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1.
BMC Cancer ; 19(1): 357, 2019 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-30991985

RESUMO

BACKGROUND: Osteosarcoma is the most common malignant bone tumor in children. Survival remains poor among histologically poor responders, and there is a need to identify them at diagnosis to avoid delivering ineffective therapy. Genetic variation contributes to a wide range of response and toxicity related to chemotherapy. The aim of this study is to use sequencing of blood cells to identify germline haplotypes strongly associated with drug resistance in osteosarcoma patients. METHODS: We used sequencing data from two patient datasets, from Inova Hospital and the NCI TARGET. We explored the effect of mutation hotspots, in the form of haplotypes, associated with relapse outcome. We then mapped the single nucleotide polymorphisms (SNPs) in these haplotypes to genes and pathways. We also performed a targeted analysis of mutations in Drug Metabolizing Enzymes and Transporter (DMET) genes associated with tumor necrosis and survival. RESULTS: We found intronic and intergenic hotspot regions from 26 genes common to both the TARGET and INOVA datasets significantly associated with relapse outcome. Among significant results were mutations in genes belonging to AKR enzyme family, cell-cell adhesion biological process and the PI3K pathways; as well as variants in SLC22 family associated with both tumor necrosis and overall survival. The SNPs from our results were confirmed using Sanger sequencing. Our results included known as well as novel SNPs and haplotypes in genes associated with drug resistance. CONCLUSION: We show that combining next generation sequencing data from multiple datasets and defined clinical data can better identify relevant pathway associations and clinically actionable variants, as well as provide insights into drug response mechanisms.


Assuntos
Células Sanguíneas/metabolismo , Neoplasias Ósseas/genética , Resistencia a Medicamentos Antineoplásicos/genética , Genômica , Mutação em Linhagem Germinativa , Osteossarcoma/genética , Alelos , Biomarcadores Tumorais , Neoplasias Ósseas/mortalidade , Frequência do Gene , Genômica/métodos , Genótipo , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Estimativa de Kaplan-Meier , Osteossarcoma/mortalidade , Polimorfismo de Nucleotídeo Único , Prognóstico
2.
Proc Natl Acad Sci U S A ; 116(12): 5819-5827, 2019 03 19.
Artigo em Inglês | MEDLINE | ID: mdl-30833390

RESUMO

Preterm birth (PTB) complications are the leading cause of long-term morbidity and mortality in children. By using whole blood samples, we integrated whole-genome sequencing (WGS), RNA sequencing (RNA-seq), and DNA methylation data for 270 PTB and 521 control families. We analyzed this combined dataset to identify genomic variants associated with PTB and secondary analyses to identify variants associated with very early PTB (VEPTB) as well as other subcategories of disease that may contribute to PTB. We identified differentially expressed genes (DEGs) and methylated genomic loci and performed expression and methylation quantitative trait loci analyses to link genomic variants to these expression and methylation changes. We performed enrichment tests to identify overlaps between new and known PTB candidate gene systems. We identified 160 significant genomic variants associated with PTB-related phenotypes. The most significant variants, DEGs, and differentially methylated loci were associated with VEPTB. Integration of all data types identified a set of 72 candidate biomarker genes for VEPTB, encompassing genes and those previously associated with PTB. Notably, PTB-associated genes RAB31 and RBPJ were identified by all three data types (WGS, RNA-seq, and methylation). Pathways associated with VEPTB include EGFR and prolactin signaling pathways, inflammation- and immunity-related pathways, chemokine signaling, IFN-γ signaling, and Notch1 signaling. Progress in identifying molecular components of a complex disease is aided by integrated analyses of multiple molecular data types and clinical data. With these data, and by stratifying PTB by subphenotype, we have identified associations between VEPTB and the underlying biology.


Assuntos
Predisposição Genética para Doença/genética , Nascimento Prematuro/genética , Metilação de DNA/genética , Feminino , Genômica/métodos , Humanos , Recém-Nascido , Masculino , Fenótipo , Polimorfismo de Nucleotídeo Único/genética , Transdução de Sinais/genética , Sequenciamento Completo do Genoma/métodos
3.
J Pharm Biomed Anal ; 165: 198-206, 2019 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-30553110

RESUMO

Stable isotope labeled (SIL) compounds have been commonly used as internal standards (IS) to ensure the accuracy and quality of liquid chromatography-mass spectrometry (LC-MS) bioanalytical assays. Recently, the application of SIL drugs and LC-MS assays to microdose absolute bioavailability (BA) studies has gained increasing attention. This approach can provide significant cost and time saving, and higher data quality compared to the accelerator mass spectrometry (AMS)-based method, since it avoids the use of radioactive drug, high-cost AMS instrumentation and complex measurement processes. It also eliminates potential metabolite interference with AMS-based assay. However, one major challenge in the application of this approach is the potential interference between the unlabeled drug, the microdose SIL drug, and the SIL-IS during LC-MS analysis. Here we report a convenient and cost-effective strategy to overcome the interference by monitoring the isotopic ion (instead of the commonly used monoisotopic ion) of the interfered compound in MS analysis. For the BMS-986205 absolute BA case study presented, significant interference was observed from the microdose IV drug [13C7,15N]-BMS-986205 to its SIL-IS, [13C7,15N, D3]-BMS-986205, since the difference of nominal molecular mass between the two compounds is only 3 mu, and there is a Cl atom in the molecules. By applying this strategy (monitoring the 37Cl ion for the analysis of the IS), a 90-fold reduction of interference was achieved, which allowed the use of a synthetically accessible SIL compound and enabled the fast progress of the absolute BA study. This strategy minimizes the number of stable isotope labels used for avoiding interference, which greatly reduces the difficulty in synthesizing the SIL compounds and generates significant time and cost savings. In addition, this strategy can also be used to reduce the MS response of the analyte, therefore, avoiding the detector saturation issue of LC-MS/MS assay for high concentration BMS-986205. A LC-MS/MS assay utilizing this strategy was successfully developed for the simultaneous analysis of BMS-986205 and [13C7, 15N]-BMS-986205 in dog plasma using [13C7,15N, D3]-BMS-986205 as the IS. The assay was successfully applied to a microdose absolute BA study of BMS-986205 in dogs. The assay was also validated in human plasma and used to support a human absolute BA study. The same strategy can also be applied to other compounds, including those not containing Cl or other elements with abundant isotopes, or other applications (e.g. selection of internal standard), and the applications were presented.


Assuntos
Acetamidas/análise , Cromatografia Líquida/métodos , Inibidores Enzimáticos/análise , Quinolinas/análise , Espectrometria de Massas em Tandem/métodos , Acetamidas/administração & dosagem , Acetamidas/farmacocinética , Animais , Disponibilidade Biológica , Cromatografia Líquida/economia , Análise Custo-Benefício , Cães , Inibidores Enzimáticos/administração & dosagem , Inibidores Enzimáticos/farmacocinética , Humanos , Marcação por Isótopo , Quinolinas/administração & dosagem , Quinolinas/farmacocinética , Espectrometria de Massas em Tandem/economia
4.
Acta Pharm Sin B ; 8(2): 252-260, 2018 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-29719786

RESUMO

In the present study, total membrane proteins from tumor cell lines including HepG2, Hep3B2, H226, Ovcar3 and N87 were extracted and digested with γLysC and trypsin. The resulting peptide lysate were pre-fractionated and subjected to untargeted quantitative proteomics analysis using a high resolution mass spectrometer. The mass spectra were processed by the MaxQuant and the protein abundances were estimated using total peak area (TPA) method. A total of 6037 proteins were identified, and the analysis resulted in the identification of 2647 membrane proteins. Of those, tumor antigens and absorption, metabolism, disposition and elimination (ADME) proteins including UDP-glucuronosyltransferase, cytochrome P450, solute carriers and ATP-binding cassette transporters were detected and disclosed significant variations among the cell lines. The principal component analysis was performed for the cluster of cell lines. The results demonstrated that H226 is closely related with N87, while Hep3B2 aligned with HepG2. The protein cluster of Ovcar3 was apart from that of other cell lines investigated. By providing for the first time quantitative untargeted proteomics analysis, the results delineated the expression profiles of membrane proteins. These findings provided a useful resource for selecting targets of choice for anticancer therapy through advancing data obtained from preclinical tumor cell line models to clinical outcomes.

5.
Br J Clin Pharmacol ; 84(6): 1335-1345, 2018 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-29469197

RESUMO

AIMS: Previous studies demonstrated direct correlation between CYP2C19 genotype and BMS-823778 clearance in healthy volunteers. The objective of the present study was to develop a physiologically-based pharmacokinetic (PBPK) model for BMS-823778 and use the model to predict PK and drug-drug interaction (DDI) in virtual populations with multiple polymorphic genes. METHODS: The PBPK model was built and verified using existing clinical data. The verified model was simulated to predict PK of BMS-823778 and significance of DDI with a strong CYP3A4 inhibitor in subjects with various CYP2C19 and UGT1A4 genotypes. RESULTS: The verified PBPK model of BMS-823778 accurately recovered observed PK in different populations. In addition, the model was able to capture the exposure differences between subjects with different CYP2C19 genotypes. PK simulation indicated higher exposures of BMS-823778 in CYP2C19 poor metabolizers who were also devoid of UGT1A4 activity, compared to those with normal UGT1A4 functionality. Moderate DDI with itraconazole was predicted in subjects with wild-type CYP2C19 or UGT1A4. However, in subjects without CYP2C19 or UGT1A4 functionality, significant DDI was predicted when BMS-823778 was coadministered with itraconazole. CONCLUSIONS: A PBPK model was developed using clinical data that accurately predicted human PK in different population with various CYP2C19 phenotypes. Simulations with the verified PBPK model indicated that UGT1A4 was probably an important clearance pathway in CYP2C19 poor metabolizers. DDI with itraconazole is likely to be dependent on the genotypes of CYP2C19 and UGT1A4.

6.
J Neurosurg ; 128(1): 86-93, 2018 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-28106497

RESUMO

OBJECTIVE Renin-angiotensin system (RAS) genetic polymorphisms are thought to play a role in cerebral aneurysm formation and rupture. The Cerebral Aneurysm Renin-Angiotensin System (CARAS) study prospectively evaluated common RAS polymorphisms and their relation to aneurysmal subarachnoid hemorrhage (aSAH). METHODS The CARAS study prospectively enrolled aSAH patients and controls at 2 academic centers in the United States. A blood sample was obtained from all patients for genetic evaluation and measurement of plasma angiotensin-converting enzyme (ACE) concentration. Common RAS polymorphisms were detected using 5' exonuclease (TaqMan) genotyping assays and restriction fragment length polymorphism analysis. RESULTS Two hundred forty-eight patients were screened, and 149 aSAH patients and 50 controls were available for analysis. There was a recessive effect of the C allele of the angiotensinogen ( AGT) C/T single-nucleotide polymorphism (SNP) (OR 1.94, 95% CI 0.912-4.12, p = 0.0853) and a dominant effect of the G allele of the angiotensin II receptor Type 2 ( AT2) G/A SNP (OR 2.11, 95% CI 0.972-4.57, p = 0.0590) on aSAH that did not reach statistical significance after adjustment for potential confounders. The ACE level was significantly lower in aSAH patients with the II genotype (17.6 ± 8.0 U/L) as compared with the ID (22.5 ± 12.1 U/L) and DD genotypes (26.6 ± 14.2 U/L) (p = 0.0195). CONCLUSIONS The AGT C/T and AT2 G/A polymorphisms were not significantly associated with aSAH after controlling for potential confounders. However, a strong trend was identified for a dominant effect of the G allele of the AT2 G/A SNP. Downregulation of the local RAS may contribute to the formation of cerebral aneurysms and subsequent presentation with aSAH. Further studies are required to elucidate the relevant pathophysiology and its potential implication in treatment of patients with aSAH.

7.
Drug Metab Dispos ; 45(12): 1215-1224, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28935657

RESUMO

Metabolites of new chemical entities can influence safety and efficacy of a molecule and often times need to be quantified in preclinical studies. However, synthetic standards of metabolites are very rarely available in early discovery. Alternate approaches such as biosynthesis need to be explored to generate these metabolites. Assessing the quantity and purity of these small amounts of metabolites with a nondestructive analytical procedure becomes crucial. Quantitative NMR becomes the method of choice for these samples. Recent advances in high-field NMR (>500 MHz) with the use of cryoprobe technology have helped to improve sensitivity for analysis of small microgram quantity of such samples. However, this type of NMR instrumentation is not routinely available in all laboratories. To analyze microgram quantities of metabolites on a routine basis with lower-resolution 400 MHz NMR instrument fitted with a broad band fluorine observe room temperature probe, a novel hybrid capillary tube setup was developed. To quantitate the metabolite in the sample, an artificial signal insertion for calculation of concentration observed (aSICCO) method that introduces an internally calibrated mathematical signal was used after acquiring the NMR spectrum. The linearity of aSICCO signal was established using ibuprofen as a model analyte. The limit of quantification of this procedure was 0.8 mM with 10 K scans that could be improved further with the increase in the number of scans. This procedure was used to quantify three metabolites-phenytoin from fosphenytoin, dextrophan from dextromethorphan, and 4-OH-diclofenac from diclofenac-and is suitable for minibiosynthesis of metabolites from in vitro systems.


Assuntos
Tubo Capilar , Espectroscopia de Ressonância Magnética/instrumentação , Anti-Inflamatórios não Esteroides/análise , Anti-Inflamatórios não Esteroides/farmacocinética , Calibragem , Cromatografia Líquida de Alta Pressão , Dextrorfano/análise , Ibuprofeno/análise , Ibuprofeno/farmacocinética , Espectroscopia de Ressonância Magnética/métodos , Fenitoína/análise , Padrões de Referência , Solventes , Espectrometria de Massas em Tandem , Temperatura Ambiente
8.
Microbiome ; 5(1): 114, 2017 09 05.
Artigo em Inglês | MEDLINE | ID: mdl-28870234

RESUMO

BACKGROUND: Effective methods are needed to collect fecal samples from children for large-scale microbiota studies. Stool collected on fecal occult blood test (FOBT) cards that can be mailed provides an effective solution; however, the quality of sequencing resulting from this method is unknown. The aim of this study is to compare microbiota metrics of 16S ribosomal RNA (rRNA) gene sequencing from stool and meconium collected on FOBT cards with stool collected in an Eppendorf tube (ET) under different conditions. METHODS: Eight stool samples from children in diapers aged 0 month-2 years and three meconium samples were collected and stored as follows: (1) ≤ 2 days at room temperature (RT) in an ET, (2) 7 days at - 80 °C in an ET, (3) 3-5 days at RT on a FOBT card, (4) 7 days at RT on a FOBT card, and (5) 7 days at - 80 °C on a FOBT card. Samples stored at - 80 °C were frozen immediately. Each specimen/condition underwent 16S rRNA gene sequencing with replicates on the Illumina MiSeq. Alpha and beta diversity measures and relative abundance of major phyla were compared between storage conditions and container (ET vs. FOBT card), with pairwise comparison between different storage conditions and the "standard" of 7 days at - 80 °C in an ET and fresh stool in an ET. RESULTS: Stool samples clustered mainly by individual diaper (P < 10-5, Adonis), rather than by storage condition (P = 0.42) or container (P = 0.16). However, meconium samples clustered more by container (P = 0.002) than by individual diaper (P = 0.009) and storage condition (P = 0.02). Additionally, there were no differences in alpha diversity measures and relative abundance of major phyla after Bonferroni correction between stool stored on a FOBT card at RT for 7 days with stool stored in an ET tube at - 80 °C; differences in alpha diversity were seen however when compared to fresh stool in an ET. Overall, based on the intraclass correlation coefficient (ICC), the different storage containers/conditions are reliable in preserving the microbial memberships and slightly less reliable in preserving the alpha diversity and relative microbial composition of infant stool. CONCLUSIONS: Acknowledging certain limitations, FOBT cards may be a useful tool in large-scale stool microbiota studies in children requiring outpatient follow-up where only small amounts of stool can be obtained, but should not be used when studying meconium.


Assuntos
Fezes/microbiologia , Microbioma Gastrointestinal , Manejo de Espécimes/métodos , Pré-Escolar , Congelamento , Microbioma Gastrointestinal/genética , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Recém-Nascido , Mecônio/microbiologia , Sangue Oculto , RNA Ribossômico 16S/genética , Temperatura Ambiente
9.
Genet Med ; 19(12): 1367-1375, 2017 12.
Artigo em Inglês | MEDLINE | ID: mdl-28617419

RESUMO

PurposeImmunodeficiency screening has been added to many state-directed newborn screening programs. The current methodology is limited to screening for severe T-cell lymphopenia disorders. We evaluated the potential of genomic sequencing to augment current newborn screening for immunodeficiency, including identification of non-T cell disorders.MethodsWe analyzed whole-genome sequencing (WGS) and clinical data from a cohort of 1,349 newborn-parent trios by genotype-first and phenotype-first approaches. For the genotype-first approach, we analyzed predicted protein-impacting variants in 329 immunodeficiency-related genes in the WGS data. As a phenotype-first approach, electronic health records were used to identify children with clinical features suggestive of immunodeficiency. Genomes of these children and their parents were analyzed using a separate pipeline for identification of candidate pathogenic variants for rare Mendelian disorders.ResultsWGS provides adequate coverage for most known immunodeficiency-related genes. 13,476 distinct variants and 8,502 distinct predicted protein-impacting variants were identified in this cohort; five individuals carried potentially pathogenic variants requiring expert clinical correlation. One clinically asymptomatic individual was found genomically to have complement component 9 deficiency. Of the symptomatic children, one was molecularly identified as having an immunodeficiency condition and two were found to have other molecular diagnoses.ConclusionNeonatal genomic sequencing can potentially augment newborn screening for immunodeficiency.


Assuntos
Síndromes de Imunodeficiência/epidemiologia , Síndromes de Imunodeficiência/genética , Triagem Neonatal , Sequenciamento Completo do Genoma , Biologia Computacional/métodos , Curadoria de Dados , Feminino , Testes Genéticos , Genótipo , Humanos , Síndromes de Imunodeficiência/diagnóstico , Recém-Nascido , Masculino , Triagem Neonatal/métodos , Fenótipo
10.
Drug Metab Dispos ; 45(6): 676-685, 2017 06.
Artigo em Inglês | MEDLINE | ID: mdl-28283499

RESUMO

Ortho Tri-Cyclen, a two-drug cocktail comprised of ethinylestradiol and norgestimate (13-ethyl-17-acetoxy-18, 19-dinor-17α-pregn-4-en-20yn-3 oxime), is commonly prescribed to avert unwanted pregnancies in women of reproductive age. In vivo, norgestimate undergoes extensive and rapid deacetylation to produce 17-deacetylnorgestimate (NGMN), an active circulating metabolite that likely contributes significantly to norgestimate efficacy. Despite being of primary significance, the metabolism and reaction phenotyping of NGMN have not been previously reported. Hence, detailed biotransformation and reaction phenotyping studies of NGMN with recombinant cytochrome P450 (P450), recombinant uridine 5'-diphospho-glucuronosyltransferases, and human liver microsomes in the presence and absence of selective P450 inhibitors were conducted. It was found that CYP3A4 plays a key role in NGMN metabolism with a fraction metabolized (fm) of 0.57. CYP2B6 and to an even lesser extent CYP2C9 were also observed to catalyze NGMN metabolism. Using this CYP3A4 fm value, the predicted plasma concentration versus time area under the curve (AUC) change in NGMN using a basic/mechanistic static model was found to be within 1.3-fold of the reported NGMN AUC changes for four modulators of CYP3A4. In addition to NGMN, we have also elucidated the biotransformation of norgestrel (NG), a downstream norgestimate and NGMN metabolite, and found that CYP3A4 and UGT1A1 have a major contribution to the elimination of NG with a combined fm value of 1. The data presented in this paper will lead to better understanding and management of NGMN-based drug-drug interactions when norgestimate is coadministered with CYP3A4 modulators.


Assuntos
Anticoncepcionais Orais Sintéticos/farmacologia , Anticoncepcionais Orais Sintéticos/farmacocinética , Norgestrel/análogos & derivados , Acetilação , Cromatografia Líquida , Anticoncepcionais Orais Sintéticos/química , Sistema Enzimático do Citocromo P-450/química , Sistema Enzimático do Citocromo P-450/metabolismo , Sistema Enzimático do Citocromo P-450/farmacologia , Interações de Medicamentos , Humanos , Cinética , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/enzimologia , Microssomos Hepáticos/metabolismo , Norgestrel/química , Norgestrel/farmacocinética , Norgestrel/farmacologia , Oximas/química , Oximas/farmacocinética , Oximas/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacologia , Espectrometria de Massas em Tandem
11.
J Neurosurg ; 126(5): 1585-1597, 2017 May.
Artigo em Inglês | MEDLINE | ID: mdl-27285537

RESUMO

OBJECTIVE Renin-angiotensin system (RAS) genetic polymorphisms are thought to play a role in cerebral aneurysm formation and rupture. The Cerebral Aneurysm Renin Angiotensin System (CARAS) study prospectively evaluated associations of common RAS polymorphisms and clinical course after aneurysmal subarachnoid hemorrhage (aSAH). METHODS The CARAS study prospectively enrolled aSAH patients at 2 academic centers in the United States. A blood sample was obtained from all patients for genetic evaluation and measurement of plasma angiotensin converting enzyme (ACE) concentration. Common RAS polymorphisms were detected using 5'exonuclease genotyping assays and pyrosequencing. Analysis of associations of RAS polymorphisms and clinical course after aSAH were performed. RESULTS A total of 166 patients were screened, and 149 aSAH patients were included for analysis. A recessive effect of allele I (insertion) of the ACE I/D (insertion/deletion) polymorphism was identified for Hunt and Hess grade in all patients (OR 2.76, 95% CI 1.17-6.50; p = 0.0206) with subsequent poor functional outcome. There was a similar effect on delayed cerebral ischemia (DCI) in patients 55 years or younger (OR 3.63, 95% CI 1.04-12.7; p = 0.0439). In patients older than 55 years, there was a recessive effect of allele A of the angiotensin II receptor Type 2 (AT2) A/C single nucleotide polymorphism (SNP) on DCI (OR 4.70, 95% CI 1.43-15.4; p = 0.0111). CONCLUSIONS Both the ACE I/D polymorphism and the AT2 A/C single nucleotide polymorphism were associated with an age-dependent risk of delayed cerebral ischemia, whereas only the ACE I/D polymorphism was associated with poor clinical grade at presentation. Further studies are required to elucidate the relevant pathophysiology and its potential implication in the treatment of patients with aSAH.

12.
Clin Ther ; 38(4): 747-53, 2016 04.
Artigo em Inglês | MEDLINE | ID: mdl-26970697

RESUMO

Our case describes the serial microbiome changes in twins discordant for necrotizing enterocolitis (NEC), who shared similar intrauterine and early environmental exposures. The key findings were that the 2 neonates had distinctly different microbiome compositions from the first stool samples collected. Also, in the twin who developed NEC there was a decrease in bacterial diversity and an increase in Proteobacteria a week before developing any clinical symptoms, suggesting an early role of the intestinal microbiome in the development of NEC. Here we briefly review the literature on the role of the intestinal microbiome in NEC and how a greater understanding of the neonatal microbiome and host interactions may help mitigate this devastating disease.


Assuntos
Enterocolite Necrosante , Microbioma Gastrointestinal , Humanos , Recém-Nascido , Gêmeos
13.
Nat Commun ; 7: 10486, 2016 Jan 19.
Artigo em Inglês | MEDLINE | ID: mdl-26781218

RESUMO

Germline mutations are the source of evolution and contribute substantially to many health-related processes. Here we use whole-genome deep sequencing data from 693 parents-offspring trios to examine the de novo point mutations (DNMs) in the offspring. Our estimate for the mutation rate per base pair per generation is 1.05 × 10(-8), well within the range of previous studies. We show that maternal age has a small but significant correlation with the total number of DNMs in the offspring after controlling for paternal age (0.51 additional mutations per year, 95% CI: 0.29, 0.73), which was not detectable in the smaller and younger parental cohorts of earlier studies. Furthermore, while the total number of DNMs increases at a constant rate for paternal age, the contribution from the mother increases at an accelerated rate with age.These observations have implications related to the incidence of de novo mutations relating to maternal age.


Assuntos
Mutação em Linhagem Germinativa , Idade Materna , Adolescente , Adulto , Análise Mutacional de DNA , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Idade Paterna , Adulto Jovem
14.
Genet Med ; 18(3): 221-30, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26334177

RESUMO

PURPOSE: To assess the potential of whole-genome sequencing (WGS) to replicate and augment results from conventional blood-based newborn screening (NBS). METHODS: Research-generated WGS data from an ancestrally diverse cohort of 1,696 infants and both parents of each infant were analyzed for variants in 163 genes involved in disorders included or under discussion for inclusion in US NBS programs. WGS results were compared with results from state NBS and related follow-up testing. RESULTS: NBS genes are generally well covered by WGS. There is a median of one (range: 0-6) database-annotated pathogenic variant in the NBS genes per infant. Results of WGS and NBS in detecting 28 state-screened disorders and four hemoglobin traits were concordant for 88.6% of true positives (n = 35) and 98.9% of true negatives (n = 45,757). Of the five infants affected with a state-screened disorder, WGS identified two whereas NBS detected four. WGS yielded fewer false positives than NBS (0.037 vs. 0.17%) but more results of uncertain significance (0.90 vs. 0.013%). CONCLUSION: WGS may help rule in and rule out NBS disorders, pinpoint molecular diagnoses, and detect conditions not amenable to current NBS assays.


Assuntos
Predisposição Genética para Doença , Genoma Humano , Triagem Neonatal/métodos , Análise de Sequência de DNA/métodos , Estudos de Coortes , Feminino , Variação Genética , Humanos , Recém-Nascido , Masculino , Sensibilidade e Especificidade
15.
Xenobiotica ; 46(1): 52-64, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26153443

RESUMO

1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes in vivo biotransformation in humans and the identification of metabolic enzymes of ASV. 2. Following a single oral dose of [(14)C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with <1% of the dose recovered in urine. Drug-related radioactivity readily appeared in circulation and the plasma radioactivity was mainly attributed to ASV. A few minor metabolites were observed in human plasma and are not expected to contribute to the pharmacological activity because of low levels. The area under the curve (AUC) values of each circulating metabolite in humans were well below their levels in animals used in the long-term toxicological studies. In bile and feces, intact ASV was a prominent radioactive peak suggesting that both metabolism and direct excretion played important roles in ASV clearance. 3. The primary metabolic pathways of ASV were hydroxylation, sulfonamide hydrolysis and the loss of isoquinoline. In vitro studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.


Assuntos
Absorção Fisiológica , Isoquinolinas/metabolismo , Sulfonamidas/metabolismo , Administração Oral , Adolescente , Adulto , Bile/metabolismo , Radioisótopos de Carbono , Citocromo P-450 CYP3A/metabolismo , Relação Dose-Resposta à Radiação , Fezes , Humanos , Hidroxilação , Isoquinolinas/administração & dosagem , Isoquinolinas/sangue , Isoquinolinas/química , Masculino , Espectrometria de Massas , Metaboloma , Pessoa de Meia-Idade , Sulfonamidas/administração & dosagem , Sulfonamidas/sangue , Sulfonamidas/química , Fatores de Tempo , Distribuição Tecidual , Adulto Jovem
16.
Drug Metab Dispos ; 44(3): 320-8, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26714763

RESUMO

Although the metabolism and disposition of diclofenac (DF) has been studied extensively, information regarding the plasma levels of its acyl-ß-d-glucuronide (DF-AG), a major metabolite, in human subjects is limited. Therefore, DF-AG concentrations were determined in plasma (acidified blood derived) of six healthy volunteers following a single oral DF dose (50 mg). Levels of DF-AG in plasma were high, as reflected by a DF-AG/DF ratio of 0.62 ± 0.21 (Cmax mean ± S.D.) and 0.84 ± 0.21 (area under the concentration-time curve mean ± S.D.). Both DF and DF-AG were also studied as substrates of different human drug transporters in vitro. DF was identified as a substrate of organic anion transporter (OAT) 2 only (Km = 46.8 µM). In contrast, DF-AG was identified as a substrate of numerous OATs (Km = 8.6, 60.2, 103.9, and 112 µM for OAT2, OAT1, OAT4, and OAT3, respectively), two organic anion-transporting polypeptides (OATP1B1, Km = 34 µM; OATP2B1, Km = 105 µM), breast cancer resistance protein (Km = 152 µM), and two multidrug resistance proteins (MRP2, Km = 145 µM; MRP3, Km = 196 µM). It is concluded that the disposition of DF-AG, once formed, can be mediated by various candidate transporters known to be expressed in the kidney (basolateral, OAT1, OAT2, and OAT3; apical, MRP2, BCRP, and OAT4) and liver (canalicular, MRP2 and BCRP; basolateral, OATP1B1, OATP2B1, OAT2, and MRP3). DF-AG is unstable in plasma and undergoes conversion to parent DF. Therefore, caution is warranted when assessing renal and hepatic transporter-mediated drug-drug interactions with DF and DF-AG.


Assuntos
Transporte Biológico/fisiologia , Diclofenaco/metabolismo , Glucuronídeos/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Transportadores de Cassetes de Ligação de ATP/metabolismo , Adulto , Interações de Medicamentos/fisiologia , Humanos , Rim/metabolismo , Fígado/metabolismo , Masculino , Proteínas de Neoplasias/metabolismo , Transportadores de Ânions Orgânicos Sódio-Independentes/metabolismo , Adulto Jovem
17.
Drug Metab Dispos ; 44(5): 617-23, 2016 May.
Artigo em Inglês | MEDLINE | ID: mdl-26669328

RESUMO

An antibody-drug conjugate (ADC) is a unique therapeutic modality composed of a highly potent drug molecule conjugated to a monoclonal antibody. As the number of ADCs in various stages of nonclinical and clinical development has been increasing, pharmaceutical companies have been exploring diverse approaches to understanding the disposition of ADCs. To identify the key absorption, distribution, metabolism, and excretion (ADME) issues worth examining when developing an ADC and to find optimal scientifically based approaches to evaluate ADC ADME, the International Consortium for Innovation and Quality in Pharmaceutical Development launched an ADC ADME working group in early 2014. This white paper contains observations from the working group and provides an initial framework on issues and approaches to consider when evaluating the ADME of ADCs.


Assuntos
Anticorpos Monoclonais/metabolismo , Imunoconjugados/metabolismo , Preparações Farmacêuticas/metabolismo , Animais , Indústria Farmacêutica/métodos , Humanos
18.
ACS Med Chem Lett ; 6(8): 850-5, 2015 Aug 13.
Artigo em Inglês | MEDLINE | ID: mdl-26288683

RESUMO

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

19.
J Pharmacol Exp Ther ; 353(2): 380-91, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25740896

RESUMO

Organic anion transporting polypeptides (OATPs) mediate hepatic drug uptake and serve as the loci of drug-drug interactions (DDIs). Consequently, there is a major need to develop animal models and refine in vitro-in vivo extrapolations. Therefore, the in vivo disposition of a model OATP substrate, [(3)H]rosuvastatin (RSV), was studied in the cynomolgus monkey and reported for the first time. After monkeys had received a 3-mg/kg oral dose, mass balance was achieved after bile duct cannulation (mean total recovery of radioactivity of 103.6%). Forty-two percent of the RSV dose was recovered in urine and bile, and the elimination pathways were similar to those reported for human subjects; 61.7%, 39.0%, and 2.9% of the dose was recovered in the feces, bile, and urine, respectively. The high levels of unchanged RSV recovered in urine and bile (26% of the dose) and the relatively low levels of metabolites observed indicated that RSV was eliminated largely by excretion. Also, for the first time, the in vitro inhibitory potential of cyclosporin A (CsA) toward cynomolgus monkey OATPs and sodium-taurocholate cotransporting polypeptide was studied in vitro (primary hepatocytes and transporter-transfected cells). It is concluded that one can study the CsA-RSV DDI in the cynomolgus monkey. For example, the in vitro IC50 values were within 2-fold (monkey versus human), and the increase (versus vehicle control) in the RSV AUC0-inf (6.3-fold) and Cmax (10.2-fold) with CsA (100 mg/kg) was similar to that reported for humans. The results further support the use of the cynomolgus monkey as a model to assess interactions involving OATP inhibition.


Assuntos
Fluorbenzenos/metabolismo , Sondas Moleculares/metabolismo , Transportadores de Ânions Orgânicos/metabolismo , Pirimidinas/metabolismo , Sulfonamidas/metabolismo , Animais , Bile/metabolismo , Transporte Biológico/efeitos dos fármacos , Ciclosporina/farmacologia , Fezes/química , Fluorbenzenos/farmacocinética , Fluorbenzenos/urina , Células HEK293 , Humanos , Marcação por Isótopo , Macaca fascicularis , Masculino , Sondas Moleculares/farmacocinética , Sondas Moleculares/urina , Transportadores de Ânions Orgânicos Dependentes de Sódio/metabolismo , Pirimidinas/farmacocinética , Pirimidinas/urina , Rosuvastatina Cálcica , Especificidade da Espécie , Sulfonamidas/farmacocinética , Sulfonamidas/urina , Simportadores/metabolismo
20.
Am J Med Genet A ; 167A(5): 1111-6, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25712426

RESUMO

Rubinstein-Taybi syndrome (RSTS) can be caused by heterozygous mutations or deletions involving CREBBP or, less commonly, EP300. To date, only 15 patients with EP300 mutations have been clinically described. Frequently reported manifestations in these patients include characteristic facial and limb features, varying degrees of neurocognitive dysfunction, and maternal preeclampsia. Other congenital anomalies are less frequently reported. We describe a child found to have a de novo EP300 mutation (c.4933C>T, predicted to result in p.Arg1645X) through research-based whole-genome sequencing of the family trio. The child's presentation involved dysmorphic features as well as unilateral renal agenesis, a myelomeningocele, and minor genitourinary anomalies. The involvement of congenital anomalies in all 16 clinically described patients with EP300 mutations (25% of which have been identified by "hypothesis free" methods, including microarray, exome, and whole-genome sequencing) is reviewed. In summary, genitourinary anomalies have been identified in 38%, cardiovascular anomalies in 25%, spinal/vertebral anomalies in 19%, other skeletal anomalies in 19%, brain anomalies in 13%, and renal anomalies in 6%. Our patient expands the phenotypic spectrum in EP300-related RSTS; this case demonstrates the evolving practice of clinical genomics related to increasing availability of genomic sequencing methods.


Assuntos
Proteína p300 Associada a E1A/genética , Mutação , Síndrome de Rubinstein-Taybi/genética , Anormalidades Urogenitais/genética , Sequência de Bases , Mapeamento Cromossômico , Exoma/genética , Feminino , Humanos , Lactente , Imagem por Ressonância Magnética , Gravidez , Radiografia , Síndrome de Rubinstein-Taybi/diagnóstico por imagem , Síndrome de Rubinstein-Taybi/etiologia , Síndrome de Rubinstein-Taybi/fisiopatologia , Deleção de Sequência , Coluna Vertebral/diagnóstico por imagem , Coluna Vertebral/fisiopatologia , Anormalidades Urogenitais/fisiopatologia
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