed that this treatment rescued 100% of the mice, increasing life expectancy from 27 to over 340 days (median survival of 199 days) in mice that normally survive about 13 days. The systemic scAAV9 therapy mediated complete correction of motor function, prevented MN death and rescued the weight loss phenotype close to normal. This study reports the most efficient rescue of SMA mice to date after a single intravenous injection of an optimized SMN-encoding scAAV9, highlighting the considerable potential of this method for the treatment of human SMA.
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Dependovirus/genética , Terapia Genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/terapia , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Animales , Modelos Animales de Enfermedad , Femenino , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Humanos , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Atrofia Muscular Espinal/patología , Fenotipo , Médula Espinal/metabolismo , Médula Espinal/patología , Análisis de Supervivencia , Resultado del TratamientoRESUMEN
OBJECTIVES: R5 viruses have long been thought to account for almost all strains present in primary HIV-1 infections (PHIs), but recent studies using sensitive phenotypic assays have revealed that 3%-6.4% of subjects also harbour CXCR4-using viruses. Phenotypic assays provide only qualitative results: the presence or absence of CXCR4-using viruses. We have therefore used ultra-deep pyrosequencing (UDS) to determine the frequency of CXCR4-using viruses among HIV-1 quasispecies. METHODS: We first screened 200 patients for HIV-1 tropism using a sensitive phenotypic assay during PHI and identified 11 infected with an R5X4 dual/mixed (D/M) virus population. We then used UDS of the V3 env region with the geno2pheno algorithm (false positive rate = 5.75) to identify the HIV-1 quasispecies. RESULTS: CXCR4-using viruses were detected in all but 1 of the 11 patients by UDS, and accounted for 0.2%-100% of the virus populations. The frequency of CXCR4-using viruses was <20% in six subjects and 100% in four subjects. Virus populations containing 100% CXCR4-using variants during PHI persisted for at least 1-2 years after the acute phase. The CCR5 Δ32 heterozygous genotype was similarly prevalent in patients infected with D/M (27%) and R5 (15%) viruses. CONCLUSIONS: UDS and the phenotype were concordant for determining HIV-1 coreceptor usage. UDS analysis indicated large differences in the percentage of CXCR4-using viruses in the HIV-1 quasispecies during PHI. Further studies should examine the impact of the proportion of CXCR4-using viruses on disease prognosis.
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Infecciones por VIH/virología , VIH-1/genética , VIH-1/fisiología , Secuenciación de Nucleótidos de Alto Rendimiento , ARN Viral/genética , Receptores CXCR4/metabolismo , Tropismo Viral , Adulto , Femenino , Variación Genética , Proteína gp120 de Envoltorio del VIH/genética , VIH-1/clasificación , Humanos , Masculino , Receptores del VIH/metabolismo , Adulto JovenRESUMEN
Co-infection with at least 2 strains of virus is the prerequisite for recombination, one of the means of genetic diversification. Little is known about the prevalence of these events in SARS-CoV-2, partly because it is difficult to detect them. We used long-read PacBio single-molecule real-time (SMRT) sequencing technology to sequence whole genomes and targeted regions for haplotyping. We identified 17 co-infections with SARS-CoV-2 strains belonging to different clades in 6829 samples sequenced between January and October, 2022 (prevalence 0.25%). There were 3 Delta/Omicron co-infections and 14 Omicron/Omicron co-infections (4 cases of 21K/21L, 1 case of 21L/22A, 2 cases of 21L/22B, 4 cases of 22A/22B, 2 cases of 22B/22C and 1 case of 22B/22E). Four of these patients (24%) also harbored recombinant minor haplotypes, including one with a recombinant virus that was selected in the viral quasispecies over the course of his chronic infection. While co-infections remain rare among SARS-CoV-2-infected individuals, long-read SMRT sequencing is a useful tool for detecting them as well as recombinant events, providing the basis for assessing their clinical impact, and a precise indicator of epidemic evolution. IMPORTANCE SARS-CoV-2 variants have been responsible for the successive waves of infection over the 3 years of pandemic. While co-infection followed by recombination is one driver of virus evolution, there have been few reports of co-infections, mainly between Delta and Omicron variants or between the first 2 Omicron variants 21K_BA.1 and 21L_BA.2. The 17 co-infections we detected during 2022 included cases with the recent clades of Omicron 22A, 22B, 22C, and 22E; 24% harbored recombinant variants. This study shows that long-read SMRT sequencing is well suited to SARS-CoV-2 genomic surveillance.
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COVID-19 , Coinfección , Humanos , COVID-19/epidemiología , SARS-CoV-2/genética , Pandemias , Recombinación GenéticaRESUMEN
BACKGROUND: Congenital secondary erythrocytoses are due to deregulation of hypoxia inducible factor resulting in overproduction of erythropoietin. The most common germline mutation identified in the hypoxia signaling pathway is the Arginine 200-Tryptophan mutant of the von Hippel-Lindau tumor suppressor gene, resulting in Chuvash polycythemia. This mutant displays a weak deficiency in hypoxia inducible factor α regulation and does not promote tumorigenesis. Other von Hippel-Lindau mutants with more deleterious effects are responsible for von Hippel-Lindau disease, which is characterized by the development of multiple tumors. Recently, a few mutations in gene for the prolyl hydroxylase domain 2 protein (PHD2) have been reported in cases of congenital erythrocytosis not associated with tumor formation with the exception of one patient with a recurrent extra-adrenal paraganglioma. DESIGN AND METHODS: Five PHD2 variants, four of which were novel, were identified in patients with erythrocytosis. These PHD2 variants were functionally analyzed and compared with the PHD2 mutant previously identified in a patient with polycythemia and paraganglioma. The capacity of PHD2 to regulate the activity, stability and hydroxylation of hypoxia inducible factor α was assessed using hypoxia-inducible reporter gene, one-hybrid and in vitro hydroxylation assays, respectively. RESULTS: This functional comparative study showed that two categories of PHD2 mutants could be distinguished: one category with a weak deficiency in hypoxia inducible factor α regulation and a second one with a deleterious effect; the mutant implicated in tumor occurrence belongs to the second category. CONCLUSIONS: As observed with germline von Hippel-Lindau mutations, there are functional differences between the PHD2 mutants with regards to hypoxia inducible factor regulation. PHD2 mutation carriers do, therefore, need careful medical follow-up, since some mutations must be considered as potential candidates for tumor predisposition.
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Mutación de Línea Germinal , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas Mutantes/metabolismo , Policitemia/genética , Policitemia/metabolismo , Procolágeno-Prolina Dioxigenasa/metabolismo , Adolescente , Adulto , Secuencia de Bases , Células Cultivadas , Femenino , Células HEK293 , Humanos , Hidrólisis , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Masculino , Persona de Mediana Edad , Procolágeno-Prolina Dioxigenasa/genética , Adulto JovenRESUMEN
Prolyl hydroxylase domain (PHD) proteins play a major role in regulating the hypoxia-inducible factor (HIF) that induces expression of genes involved in angiogenesis, erythropoiesis, and cell metabolism, proliferation, and survival. Germ-line mutations in the prolyl hydroxylase domain 2 gene (PHD2) have been reported in patients with familial erythrocytosis but not in association with tumors. We describe a patient with erythrocytosis and recurrent paraganglioma who carries a newly discovered PHD2 mutation. This mutation affects PHD2 function and stabilizes HIF-alpha proteins. In addition, we demonstrate loss of heterozygosity of PHD2 in the tumor, suggesting that PHD2 could be a tumor-suppressor gene.
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Mutación de Línea Germinal , Pérdida de Heterocigocidad , Neoplasias del Mediastino/genética , Paraganglioma/genética , Policitemia/genética , Procolágeno-Prolina Dioxigenasa/genética , Adulto , Femenino , Proteína de la Hemocromatosis , Antígenos de Histocompatibilidad Clase I/genética , Homocigoto , Humanos , Prolina Dioxigenasas del Factor Inducible por Hipoxia , Masculino , Proteínas de la Membrana/genética , Neoplasias Primarias Secundarias/genética , Linaje , Policitemia/congénito , Policitemia/diagnóstico , Procolágeno-Prolina Dioxigenasa/metabolismo , Análisis de Secuencia de ADNRESUMEN
BACKGROUND: Patients on antiretroviral therapy could benefit from HIV-1 DNA resistance genotyping for exploring virological failure with low viral load or to guide treatment simplification. Few new generation sequencing data are available. OBJECTIVE: To check that the automated deep sequencing Sentosa platform (Vela DX) detected minority resistant variants well enough for HIV DNA genotyping. STUDY DESIGN: We evaluated the Sentosa SQ HIV genotyping assay with automated extraction on 40 DNA longitudinal samples from treatment-experienced patients by comparison with Sanger sequencing. HIV drug resistance was interpreted using the ANRS algorithm (v29) at the threshold of 20 % and 3 %. RESULTS: The Sentosa SQ HIV genotyping assay was 100 % successful to amplify and sequence PR and RT and 86 % to amplify and sequence IN when the HIV DNA load was >2.5 log copies/million cells. The Sentosa and Sanger sequencing were concordant for predicting PR-RT resistance at the threshold of 20 % in 14/18 samples successfully sequenced. A higher level of resistance was predicted by Sentosa in three samples and by Sanger in one sample. The prevalence of resistance was 7 % to PI, 59 % to NRTI, 31 % to NNRTI and 20 % to integrase inhibitors using the Sentosa SQ genotyping assay at the threshold of 3 %. Seven additional mutations <20 % were detected using the Sentosa assay. CONCLUSION: Automated DNA extraction and sequencing using the Sentosa SQ HIV genotyping assay accurately predicted HIV DNA drug resistance by comparison with Sanger. Prospective studies are needed to evaluate the clinical interest of HIV DNA genotyping.
Asunto(s)
Farmacorresistencia Viral/genética , Técnicas de Genotipaje/métodos , VIH-1/genética , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Automatización de Laboratorios , Infecciones por VIH/virología , VIH-1/efectos de los fármacos , Humanos , Estudios Longitudinales , Mutación , ARN Viral/sangre , Carga ViralRESUMEN
Recombinant strains of hepatitis E virus (HEV) with insertions of human genomic fragments or HEV sequence duplications in the sequence encoding the polyproline region (PPR) were previously described in chronically infected patients. Such genomic rearrangements confer a replicative advantage in vitro but little is known about their frequency, location, or origin. As the sequences of only a few virus genomes are available, we analyzed the complete genomes of 114 HEV genotype 3 strains from immunocompromised (n = 85) and immunocompetent (n = 29) patients using the single molecular real-time sequencing method to determine the frequency, location, and origin of inserted genomic fragments, plus the proportions of variants with genomic rearrangements in each virus quasispecies. We also examined the amino acid compositions and post-translational modifications conferred by these rearrangements by comparing them to sequences without human gene insertions or HEV gene duplications. We found genomic rearrangements in 7/114 (6.1%) complete genome sequences (4 HEV-3f, 1 HEV-3e, 1 HEV-3 h, and 1 HEV-3chi-new), all from immunocompromised patients, and 3/7 were found at the acute phase of infection. Six of the seven patients harbored virus-host recombinant variants, including one patient with two different recombinant variants. We also detected three recombinant variants with genome duplications of the PPR or PPR + X domains in a single patient. All the genomic rearrangements (seven human fragment insertions of varying origins and three HEV genome duplications) occurred in the PPR. The sequences with genomic rearrangements had specific characteristics: increased net load (p < 0.001) and more ubiquitination (p < 0.001), phosphorylation (p < 0.001), and acetylation (p < 0.001) sites. The human fragment insertions and HEV genome duplications had slightly different characteristics. We believe this is the first description of HEV strains with genomic rearrangements in patients at the acute phase of infection; perhaps these strains are directly transmitted. Clearly, genomic rearrangements produce a greater net load with duplications and insertions having different features. Further studies are needed to clarify the mechanisms by which such modifications influence HEV replication.
RESUMEN
OBJECTIVE: To study the long-term evolution of the transmitted CXCR4-using viruses. CCR5-using viruses (R5 viruses) predominate during primary HIV-1 infections (PHI) while CXCR4-using viruses are isolated in less than 10% of PHI. DESIGN: Six patients infected with an R5X4 virus, detected by a sensitive phenotypic assay during PHI, were matched with six patients infected with a pure R5 virus for sex, Fiebig stage, time of antiretroviral initiation and duration of follow-up. METHODS: We used MiSeq ultra-deep sequencing to determine the composition of the virus quasispecies during PHI and at the end of follow-up (median time of follow-up: 12.5 years). RESULTS: X4 viruses were detected by genetic analysis in three of six samples from the R5X4 group, accounting for 1.3-100% of the virus quasispecies, during PHI, and in four of six samples (accounting for 6.7-100%) at the end of follow-up. No X4 virus was detected in the R5 group during PHI and in only one patient (accounting for 1.2%) at the end of follow-up. The complexity of the virus quasispecies at the stage of PHI was higher in the R5X4 group than in the R5 group. Complexity increased from PHI to the end of follow-up in the R5 group but remained stable in the R5X4 group. CONCLUSION: CXCR4-using viruses persisted in the peripheral blood mononuclear cells of several patients on suppressive antiretroviral therapy for a median duration of 12.5 years after PHI. The genetic complexity of HIV-1 evolved differently post-PHI in patients infected with R5X4 viruses from those infected with R5 viruses.