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BACKGROUND: Four severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) variants predominated in the United States since 2021. Understanding disease severity related to different SARS-CoV-2 variants remains limited. METHOD: Viral genome analysis was performed on SARS-CoV-2 clinical isolates circulating March 2021 through March 2022 in Cleveland, Ohio. Major variants were correlated with disease severity and patient outcomes. RESULTS: In total 2779 patients identified with either Alpha (n 1153), Gamma (n 122), Delta (n 808), or Omicron variants (n 696) were selected for analysis. No difference in frequency of hospitalization, intensive care unit (ICU) admission, and death were found among Alpha, Gamma, and Delta variants. However, patients with Omicron infection were significantly less likely to be admitted to the hospital, require oxygen, or admission to the ICU (2 12.8, P .001; 2 21.6, P .002; 2 9.6, P .01, respectively). In patients whose vaccination status was known, a substantial number had breakthrough infections with Delta or Omicron variants (218/808 [26.9] and 513/696 [73.7], respectively). In breakthrough infections, hospitalization rate was similar regardless of variant by multivariate analysis. No difference in disease severity was identified between Omicron subvariants BA.1 and BA.2. CONCLUSIONS: Disease severity associated with Alpha, Gamma, and Delta variants is comparable while Omicron infections are significantly less severe. Breakthrough disease is significantly more common in patients with Omicron infection.
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COVID-19 , Humanos , SARS-CoV-2/genética , Gravedad del Paciente , Infección IrruptivaRESUMEN
SARS-CoV-2 mutation is minimized through a proofreading function encoded by NSP-14. Most estimates of the SARS-CoV-2 mutation rate are derived from population based sequence data. Our understanding of SARS-CoV-2 evolution might be enhanced through analysis of intra-host viral mutation rates in specific populations. Viral genome analysis was performed between paired samples and mutations quantified at allele frequencies (AF) ≥ 0.25, ≥ 0.5 and ≥ 0.75. Mutation rate was determined employing F81 and JC69 evolution models and compared between isolates with (ΔNSP-14) and without (wtNSP-14) non-synonymous mutations in NSP-14 and by patient comorbidity. Forty paired samples with median interval of 13 days [IQR 8.5-20] were analyzed. The estimated mutation rate by F81 modeling was 93.6 (95%CI 90.8-96.4], 40.7 (95%CI 38.9-42.6) and 34.7 (95%CI 33.0-36.4) substitutions/genome/year at AF ≥ 0.25, ≥ 0.5, ≥ 0.75 respectively. Mutation rate in ΔNSP-14 were significantly elevated at AF ≥ 0.25 vs wtNSP-14. Patients with immune comorbidities had higher mutation rate at all allele frequencies. Intra-host SARS-CoV-2 mutation rates are substantially higher than those reported through population analysis. Virus strains with altered NSP-14 have accelerated mutation rate at low AF. Immunosuppressed patients have elevated mutation rate at all AF. Understanding intra-host virus evolution will aid in current and future pandemic modeling.
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COVID-19 , Humanos , COVID-19/epidemiología , Tasa de Mutación , SARS-CoV-2/genética , Pandemias , Mutación , Genoma Viral/genéticaRESUMEN
Mutations in the genome of SARS-CoV-2 can affect the performance of molecular diagnostic assays. In some cases, such as S-gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here, we describe partial ORF1ab gene target failure (pOGTF) on the cobas SARS-CoV-2 assays, defined by a ≥2-thermocycle delay in detection of the ORF1ab gene compared to that of the E-gene. We demonstrate that pOGTF is 98.6% sensitive and 99.9% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may affect transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly, increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.
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COVID-19 , SARS-CoV-2 , Secuencia de Bases , Humanos , Mutación , SARS-CoV-2/genéticaRESUMEN
FLT3-internal tandem duplication occurs in 20-30% of acute myeloid leukemia and confers an adverse prognosis with its allelic ratio being a key risk stratifier. The US Food and Drug Administration recently approved FLT3 inhibitors midostaurin and gilteritinib in FLT3 mutation-positive acute myeloid leukemia. Historically, FLT3 was tested by fragment analysis, which has become the standard method endorsed by international guidelines. However, next generation sequencing is increasingly used at acute myeloid leukemia diagnosis given its ability to simultaneously evaluate multiple clinically informative markers. As FLT3-internal tandem duplication detection was known to be challenging by next generation sequencing and the results carry profound prognostic and therapeutic implications, it is important to thoroughly examine its performance in FLT3-internal tandem duplication detection and allelic ratio classification. In a comparative study with fragment analysis, we retrospectively reviewed our experience using a custom-designed, hybridization capture-based, targeted next generation sequencing panel. Among 7902 cases, FLT3-internal tandem duplication was detected in 335 with variable sizes (3-231 bp) and insertion sites. Fragment analysis was also performed in 402 cases, demonstrating 100% concordance in FLT3-internal tandem duplication detection. In 136 dual-tested, positive cases, 128/136 (94%) exhibited concordant high/low allelic ratio classifications. The remaining 6% showed borderline low allelic ratio by next generation sequencing. The two methods were concordant in FLT3-tyrosine kinase domain mutation detection at the hotspot D835/I836 targeted by fragment analysis. Furthermore, seven mutations which may benefit from FLT3 inhibitor therapy were detected by next generation sequencing, in regions not covered by fragment analysis. Our study demonstrates that using a hybridization capture-based chemistry and optimized bioinformatics pipeline, next generation sequencing can reliably detect FLT3-internal tandem duplication and classify its allelic ratio for acute myeloid leukemia risk stratification. Next generation sequencing also exhibits superior comprehensiveness in FLT3 mutation detection and may further improve personalized, targeted therapy in acute myeloid leukemia.
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Biomarcadores de Tumor/genética , Análisis Mutacional de ADN , Secuenciación de Nucleótidos de Alto Rendimiento , Leucemia Mieloide Aguda/genética , Mutación , Secuencias Repetidas en Tándem , Tirosina Quinasa 3 Similar a fms/genética , Biología Computacional , Predisposición Genética a la Enfermedad , Humanos , Fenotipo , Valor Predictivo de las Pruebas , Reproducibilidad de los Resultados , Estudios RetrospectivosRESUMEN
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
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This study piloted a pan-solid-tumor next generation sequence (NGS)-based laboratory developed test as a diagnostic aid in melanocytic tumors. 31 cases (4 "epithelioid" nevi, 5 blue nevi variants, 7 Spitz tumors [3 benign and 4 malignant] and 15 melanomas) were evaluated. All tumors [median diameter 7 mm (range 4-15 mm); median thickness 2.25 mm (range 0.25-12 mm)] yielded satisfactory results. The number of small nucleotide variants/tumor was significantly different between melanoma (median 18/tumor, range 4-71) and all other lesions (median 8/tumor, range 3-17) (P < 0.004) and malignant (median 16/tumor, range 4-71) vs benign lesions (median 7/tumor, range 3-14) (P = 0.01). BRAF, MET, NTRK1, and ROS fusions only occurred in benign Spitz tumors; EML4 fusion, BRAF, MAP2K1 and TERT mutations occurred in malignant Spitz tumors and/or melanoma. Amplifications and NRAS and NF1 mutations only occurred in melanoma. Most melanomas contained >1 pathogenic alteration. Developed NGS-based criteria correctly classified all malignant lesions in this series. 10/12 cases showed concordance with FISH; consensus diagnosis agreed with NGS classification in FISH-non-concordant cases. This pilot study suggests that NGS may be an effective diagnostic adjunct comparable to FISH, but further studies with larger numbers of cases are needed.
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Secuenciación de Nucleótidos de Alto Rendimiento/clasificación , Melanocitos/metabolismo , Melanocitos/patología , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Consenso , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Hibridación Fluorescente in Situ/métodos , Hibridación Fluorescente in Situ/estadística & datos numéricos , Lactante , Masculino , Melanoma/genética , Melanoma/patología , Persona de Mediana Edad , Nevo Azul/genética , Nevo Azul/patología , Nevo de Células Epitelioides y Fusiformes/genética , Nevo de Células Epitelioides y Fusiformes/patología , Nucleótidos/genética , Proyectos Piloto , Carga Tumoral/genética , Adulto JovenRESUMEN
BACKGROUND AND AIMS: In an era of precision medicine, customized genotyping of GI stromal tumors by screening for driver mutations will become the standard of care. The fidelity of genotype concordance between paired cytology smears and surgical pathology specimens is unknown. In patients with either primary or metastatic sporadic disease, we sought to determine the frequency of KIT and PDGFRA pathogenic alterations within such specimens, imatinib sensitivity, and the concordance of pathogenic alterations between paired specimens. METHODS: DNA obtained from cytology smears from 36 patients, 24 of whom had paired surgical pathology specimens, underwent targeted next-generation sequencing by using a custom panel to evaluate somatic mutations within KIT (exon 2, 9, 10, 11, 13, 14, 15, 17, 18) and PDGFRA (exon 12, 14, 15, 18) genes. Patients with KIT and PDGRFA wild-type genes completed the Qiagen Human Comprehensive Cancer GeneRead DNAseq Targeted Array V2. RESULTS: Genotyping revealed KIT and PDGFRA mutations in 68% and 15% of patients. The wild-type population did not harbor mutations in BRAF, RAS family, SDHB, SETD2, or NF1. Imatinib sensitivity based on the oncogenic kinase mutation prevalence was estimated to be 68%. Mutational concordance between paired cytology and surgical pathology specimens was 96%. CONCLUSIONS: Our data have demonstrated the ability to stratify either primary or metastatic gastrointestinal stromal tumors by mutational subtype using a targeted next-generation sequencing 2 gene mutation panel. We highlight the ability to use cytology specimens obtained via minimally invasive techniques as a surrogate to surgical specimens given the high mutational landscape concordance between paired specimens.
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ADN de Neoplasias/análisis , Neoplasias Gastrointestinales/genética , Tumores del Estroma Gastrointestinal/genética , Técnicas de Genotipaje , Proteínas Proto-Oncogénicas c-kit/genética , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genética , Antineoplásicos/uso terapéutico , Quimioterapia Adyuvante , Análisis Citogenético , Resistencia a Antineoplásicos/genética , Biopsia por Aspiración con Aguja Fina Guiada por Ultrasonido Endoscópico , Neoplasias Gastrointestinales/patología , Neoplasias Gastrointestinales/terapia , Tumores del Estroma Gastrointestinal/patología , Tumores del Estroma Gastrointestinal/terapia , Secuenciación de Nucleótidos de Alto Rendimiento , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Mesilato de Imatinib/uso terapéutico , Neurofibromina 1/genética , Medicina de Precisión , Proteínas Proto-Oncogénicas B-raf/genética , Radiología Intervencionista , Succinato Deshidrogenasa/genética , Tomografía Computarizada por Rayos X , Proteínas ras/genéticaRESUMEN
The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence.
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Ascomicetos/genética , Péptido Sintasas/genética , Enfermedades de las Plantas , Sintasas Poliquetidas/genética , Polimorfismo de Nucleótido Simple/genética , Ascomicetos/patogenicidad , Secuencia de Bases , Evolución Molecular , Variación Genética , Genoma Fúngico , Filogenia , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/parasitología , Virulencia/genéticaRESUMEN
Antimicrobials have been used extensively as growth promoters (AGPs) in agricultural animal production. However, the specific mechanism of action for AGPs has not yet been determined. The work presented here was to determine and characterize the microbiome of pigs receiving one AGP, tylosin, compared with untreated pigs. We hypothesized that AGPs exerted their growth promoting effect by altering gut microbial population composition. We determined the fecal microbiome of pigs receiving tylosin compared with untreated pigs using pyrosequencing of 16S rRNA gene libraries. The data showed microbial population shifts representing both microbial succession and changes in response to the use of tylosin. Quantitative and qualitative analyses of sequences showed that tylosin caused microbial population shifts in both abundant and less abundant species. Our results established a baseline upon which mechanisms of AGPs in regulation of health and growth of animals can be investigated. Furthermore, the data will aid in the identification of alternative strategies to improve animal health and consequently production.
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Antiinfecciosos/farmacología , Intestinos/microbiología , Tilosina/farmacología , Animales , Bacterias/efectos de los fármacos , Biodiversidad , Biología Computacional/métodos , Tracto Gastrointestinal/efectos de los fármacos , Tracto Gastrointestinal/microbiología , Biblioteca de Genes , Metagenoma , Metagenómica , Reacción en Cadena de la Polimerasa , ARN Ribosómico 16S/genética , Análisis de Secuencia de ADN , PorcinosRESUMEN
Phosphorus, in its orthophosphate form (P(i)), is one of the most limiting macronutrients in soils for plant growth and development. However, the whole-genome molecular mechanisms contributing to plant acclimation to P(i) deficiency remain largely unknown. White lupin (Lupinus albus) has evolved unique adaptations for growth in P(i)-deficient soils, including the development of cluster roots to increase root surface area. In this study, we utilized RNA-Seq technology to assess global gene expression in white lupin cluster roots, normal roots, and leaves in response to P(i) supply. We de novo assembled 277,224,180 Illumina reads from 12 complementary DNA libraries to build what is to our knowledge the first white lupin gene index (LAGI 1.0). This index contains 125,821 unique sequences with an average length of 1,155 bp. Of these sequences, 50,734 were transcriptionally active (reads per kilobase per million reads ≥ 3), representing approximately 7.8% of the white lupin genome, using the predicted genome size of Lupinus angustifolius as a reference. We identified a total of 2,128 sequences differentially expressed in response to P(i) deficiency with a 2-fold or greater change and P ≤ 0.05. Twelve sequences were consistently differentially expressed due to P(i) deficiency stress in three species, Arabidopsis (Arabidopsis thaliana), potato (Solanum tuberosum), and white lupin, making them ideal candidates to monitor the P(i) status of plants. Additionally, classic physiological experiments were coupled with RNA-Seq data to examine the role of cytokinin and gibberellic acid in P(i) deficiency-induced cluster root development. This global gene expression analysis provides new insights into the biochemical and molecular mechanisms involved in the acclimation to P(i) deficiency.
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Aclimatación/genética , Lupinus/genética , Fosfatos/farmacología , Fósforo/farmacología , Transcriptoma/efectos de los fármacos , Análisis por Conglomerados , Ecosistema , Perfilación de la Expresión Génica/métodos , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Lupinus/crecimiento & desarrollo , Lupinus/metabolismo , MicroARNs/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Oxidorreductasas/genética , Fosfatos/metabolismo , Fósforo/metabolismo , Proteínas de Plantas/clasificación , Proteínas de Plantas/genética , Raíces de Plantas/genética , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Análisis de Secuencia de ARN , Suelo/químicaRESUMEN
Therapy-related myeloid neoplasms (tMN) are complications of cytotoxic therapies. Risk of tMN is high in recipients of autologous hematopoietic stem cell transplantation (aHSCT). Acquisition of genomic mutations represents a key pathogenic driver but the origins, timing and dynamics, particularly in the context of preexisting or emergent clonal hematopoiesis (CH), have not been sufficiently clarified. We studied a cohort of 1507 patients undergoing aHSCT and a cohort of 263 patients who developed tMN without aHSCT to determine clinico-molecular features unique to post-aHSCT tMN. We show that tMN occurs in up to 2.3% of patients at median of 2.6 years post-AHSCT. Age ≥ 60 years, male sex, radiotherapy, high treatment burden ( ≥ 3 lines of chemotherapy), and graft cellularity increased the risk of tMN. Time to evolution and overall survival were shorter in post-aHSCT tMN vs. other tMN, and the earlier group's mutational pattern was enriched in PPM1D and TP53 lesions. Preexisting CH increased the risk of adverse outcomes including post-aHSCT tMN. Particularly, antecedent lesions affecting PPM1D and TP53 predicted tMN evolution post-transplant. Notably, CH-derived tMN had worse outcomes than non CH-derived tMN. As such, screening for CH before aHSCT may inform individual patients' prognostic outcomes and influence their prospective treatment plans. Presented in part as an oral abstract at the 2022 American Society of Hematology Annual Meeting, New Orleans, LA, 2022.
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Hematopoyesis Clonal , Trasplante de Células Madre Hematopoyéticas , Mutación , Neoplasias Primarias Secundarias , Trasplante Autólogo , Humanos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Masculino , Persona de Mediana Edad , Femenino , Trasplante Autólogo/efectos adversos , Adulto , Neoplasias Primarias Secundarias/etiología , Neoplasias Primarias Secundarias/genética , Neoplasias Primarias Secundarias/terapia , Anciano , Pronóstico , Trastornos Mieloproliferativos/terapia , Trastornos Mieloproliferativos/etiología , Trastornos Mieloproliferativos/genética , Trastornos Mieloproliferativos/patología , Adulto Joven , Adolescente , Proteína Fosfatasa 2C/genética , Proteína p53 Supresora de Tumor/genética , Estudios de Seguimiento , Linfoma/terapia , Linfoma/etiología , Linfoma/genética , Tasa de SupervivenciaRESUMEN
BACKGROUND: Next generation sequencing provides new opportunities to explore transcriptomes. However, challenges remain for accurate differentiation of homoeoalleles and paralogs, particularly in polyploid organisms with no supporting genome sequence. In this study, RNA-Seq was employed to generate and characterize the first gene expression atlas for hexaploid oat. RESULTS: The software packages Trinity and Oases were used to produce a transcript assembly from nearly 134 million 100-bp paired-end reads from developing oat seeds. Based on the quality-parameters employed, Oases assemblies were superior. The Oases 67-kmer assembly, denoted dnOST (de novo Oat Seed Transcriptome), is over 55 million nucleotides in length and the average transcript length is 1,043 nucleotides. The 74.8× sequencing depth was adequate to differentiate a large proportion of putative homoeoalleles and paralogs. To assess the robustness of dnOST, we successfully identified gene transcripts associated with the biosynthetic pathways of three compounds with health-promoting properties (avenanthramides, tocols, ß-glucans), and quantified their expression. CONCLUSIONS: To our knowledge, this study provides the first direct performance comparison between two major assemblers in a polyploid organism. The workflow we developed provides a useful guide for comparable analyses in other organisms. The transcript assembly developed here is a major advance. It expands the number of oat ESTs 3-fold, and constitutes the first comprehensive transcriptome study in oat. This resource will be a useful new tool both for analysis of genes relevant to nutritional enhancement of oat, and for improvement of this crop in general.
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Avena/genética , Perfilación de la Expresión Génica , Anotación de Secuencia Molecular , Poliploidía , Semillas/genética , Avena/crecimiento & desarrollo , Secuenciación de Nucleótidos de Alto Rendimiento , Repeticiones de Microsatélite/genética , Semillas/crecimiento & desarrolloRESUMEN
BACKGROUND: The late blight pathogen Phytophthora infestans can attack both potato foliage and tubers. Although interaction transcriptome dynamics between potato foliage and various pathogens have been reported, no transcriptome study has focused specifically upon how potato tubers respond to pathogen infection. When inoculated with P. infestans, tubers of nontransformed 'Russet Burbank' (WT) potato develop late blight disease while those of transgenic 'Russet Burbank' line SP2211 (+RB), which expresses the potato late blight resistance gene RB (Rpi-blb1), do not. We compared transcriptome responses to P. infestans inoculation in tubers of these two lines. RESULTS: We demonstrated the practicality of RNA-seq to study tetraploid potato and present the first RNA-seq study of potato tuber diseases. A total of 483 million paired end Illumina RNA-seq reads were generated, representing the transcription of around 30,000 potato genes. Differentially expressed genes, gene groups and ontology bins that exhibited differences between the WT and +RB lines were identified. P. infestans transcripts, including those of known effectors, were also identified. CONCLUSION: Faster and stronger activation of defense related genes, gene groups and ontology bins correlate with successful tuber resistance against P. infestans. Our results suggest that the hypersensitive response is likely a general form of resistance against the hemibiotrophic P. infestans-even in potato tubers, organs that develop below ground.
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Interacciones Huésped-Patógeno , Phytophthora infestans/fisiología , Tubérculos de la Planta/inmunología , Tubérculos de la Planta/microbiología , Análisis de Secuencia de ARN , Solanum tuberosum/inmunología , Solanum tuberosum/microbiología , Resistencia a la Enfermedad/genética , Perfilación de la Expresión Génica , Genómica , Genotipo , Especificidad de Órganos , Phytophthora infestans/genética , Enfermedades de las Plantas/microbiología , Tubérculos de la Planta/genética , Plantas Modificadas Genéticamente , ARN Mensajero/genética , ARN Mensajero/metabolismo , Solanum tuberosum/genéticaRESUMEN
Background: DNA hypermethylation and instability due to inactivation mutations in Ten-eleven translocation 2 (TET2) is a key biomarker of hematological malignancies. This study aims at characterizing two intronic noncanonical splice-site variants, c.3954+5_3954+8delGTTT and c.3954+5G>A. Methods: We used in silico prediction tools, reverse transcription (RT)-PCR, and Sanger sequencing on blood/bone marrow-derived RNA specimens to determine the aberrant splicing. Results: In silico prediction of both variants exhibited reduced splicing strength at the TET2 intron 7 splicing donor site. RT-PCR and Sanger sequencing identified a 62-bp deletion at the exon 7, producing a frameshift mutation, p.Cys1298*. Conclusion: This study provides functional evidence for two intronic TET2 variants that cause alternative splicing and frameshift mutation.
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(1) Background: EWS fusion genes are associated with Ewing sarcoma and other Ewing family tumors including desmoplastic small round tumor, DSRCT. We utilize a clinical genomics workflow to reveal real-world frequencies of EWS fusion events, cataloging events that are similar, or divergent at the EWS breakpoint. (2) Methods: EWS fusion events from our next-generation sequencing panel (NGS) samples were first sorted by breakpoint or fusion junctions to map out the frequency of breakpoints. Fusion results were illustrated as in-frame fusion peptides involving EWS and a partner gene. (3) Results: From 2471 patient pool samples for fusion analysis at the Cleveland Clinic Molecular Pathology Laboratory, we identified 182 fusion samples evolved with the EWS gene. They are clustered in several breakpoints: chr22:29683123 (65.9%), and chr22:29688595 (2.7%). About 3/4 of Ewing sarcoma and DSRCT tumors have an identical EWS breakpoint motif at Exon 7 (SQQSSSYGQQ-) fused to a specific part of FLI1 (NPSYDSVRRG or-SSLLAYNTSS), ERG (NLPYEPPRRS), FEV (NPVGDGLFKD) or WT1 (SEKPYQCDFK). Our method also worked with Caris transcriptome data, too. Our primary clinical utility is to use this information to identify neoantigens for therapeutic purposes. (4) Conclusions and future perspectives: our method allows interpretation of what peptides result from the in-frame translation of EWS fusion junctions. These sequences, coupled with HLA-peptide binding data, are used to identify potential sequences of cancer-specific immunogenic peptides for Ewing sarcoma or DSRCT patients. This information may also be useful for immune monitoring (e.g., circulating T-cells with fusion-peptide specificity) to detect vaccine candidates, responses, or residual disease.
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BACKGROUND: Key criteria in the diagnostic workup and risk stratification for myeloproliferative neoplasms (MPN) include molecular testing for JAK2V617F and other mutant alleles. Multiple methods for quantitatively detecting nucleotide sequence changes exist, but the lower limit of detection can limit identification of the low-level allele fraction of a variant. We evaluated a recently developed blocker displacement amplification (BDA)-based quantitative PCR platform for detection and quantitation of JAK2V617F variant allele fraction (VAF). METHODS: Clinical samples were tested using BDA, next-generation sequencing (NGS), and droplet digital PCR (ddPCR) in a head-to-head comparison of sensitivity and specificity in detecting the JAK2V617F variant. In total, 112 human genomic DNA specimens previously tested for JAK2V617F gene mutation status with NGS were analyzed, including 12 samples with low-level variants with VAF ≤2%, 6 samples with VAF >2%, and 94 samples with no variant previously identified by NGS. RESULTS: BDA and ddPCR results correlated well across a range of VAFs, with both methods identifying the JAK2V617F variant down to at least 0.05% VAF. NGS of routine sequencing depth was less sensitive, identifying JAK2V617F only at 0.6% VAF. CONCLUSIONS: BDA can provide a cost-effective alternative means to identify low-level variants using instrumentation commonly found in laboratories.
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Trastornos Mieloproliferativos , Humanos , Trastornos Mieloproliferativos/diagnóstico , Trastornos Mieloproliferativos/genética , Mutación , Reacción en Cadena de la Polimerasa/métodos , Alelos , Técnicas de Amplificación de Ácido NucleicoRESUMEN
The human salivary proteome is extremely complex, including proteins from salivary glands, serum, and oral microbes. Much has been learned about the host component, but little is known about the microbial component. Here we report a metaproteomic analysis of salivary supernatant pooled from six healthy subjects. For deep interrogation of the salivary proteome, we combined protein dynamic range compression (DRC), multidimensional peptide fractionation, and high-mass accuracy MS/MS with a novel two-step peptide identification method using a database of human proteins plus those translated from oral microbe genomes. Peptides were identified from 124 microbial species as well as uncultured phylotypes such as TM7. Streptococcus, Rothia, Actinomyces, Prevotella, Neisseria, Veilonella, Lactobacillus, Selenomonas, Pseudomonas, Staphylococcus, and Campylobacter were abundant among the 65 genera from 12 phyla represented. Taxonomic diversity in our study was broadly consistent with metagenomic studies of saliva. Proteins mapped to 20 KEGG pathways, with carbohydrate metabolism, amino acid metabolism, energy metabolism, translation, membrane transport, and signal transduction most represented. The communities sampled appear to be actively engaged in glycolysis and protein synthesis. This first deep metaproteomic catalog from human salivary supernatant provides a baseline for future studies of shifts in microbial diversity and protein activities potentially associated with oral disease.
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Proteínas Bacterianas/química , Metagenómica/métodos , Péptidos/química , Proteoma/análisis , Proteómica/métodos , Saliva/química , Proteínas Bacterianas/metabolismo , Bases de Datos de Proteínas , Humanos , Redes y Vías Metabólicas , Péptidos/metabolismo , Filogenia , Proteoma/química , Proteoma/metabolismo , Saliva/metabolismo , Saliva/microbiologíaRESUMEN
In 2020, the U.S. Food and Drug Administration (FDA) enabled manufacturers to request emergency use authorization (EUA) to facilitate the rapid authorization of in vitro diagnostic (IVD) platforms for the detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Uncommon SARS-CoV-2 point mutations could cause nucleocapsid (N) gene target failure (NGTF) when using first-generation Xpert Xpress assays, so improvements were designed and implemented. In response to NGTF reports and with consideration of viral genomic information in public databases, the Xpress assays were redesigned to mitigate the impact of SARS-CoV-2 mutations on qualitative assay performance. The second-generation assays include a third gene target (RNA-dependent RNA polymerase [RdRp]) and redundant oligonucleotide probes for the N2 target. First- and second-generation assay performances were evaluated using a challenge set of samples. A second-generation assay with updated oligonucleotide chemistry received FDA EUA in September 2021. A prototype assay with oligonucleotide chemistry similar to that of the second-generation assay with FDA EUA successfully detected all three gene targets (N2, envelope [E], and RdRp) in all challenge samples (100%; 50/50), including variants with N gene mutations (g.29197C>T or g.29200C>T), which caused NGTF in the first-generation assays. Investigation and reporting of IVD target failures, public sharing of viral genomic sequence data, and the FDA EUA pathway were essential components in facilitating a short cycle time from the identification of mutations that impact the performance of an IVD assay to the design and implementation of an improved IVD assay. IMPORTANCE The SARS-CoV-2 genome has mutated during the coronavirus disease 2019 (COVID-19) pandemic. Some of these mutations have impacted the performance of nucleic acid amplification tests like PCR, which are commonly used as diagnostic tools to detect an infection. The U.S. Food and Drug Administration (FDA) emergency use authorization (EUA) process enables the rapid reformulation and regulatory authorization of improved PCRs. In our experience, the identification of SARS-CoV-2 mutations that impact PCR performance, the subsequent development of improved PCR chemistry, and the use of the FDA EUA regulatory pathway led to improved diagnostic performance during the SARS-CoV-2 pandemic that is able to keep pace with the rapidly evolving genome of SARS-CoV-2.
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COVID-19 , SARS-CoV-2 , Humanos , SARS-CoV-2/genética , COVID-19/diagnóstico , Prueba de COVID-19 , Técnicas de Laboratorio Clínico , Mutación , GenómicaRESUMEN
Mutations in the viral genome of SARS-CoV-2 can impact the performance of molecular diagnostic assays. In some cases, such as S gene target failure, the impact can serve as a unique indicator of a particular SARS-CoV-2 variant and provide a method for rapid detection. Here we describe partial ORF1ab gene target failure (pOGTF) on the cobas ® SARS-CoV-2 assays, defined by a ≥2 thermocycles delay in detection of the ORF1ab gene compared to the E gene. We demonstrate that pOGTF is 97% sensitive and 99% specific for SARS-CoV-2 lineage BA.2.12.1, an emerging variant in the United States with spike L452Q and S704L mutations that may impact transmission, infectivity, and/or immune evasion. Increasing rates of pOGTF closely mirrored rates of BA.2.12.1 sequences uploaded to public databases, and, importantly increasing local rates of pOGTF also mirrored increasing overall test positivity. Use of pOGTF as a proxy for BA.2.12.1 provides faster tracking of the variant than whole-genome sequencing and can benefit laboratories without sequencing capabilities.