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BACKGROUND: X-chromosome inactivation (XCI) is an epigenetic process that occurs during early development in mammalian females by randomly silencing one of two copies of the X chromosome in each cell. The preferential inactivation of either the maternal or paternal copy of the X chromosome in a majority of cells results in a skewed or non-random pattern of X inactivation and is observed in over 25% of adult females. Identifying skewed X inactivation is of clinical significance in patients with suspected rare genetic diseases due to the possibility of biased expression of disease-causing genes present on the active X chromosome. The current clinical test for the detection of skewed XCI relies on the methylation status of the methylation-sensitive restriction enzyme (Hpall) binding site present in proximity of short tandem polymorphic repeats on the androgen receptor (AR) gene. This approach using one locus results in uninformative or inconclusive data for 10-20% of tests. Further, recent studies have shown inconsistency between methylation of the AR locus and the state of inactivation of the X chromosome. Herein, we develop a method for estimating X inactivation status, using exome and transcriptome sequencing data derived from blood in 227 female samples. We built a reference model for evaluation of XCI in 135 females from the GTEx consortium. We tested and validated the model on 11 female individuals with different types of undiagnosed rare genetic disorders who were clinically tested for X-skew using the AR gene assay and compared results to our outlier-based analysis technique. RESULTS: In comparison to the AR clinical test for identification of X inactivation, our method was concordant with the AR method in 9 samples, discordant in 1, and provided a measure of X inactivation in 1 sample with uninformative clinical results. We applied this method on an additional 81 females presenting to the clinic with phenotypes consistent with different hereditary disorders without a known genetic diagnosis. CONCLUSIONS: This study presents the use of transcriptome and exome sequencing data to provide an accurate and complete estimation of X-inactivation and skew status in a cohort of female patients with different types of suspected rare genetic disease.
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Exoma , Inativação do Cromossomo X , Adulto , Humanos , Feminino , Transcriptoma , Sequenciamento do Exoma , Cromossomos Humanos X/genéticaRESUMO
Tumors resembling tenosynovial giant cell tumor (TGCT) but additionally forming chondroid matrix (C-TGCT) are rare and most often involve the temporomandibular joint (TMJ). We studied 21 tumors consisting of synoviocytes (large, eosinophilic mononuclear cells containing hemosiderin) and chondroid matrix to better understand these unusual neoplasms. The tumors occurred in 10 males and 11 females, ranging from 31-80 years (median, 50 years) and involved the temporomandibular joint region (16), extremities (4), and spine (1). As in conventional TGCT, all were composed of synoviocytes, small histiocytes, foamy macrophages, siderophages and osteoclast-like giant cells in variably hyalinized background. Expansile nodules of large, moderately atypical synoviocytes were present, in addition to "chondroblastoma-like", "chondroma-like", or "phosphaturic mesenchymal tumor-like" calcified matrix. The synoviocytes expressed clusterin (17/19) and less often desmin (3/15). The tumors were frequently CSF1-positive by CISH (8/13) but at best weakly positive for CSF1 by IHC (0/3). Background small histiocytes were CD163-positive (12/12). All were FGF23-negative (0/10). Cells within lacunae showed a synoviocytic phenotype (clusterin-positive; S100 protein and ERG-negative). RNA-seq was successful in 13 cases; fusions were present in 7 tumors, including FN1::TEK (5 cases), FN1::PRG4 (2 cases), MALAT1::FN1, PDGFRA::USP35 and TIMP3::ZCCHC7 (1 case each). Three tumors contained more than one fusion (FN1::PRG4 with TIMP3::ZCCHC7, FN1::TEK with FN1::PRG4, and FN1::TEK with MALAT1::FN1). Clinical follow up (17 patients; median follow up duration 38 months; range 4-173 months) showed 13 (76%) to be alive without evidence of disease and 4 (24%) to be alive with persistent/recurrent local disease. No metastases or deaths from disease were observed. We conclude that these unusual tumors represent a distinct category of synoviocytic neoplasia, which we term "chondroid synoviocytic neoplasm", rather than simply ordinary TGCT with cartilage. Despite potentially worrisome morphologic features, they appear to behave in at most a locally aggressive fashion.
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INTRODUCTION: Adamantinoma-like Ewing sarcoma (ALES) is a rare aggressive malignancy occasionally diagnosed in the thyroid gland. ALES shows basaloid cytomorphology, expresses keratins, p63, p40, frequently CD99, and harbours the t(11;22) EWSR1::FLI1 translocation. There is debate on whether ALES resembles more sarcoma or carcinoma. METHODS: We performed RNA sequencing from two ALES cases and compared findings with skeletal Ewing's sarcomas and nonneoplastic thyroid tissue. ALES was investigated by in situ hybridization (ISH) for high-risk human papillomavirus (HPV) DNA and immunohistochemistry for the following antigens: keratin 7, keratin 20, keratin 5, keratins (AE1/AE3 and CAM5.2), CD45, CD20, CD5, CD99, chromogranin, synaptophysin, calcitonin, thyroglobulin, PAX8, TTF1, S100, p40, p63, p16, NUT, desmin, ER, FLI1, INI1, and myogenin. RESULTS: An uncommon EWSR1::FLI transcript with retained EWSR1 exon 8 was detected in both ALES cases. Regulators of EWSR1::FLI1 splicing (HNRNPH1, SUPT6H, SF3B1) necessary for production of a functional fusion oncoprotein, as well as 53 genes (including TNNT1, NKX2.2) activated downstream to the EWSR1::FLI1 cascade, were overexpressed. Eighty-six genes were uniquely overexpressed in ALES, most of which were related to squamous differentiation. Immunohistochemically, ALES strongly expressed keratins 5, AE1/AE3 and CAM5.2, p63, p40, p16, and focally CD99. INI1 was retained. The remaining immunostains and HPV DNA ISH were negative. CONCLUSION: Comparative transcriptomic profiling reveals overlapping features of ALES with skeletal Ewing's sarcoma and an epithelial carcinoma, as evidenced by immunohistochemical expression of keratin 5, p63, p40, CD99, the transcriptome profile, and detection of EWSR1::FLI1 fusion transcript by RNA sequencing.
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Adamantinoma , Carcinoma , Infecções por Papillomavirus , Sarcoma de Ewing , Humanos , Sarcoma de Ewing/diagnóstico , Sarcoma de Ewing/genética , Adamantinoma/diagnóstico , Adamantinoma/genética , Adamantinoma/química , Glândula Tireoide/patologia , Transcriptoma , Queratina-5/metabolismo , Proteína EWS de Ligação a RNA/genética , Proteína EWS de Ligação a RNA/metabolismo , Fatores de Transcrição/genética , Proteínas de Fusão Oncogênica/genética , Proteínas de Fusão Oncogênica/metabolismoRESUMO
Primary aneurysmal bone cyst (ABC) is a benign multiloculated cystic lesion of bone that is defined cytogenetically by USP6 gene rearrangements. Rearrangements involving USP6 are promoter swaps, usually generated by fusion of the noncoding upstream exons of different partner genes with exon 1 or 2 of USP6, thus leading to transcriptional upregulation of full-length USP6 coding sequence. Testing for USP6 rearrangements is used diagnostically to distinguish it from secondary ABC and other giant cell-rich primary bone tumors. In this report, we present a case of a 16-year-old male with a primary ABC of the left distal femur. USP6 break apart fluorescence in situ hybridization was positive for a rearrangement and conventional chromosome analysis identified a reciprocal X;17 translocation. In order to identify the putative USP6 fusion partner, we performed RNA sequencing and uncovered a novel USP9X-USP6 promoter swap fusion. This result was confirmed by reverse transcription-polymerase chain reaction (RT-PCR) and by mate pair sequencing thus showing the utility of these alternative methodologies in identifying novel fusion candidates. Ubiquitin-specific protease 9X (USP9X), like USP6, encodes a highly conserved substrate-specific deubiquitylating enzyme. USP9X is highly expressed in a number of tissue types and acts as both an oncogene and tumor suppressor in several human cancers. We conclude that oncogenic activation of USP6 via USP9X promoter exchange represents a novel driver of primary ABC formation.
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Cistos Ósseos Aneurismáticos/diagnóstico , Cistos Ósseos Aneurismáticos/genética , Predisposição Genética para Doença , Proteínas de Fusão Oncogênica/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Ubiquitina Tiolesterase/genética , Adolescente , Biomarcadores Tumorais , Biópsia , Bandeamento Cromossômico , Biologia Computacional/métodos , Estudos de Associação Genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Hibridização in Situ Fluorescente , Imageamento por Ressonância Magnética , Masculino , Tomografia Computadorizada por Raios XRESUMO
BACKGROUND: RNA-seq is a well-established method for studying the transcriptome. Popular methods for library preparation in RNA-seq such as Illumina TruSeq® RNA v2 kit use a poly-A pulldown strategy. Such methods can cause loss of coverage at the 5' end of genes, impacting the ability to detect fusions when used on degraded samples. The goal of this study was to quantify the effects RNA degradation has on fusion detection when using poly-A selected mRNA and to identify the variables involved in this process. RESULTS: Using both artificially and naturally degraded samples, we found that there is a reduced ability to detect fusions as the distance of the breakpoint from the 3' end of the gene increases. The median transcript coverage decreases exponentially as a function of the distance from the 3' end and there is a linear relationship between the coverage decay rate and the RNA integrity number (RIN). Based on these findings we developed plots that show the probability of detecting a gene fusion ("sensitivity") as a function of the distance of the fusion breakpoint from the 3' end. CONCLUSIONS: This study developed a strategy to assess the impact that RNA degradation has on the ability to detect gene fusions by RNA-seq.
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Estabilidade de RNA , RNA/genética , Recombinação Genética , Linhagem Celular Tumoral , Pontos de Quebra do Cromossomo , Proteínas de Fusão bcr-abl/genética , Biblioteca Gênica , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , RNA/metabolismo , RNA Mensageiro/genética , Análise de Sequência de RNARESUMO
Sweat gland neoplasms represent a challenging area of dermatopathology, as they are relatively uncommon and often histopathologically complex. Recent studies have uncovered distinct immunohistochemical and molecular profiles in several sweat gland neoplasms, including digital papillary adenocarcinoma (DPA), papillary eccrine adenoma/tubular apocrine adenoma (PEA/TAA), poroid family tumors (PFT)/porocarcinoma, and clear cell hidradenoma (CCH)/clear cell hidradenocarcinoma (CCHCa). To further evaluate the diagnostic utility of ancillary studies in various sweat gland neoplasms, we performed an independent validation study in a cohort of patients with acral and non-acral tumors (9 DPA, 8 PEA/TAA, 13 PFT, 5 porocarcinoma, 23 CCH, 7 CCHCa, 6 sweat gland carcinoma not otherwise specified). p63 immunohistochemistry (IHC) demonstrated a myoepithelial pattern in 8/8 DPA and 4 of 4 tested PEA/TAA cases, and showed a ductal pattern in all tested PFT/porocarcinoma and CCH/CCHCa cases (42/42). All PEA/TAA (8/8) cases were positive for BRAF V600E IHC. 5 of 12 tested PFT and 5/5 porocarcinoma cases showed either positive staining with NUT IHC or harbored YAP1::NUTM1 fusion gene by RNA sequencing. MAML2 fluorescence in situ hybridization (FISH) was positive in all CCH and CCHCa cases (23/23 and 7/7, respectively). Our results further support the usefulness of appropriate ancillary studies in precise classification of sweat gland tumors, which may be routinely applied in diagnostic pathology practice when morphologic evaluation is in doubt.
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Biomarcadores Tumorais , Imuno-Histoquímica , Neoplasias das Glândulas Sudoríparas , Humanos , Neoplasias das Glândulas Sudoríparas/patologia , Neoplasias das Glândulas Sudoríparas/genética , Neoplasias das Glândulas Sudoríparas/diagnóstico , Neoplasias das Glândulas Sudoríparas/classificação , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/genética , Masculino , Feminino , Pessoa de Meia-Idade , Idoso , Adulto , Idoso de 80 Anos ou mais , Reprodutibilidade dos Testes , Hibridização in Situ Fluorescente , Fatores de Transcrição/análise , Valor Preditivo dos TestesRESUMO
Genome sequencing (GS) is a powerful clinical tool used for the comprehensive diagnosis of germline disorders. GS library preparation typically involves mechanical DNA fragmentation, end repair, and bead-based library size selection followed by adapter ligation, which can require a large amount of input genomic DNA. Tagmentation using bead-linked transposomes can simplify the library preparation process and reduce the DNA input requirement. Here we describe the clinical validation of tagmentation-based PCR-free GS as a clinical test for rare germline disorders. Compared with the Genome-in-a-Bottle Consortium benchmark variant sets, GS had a recall >99.7% and a precision of 99.8% for single nucleotide variants and small insertion-deletions. GS also exhibited 100% sensitivity for clinically reported sequence variants and the copy number variants examined. Furthermore, GS detected mitochondrial sequence variants above 5% heteroplasmy and showed reliable detection of disease-relevant repeat expansions and SMN1 homozygous loss. Our results indicate that while lowering DNA input requirements and reducing library preparation time, GS enables uniform coverage across the genome as well as robust detection of various types of genetic alterations. With the advantage of comprehensive profiling of multiple types of genetic alterations, GS is positioned as an ideal first-tier diagnostic test for germline disorders.
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DNA , Doenças Raras , Humanos , Sequência de Bases , Mapeamento Cromossômico , Análise de Sequência de DNA/métodos , Biblioteca Gênica , Sequenciamento de Nucleotídeos em Larga Escala/métodosRESUMO
Polymorphous low-grade neuroepithelial tumor of the young (PLNTY) is a recently described epileptogenic tumor characterized by oligodendroglioma-like components, aberrant CD34 expression, and frequent mitogen-activated protein kinase (MAPK) pathway activation. We molecularly profiled 13 cases with diagnostic histopathological features of PLNTY (10 female; median age, 16 years; range, 5-52). Patients frequently presented with seizures (9 of 12 with available history) and temporal lobe tumors (9 of 13). MAPK pathway activating alterations were identified in all 13 cases. Fusions were present in the 7 youngest patients: FGFR2-CTNNA3 (n = 2), FGFR2-KIAA1598 (FGFR2-SHTN1) (n = 1), FGFR2-INA (n = 1), FGFR2-MPRIP (n = 1), QKI-NTRK2 (n = 1), and KIAA1549-BRAF (n = 1). BRAF V600E mutation was present in 6 patients (17 years or older). Two fusion-positive cases additionally harbored TP53/RB1 abnormalities suggesting biallelic inactivation. Copy number changes predominantly involving whole chromosomes were observed in all 10 evaluated cases, with losses of chromosome 10q occurring with FGFR2-KIAA1598 (SHTN1)/CTNNA3 fusions. The KIAA1549-BRAF and QKI-NTRK2 fusions were associated respectively with a 7q34 deletion and 9q21 duplication. This study shows that despite its name, PLNTY also occurs in older adults, who frequently show BRAF V600E mutation. It also expands the spectrum of the MAPK pathway activating alterations associated with PLNTY and demonstrates recurrent chromosomal copy number changes consistent with chromosomal instability.
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Glicoproteínas de Membrana/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Neoplasias Neuroepiteliomatosas/metabolismo , Receptor trkB/metabolismo , Convulsões/metabolismo , Adulto , Aneuploidia , Cromossomos Humanos Par 9/metabolismo , Feminino , Fusão Gênica/fisiologia , Humanos , Recidiva , Convulsões/genética , Fatores de Transcrição/metabolismoRESUMO
Tumor mutation burden (TMB) is an emerging biomarker of immunotherapy response. RNA sequencing in FFPE tissue samples was used for determining TMB in microsatellite-stable (MSS) and microsatellite instability-high (MSI-H) tumors in patients with colorectal or endometrial cancer. Tissue from tumors and paired normal tissue from 46 MSI-H and 12 MSS cases were included. Of the MSI-H tumors, 29 had defective DNA mismatch-repair mutations, and 17 had MLH1 promoter hypermethylation. TMB was measured using the expressed somatic nucleotide variants (eTMB). A method of accurate measurement of eTMB was developed that removes FFPE-derived artifacts by leveraging mutation signatures. There was a significant difference in the median eTMB values observed between MSI-H and MSS cases: 27.3 versus 6.7 mutations/megabase (mut/Mb) (P = 3.5 × 10-9). Among tumors with defective DNA-mismatch repair, those with mismatch-repair mutations had a significantly higher median eTMB than those with hypermethylation: 28.1 versus 17.5 mut/Mb (P = 0.037). Multivariate analysis showed that MSI status, tumor type (endometrial or colorectal), and age were significantly associated with eTMB. Additionally, using whole-exome sequencing in a subset of these patients, it was determined that DNA TMB correlated well with eTMB (Spearman correlation coefficient, 0.83). These results demonstrate that RNA sequencing can be used for measuring eTMB in FFPE tumor specimens.
Assuntos
Biomarcadores Tumorais/genética , Neoplasias Colorretais/patologia , Reparo de Erro de Pareamento de DNA/genética , Neoplasias do Endométrio/patologia , Mutação , RNA-Seq/métodos , Adulto , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/genética , Neoplasias do Endométrio/genética , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Prognóstico , Estudos RetrospectivosRESUMO
Gestational trophoblastic disease (GTD) is a heterogeneous group of lesions arising from placental tissue. Epithelioid trophoblastic tumor (ETT), derived from chorionic-type trophoblast, is the rarest form of GTD with only approximately 130 cases described in the literature. Due to its morphologic mimicry of epithelioid smooth muscle tumors and carcinoma, ETT can be misdiagnosed. To date, molecular characterization of ETTs is lacking. Furthermore, ETT is difficult to treat when disease spreads beyond the uterus. Here using RNA-Seq analysis in a cohort of ETTs and other gestational trophoblastic lesions we describe the discovery of LPCAT1-TERT fusion transcripts that occur in ETTs and coincide with underlying genomic deletions. Through cell-growth assays we demonstrate that LPCAT1-TERT fusion proteins can positively modulate cell proliferation and therefore may represent future treatment targets. Furthermore, we demonstrate that TERT upregulation appears to be a characteristic of ETTs, even in the absence of LPCAT1-TERT fusions, and that it appears linked to copy number gains of chromosome 5. No evidence of TERT upregulation was identified in other trophoblastic lesions tested, including placental site trophoblastic tumors and placental site nodules, which are thought to be the benign chorionic-type trophoblast counterpart to ETT. These findings indicate that LPCAT1-TERT fusions and copy-number driven TERT activation may represent novel markers for ETT, with the potential to improve the diagnosis, treatment, and outcome for women with this rare form of GTD.
Assuntos
1-Acilglicerofosfocolina O-Aciltransferase/genética , Células Epitelioides/patologia , Doença Trofoblástica Gestacional/etiologia , Proteínas de Fusão Oncogênica/genética , Telomerase/genética , Neoplasias Trofoblásticas/patologia , Neoplasias Uterinas/patologia , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo , Adulto , Biomarcadores Tumorais/genética , Proliferação de Células , Células Epitelioides/metabolismo , Feminino , Doença Trofoblástica Gestacional/patologia , Humanos , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/metabolismo , Gravidez , Telomerase/metabolismo , Neoplasias Trofoblásticas/genética , Neoplasias Trofoblásticas/metabolismo , Neoplasias Uterinas/genética , Neoplasias Uterinas/metabolismoRESUMO
Uterine inflammatory myofibroblastic tumors (IMTs) have been reported in association with pregnancy and, in some instances, secondarily involve the placenta. The clinicopathological spectrum of these tumors in the setting of pregnancy is not well defined. We investigated the clinical, morphologic, immunohistochemical, molecular cytogenetic, and genetic features of 6 uterine IMTs occurring in pregnant women. Each tumor was discovered at parturition, and none was identified by prenatal ultrasound. Patient age ranged from 25 to 41â¯years (mean 31.5). Tumor size ranged from 1.5 to 9â¯cm (mean 4.7). Four of 6 had usual IMT features, with at least focal deciduoid change in 3. Necrosis was identified in 3 tumors; and multinucleated cells, in 3 tumors. Sex hormone receptor expression was consistent with estrogen receptor negative or focally weakly positive and progesterone receptor diffusely moderately or moderately to strongly positive in all 6 tumors. ALK immunohistochemistry was strongly positive in 5 tumors, and all of these had an ALK rearrangement detected by break-apart fluorescence in situ hybridization. Subsequent RNA sequencing of these 5 tumors identified a TIMP3-ALK fusion in 4 and a THBS1-ALK in 1. In the ALK-negative tumor, RNA sequencing detected a novel TIMP3-RET fusion that was confirmed by RET break-apart fluorescence in situ hybridization. Follow-up was available for 2 of 6 patients 5 and 19â¯months after diagnosis. Neither patient developed recurrence. ALK immunohistochemistry will distinguish most uterine IMTs, but if ALK expression and gene studies are negative, in the appropriate morphologic context, evaluation of other tyrosine kinase genes known to be more commonly altered in extrauterine IMTs such as ROS1, NTRK3, PDGFRß, and RET may be necessary for diagnostic confirmation.
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Biomarcadores Tumorais/genética , Fusão Gênica , Miofibroblastos/patologia , Neoplasias de Tecido Fibroso/genética , Placenta/patologia , Complicações Neoplásicas na Gravidez/genética , Proteínas Proto-Oncogênicas c-ret/genética , Inibidor Tecidual de Metaloproteinase-3/genética , Neoplasias Uterinas/genética , Adulto , Quinase do Linfoma Anaplásico/genética , Feminino , Rearranjo Gênico , Predisposição Genética para Doença , Humanos , Necrose , Neoplasias de Tecido Fibroso/patologia , Neoplasias de Tecido Fibroso/terapia , Fenótipo , Gravidez , Complicações Neoplásicas na Gravidez/patologia , Complicações Neoplásicas na Gravidez/terapia , Resultado do Tratamento , Carga Tumoral , Neoplasias Uterinas/patologia , Neoplasias Uterinas/terapiaRESUMO
The Student Council of the International Society for Computational Biology (ISCB-SC) is a student-focused organization for researchers from all early career levels of training (undergraduates, masters, PhDs and postdocs) that organizes bioinformatics and computational biology activities across the globe. Among its activities, the ISCB-SC organizes several symposia in different continents, many times, with the help of the Regional Student Groups (RSGs) that are based on each region. In this editorial we highlight various key moments and learned lessons from the 14th Student Council Symposium (SCS, Chicago, USA), the 5th European Student Council Symposium (ESCS, Athens, Greece) and the 3rd Latin American Student Council Symposium (LA-SCS, Viña del Mar, Chile).
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Biologia Computacional , Liderança , Estudantes , Chile , Humanos , PesquisadoresRESUMO
We assessed the performance characteristics of an RNA sequencing (RNA-Seq) assay designed to detect gene fusions in 571 genes to help manage patients with cancer. Polyadenylated RNA was converted to cDNA, which was then used to prepare next-generation sequencing libraries that were sequenced on an Illumina HiSeq 2500 instrument and analyzed with an in-house developed bioinformatic pipeline. The assay identified 38 of 41 gene fusions detected by another method, such as fluorescence in situ hybridization or RT-PCR, for a sensitivity of 93%. No false-positive gene fusions were identified in 15 normal tissue specimens and 10 tumor specimens that were negative for fusions by RNA sequencing or Mate Pair NGS (100% specificity). The assay also identified 22 fusions in 17 tumor specimens that had not been detected by other methods. Eighteen of the 22 fusions had not previously been described. Good intra-assay and interassay reproducibility was observed with complete concordance for the presence or absence of gene fusions in replicates. The analytical sensitivity of the assay was tested by diluting RNA isolated from gene fusion-positive cases with fusion-negative RNA. Gene fusions were generally detectable down to 12.5% dilutions for most fusions and as little as 3% for some fusions. This assay can help identify fusions in patients with cancer; these patients may in turn benefit from both US Food and Drug Administration-approved and investigational targeted therapies.
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Neoplasias/genética , Fusão Oncogênica/genética , Análise de Sequência de RNA/métodos , Regulação Neoplásica da Expressão Gênica , Humanos , Limite de Detecção , Estabilidade de RNA/genética , RNA Neoplásico/genética , RNA Neoplásico/normas , Reprodutibilidade dos Testes , Sensibilidade e EspecificidadeRESUMO
Significant barriers, such as lack of professional guidelines, specialized training for interpretation of pharmacogenomics (PGx) data, and insufficient evidence to support clinical utility, prevent preemptive PGx testing from being widely clinically implemented. The current study, as a pilot project for the Right Drug, Right Dose, Right Time-Using Genomic Data to Individualize Treatment Protocol, was designed to evaluate the impact of preemptive PGx and to optimize the workflow in the clinic setting. We used an 84-gene next-generation sequencing panel that included SLCO1B1, CYP2C19, CYP2C9, and VKORC1 together with a custom-designed CYP2D6 testing cascade to genotype the 1013 subjects in laboratories approved by the Clinical Laboratory Improvement Act. Actionable PGx variants were placed in patient's electronic medical records where integrated clinical decision support rules alert providers when a relevant medication is ordered. The fraction of this cohort carrying actionable PGx variant(s) in individual genes ranged from 30% (SLCO1B1) to 79% (CYP2D6). When considering all five genes together, 99% of the subjects carried an actionable PGx variant(s) in at least one gene. Our study provides evidence in favor of preemptive PGx testing by identifying the risk of a variant being present in the population we studied.
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Citocromo P-450 CYP2D6/genética , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Farmacogenética/métodos , Medicina de Precisão/métodos , Alelos , Citocromo P-450 CYP2C19/genética , Citocromo P-450 CYP2C19/metabolismo , Citocromo P-450 CYP2D6/metabolismo , Ativação Enzimática , Duplicação Gênica , Humanos , FenótipoRESUMO
Current clinical laboratory practice guidelines for next-generation sequencing (NGS) do not provide definitive guidance on confirming NGS variants. Sanger confirmation of NGS results can be inefficient, redundant, and expensive. We evaluated the accuracy of NGS-detected single-nucleotide variants (SNVs) and insertion/deletion variants (indels) and the necessity of NGS variant confirmation using four NGS target-capture gene panels covering 117 genes, 568 Kbp, and 77 patient DNA samples. Unique NGS-detected variants (1080 SNVs and 124 indels) underwent Sanger confirmation and/or were compared to data from the 1000 Genomes Project (1000G). Recurrent variants in unrelated samples resulted in 919 comparisons between NGS and Sanger, with 100% concordance. In a second comparison, 762 unique NGS results (736 SNVs, 26 indels) from seven 1000G samples were found to have 97.1% concordance with 1000G phase 1 data. Sanger sequencing and 1000G phase 3 data confirmed the accuracy of the NGS results for all 1000G phase 1 discrepancies. In all samples, the depth of coverage exceeded 100× in >99.7% of bases in the target regions. In conclusion, confirmatory analysis by Sanger sequencing of SNVs detected via capture-based NGS testing that meets appropriate quality thresholds is unnecessarily redundant. In contrast, Sanger sequencing for indels may be required for defining the correct genomic location, and Sanger may be used for quality-assurance purposes.
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Variação Genética/genética , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala/normas , Técnicas de Diagnóstico Molecular/métodos , Análise de Sequência de DNA/métodos , HumanosRESUMO
Hepatitis C virus (HCV) is the leading cause of chronic liver disease in humans. The envelope proteins of HCV are potential candidates for vaccine development. The absence of three-dimensional (3D) structures for the functional domain of HCV envelope proteins [E1.E2] monomer complex has hindered overall understanding of the virus infection, and also structure-based drug design initiatives. In this study, we report a 3D model containing both E1 and E2 proteins of HCV using the recently published structure of the core domain of HCV E2 and the functional part of E1, and investigate immunogenic implications of the model. HCV [E1.E2] molecule is modeled by using aa205-319 of E1 to aa421-716 of E2. Published experimental data were used to further refine the [E1.E2] model. Based on the model, we predict 77 exposed residues and several antigenic sites within the [E1.E2] that could serve as vaccine epitopes. This study identifies eight peptides which have antigenic propensity and have two or more sequentially exposed amino acids and 12 singular sites are under negative selection pressure that can serve as vaccine or therapeutic targets. Our special interest is 285FLVGQLFTFSPRRHW299 which has five negatively selected sites (L286, V287, G288, T292, and G303) with three of them sequential and four amino acids exposed (F285, L286, T292, and R296). This peptide in the E1 protein maps to dengue envelope vaccine target identified previously by our group. Our model provides for the first time an overall view of both the HCV envelope proteins thereby allowing researchers explore structure-based drug design approaches.