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1.
Genome Med ; 11(1): 30, 2019 05 17.
Artigo em Inglês | MEDLINE | ID: mdl-31101064

RESUMO

BACKGROUND: Exome sequencing (ES) has been successfully applied in clinical detection of single nucleotide variants (SNVs) and small indels. However, identification of copy number variants (CNVs) using ES data remains challenging. The purpose of this study is to understand the contribution of CNVs and copy neutral runs of homozygosity (ROH) in molecular diagnosis of patients referred for ES. METHODS: In a cohort of 11,020 consecutive ES patients, an Illumina SNP array analysis interrogating mostly coding SNPs was performed as a quality control (QC) measurement and for CNV/ROH detection. Among these patients, clinical chromosomal microarray analysis (CMA) was performed at Baylor Genetics (BG) on 3229 patients, either before, concurrently, or after ES. We retrospectively analyzed the findings from CMA and the QC array. RESULTS: The QC array can detect ~ 70% of pathogenic/likely pathogenic CNVs (PCNVs) detectable by CMA. Out of the 11,020 ES cases, the QC array identified PCNVs in 327 patients and uniparental disomy (UPD) disorder-related ROH in 10 patients. The overall PCNV/UPD detection rate was 5.9% in the 3229 ES patients who also had CMA at BG; PCNV/UPD detection rate was higher in concurrent ES and CMA than in ES with prior CMA (7.2% vs 4.6%). The PCNVs/UPD contributed to the molecular diagnoses in 17.4% (189/1089) of molecularly diagnosed ES cases with CMA and were estimated to contribute in 10.6% of all molecularly diagnosed ES cases. Dual diagnoses with both PCNVs and SNVs were detected in 38 patients. PCNVs affecting single recessive disorder genes in a compound heterozygous state with SNVs were detected in 4 patients, and homozygous deletions (mostly exonic deletions) were detected in 17 patients. A higher PCNV detection rate was observed for patients with syndromic phenotypes and/or cardiovascular abnormalities. CONCLUSIONS: Our clinical genomics study demonstrates that detection of PCNV/UPD through the QC array or CMA increases ES diagnostic rate, provides more precise molecular diagnosis for dominant as well as recessive traits, and enables more complete genetic diagnoses in patients with dual or multiple molecular diagnoses. Concurrent ES and CMA using an array with exonic coverage for disease genes enables most effective detection of both CNVs and SNVs and therefore is recommended especially in time-sensitive clinical situations.

2.
Prenat Diagn ; 2018 Oct 25.
Artigo em Inglês | MEDLINE | ID: mdl-30357877

RESUMO

OBJECTIVE: To gather additional data on the ability to detect subchromosomal abnormalities of various sizes in single fetal cells isolated from maternal blood, using low-coverage shotgun next-generation sequencing for cell-based noninvasive prenatal testing (NIPT). METHOD: Fetal trophoblasts were recovered from approximately 30 mL of maternal blood using maternal white blood cell depletion, density-based cell separation, immunofluorescence staining, and high-resolution scanning. These trophoblastic cells were picked as single cells and underwent whole genome amplification for subsequent genome-wide copy number analysis and genotyping to confirm the fetal origin of the cells. RESULTS: Applying our fetal cell isolation method to a series of 125 maternal blood samples, we detected on average 4.17 putative fetal cells/sample. The series included 15 cases with clinically diagnosed fetal aneuploidies and five cases with subchromosomal abnormalities. This method was capable of detecting findings that were 1 to 2 Mb in size, and all were concordant with the microarray or karyotype data obtained on a fetal sample. A minority of fetal cells showed evidence of genome degradation likely related to apoptosis. CONCLUSION: We demonstrate that this cell-based NIPT method has the capacity to reliably diagnose fetal chromosomal abnormalities down to 1 to 2 Mb in size.

3.
Genome Med ; 10(1): 74, 2018 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-30266093

RESUMO

BACKGROUND: Exome sequencing is now being incorporated into clinical care for pediatric and adult populations, but its integration into prenatal diagnosis has been more limited. One reason for this is the paucity of information about the clinical utility of exome sequencing in the prenatal setting. METHODS: We retrospectively reviewed indications, results, time to results (turnaround time, TAT), and impact of exome results for 146 consecutive "fetal exomes" performed in a clinical diagnostic laboratory between March 2012 and November 2017. We define a fetal exome as one performed on a sample obtained from a fetus or a product of conception with at least one structural anomaly detected by prenatal imaging or autopsy. Statistical comparisons were performed using Fisher's exact test. RESULTS: Prenatal exome yielded an overall molecular diagnostic rate of 32% (n = 46/146). Of the 46 molecular diagnoses, 50% were autosomal dominant disorders (n = 23/46), 41% were autosomal recessive disorders (n = 19/46), and 9% were X-linked disorders (n = 4/46). The molecular diagnostic rate was highest for fetuses with anomalies affecting multiple organ systems and for fetuses with craniofacial anomalies. Out of 146 cases, a prenatal trio exome option designed for ongoing pregnancies was performed on 62 fetal specimens, resulting in a diagnostic yield of 35% with an average TAT of 14 days for initial reporting (excluding tissue culture time). The molecular diagnoses led to refined recurrence risk estimates, altered medical management, and informed reproductive planning for families. CONCLUSION: Exome sequencing is a useful diagnostic tool when fetal structural anomalies suggest a genetic etiology, but other standard prenatal genetic tests did not provide a diagnosis.

4.
JAMA Pediatr ; 171(12): e173438, 2017 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-28973083

RESUMO

Importance: While congenital malformations and genetic diseases are a leading cause of early infant death, to our knowledge, the contribution of single-gene disorders in this group is undetermined. Objective: To determine the diagnostic yield and use of clinical exome sequencing in critically ill infants. Design, Setting, and Participants: Clinical exome sequencing was performed for 278 unrelated infants within the first 100 days of life who were admitted to Texas Children's Hospital in Houston, Texas, during a 5-year period between December 2011 and January 2017. Exome sequencing types included proband exome, trio exome, and critical trio exome, a rapid genomic assay for seriously ill infants. Main Outcomes and Measures: Indications for testing, diagnostic yield of clinical exome sequencing, turnaround time, molecular findings, patient age at diagnosis, and effect on medical management among a group of critically ill infants who were suspected to have genetic disorders. Results: The mean (SEM) age for infants participating in the study was 28.5 (1.7) days; of these, the mean (SEM) age was 29.0 (2.2) days for infants undergoing proband exome sequencing, 31.5 (3.9) days for trio exome, and 22.7 (3.9) days for critical trio exome. Clinical indications for exome sequencing included a range of medical concerns. Overall, a molecular diagnosis was achieved in 102 infants (36.7%) by clinical exome sequencing, with relatively low yield for cardiovascular abnormalities. The diagnosis affected medical management for 53 infants (52.0%) and had a substantial effect on informed redirection of care, initiation of new subspecialist care, medication/dietary modifications, and furthering life-saving procedures in select patients. Critical trio exome sequencing revealed a molecular diagnosis in 32 of 63 infants (50.8%) at a mean (SEM) of 33.1 (5.6) days of life with a mean (SEM) turnaround time of 13.0 (0.4) days. Clinical care was altered by the diagnosis in 23 of 32 patients (71.9%). The diagnostic yield, patient age at diagnosis, and medical effect in the group that underwent critical trio exome sequencing were significantly different compared with the group who underwent regular exome testing. For deceased infants (n = 81), genetic disorders were molecularly diagnosed in 39 (48.1%) by exome sequencing, with implications for recurrence risk counseling. Conclusions and Relevance: Exome sequencing is a powerful tool for the diagnostic evaluation of critically ill infants with suspected monogenic disorders in the neonatal and pediatric intensive care units and its use has a notable effect on clinical decision making.


Assuntos
Doenças Genéticas Inatas/diagnóstico , Unidades de Terapia Intensiva Pediátrica , Sequenciamento Completo do Exoma/métodos , Adulto , Cuidados Críticos/métodos , Gerenciamento Clínico , Exoma , Aconselhamento Genético/métodos , Doenças Genéticas Inatas/genética , Doenças Genéticas Inatas/terapia , Humanos , Lactente , Cuidado do Lactente/métodos , Recém-Nascido , Tempo de Internação/estatística & dados numéricos , Estudos Retrospectivos , Texas
5.
Am J Med Genet A ; 173(10): 2680-2689, 2017 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-28815871

RESUMO

DNA alterations in the 1q43-q44 region are associated with syndromic neurodevelopmental disorders characterized by global developmental delay, intellectual disability, dysmorphic features, microcephaly, seizures, and agenesis of the corpus callosum. HNRNPU is located within the 1q43-q44 region and mutations in the gene have been reported in patients with early infantile epileptic encephalopathy. Here, we report on the clinical presentation of four patients with de novo heterozygous HNRNPU loss-of-function mutations detected by clinical whole exome sequencing: c.651_660del (p.Gly218Alafs*118), c.1089G>A (p.Trp363*), c.1714C>T (p.Arg572*), and c.2270_2271del (p.Pro757Argfs*7). All patients shared similar clinical features as previously reported including seizures, global developmental delay, intellectual disability, variable neurologic regression, behavior issues, and dysmorphic facial features. Features including heart defects and kidney abnormalities were not reported in our patients. These findings expands the clinical spectrum of HNRNPU-related disorder and shows that HNRNPU contributes to a subset of the clinical phenotypes associated with the contiguous 1q43-q44 deletion syndrome.


Assuntos
Deleção Cromossômica , Ribonucleoproteínas Nucleares Heterogêneas Grupo U/genética , Transtornos do Neurodesenvolvimento/genética , Criança , Feminino , Haploinsuficiência , Humanos , Lactente , Masculino , Transtornos do Neurodesenvolvimento/patologia , Linhagem , Fenótipo
6.
Nat Genet ; 49(4): 613-617, 2017 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-28288113

RESUMO

ABL1 is a proto-oncogene well known as part of the fusion gene BCR-ABL1 in the Philadelphia chromosome of leukemia cancer cells. Inherited germline ABL1 changes have not been associated with genetic disorders. Here we report ABL1 germline variants cosegregating with an autosomal dominant disorder characterized by congenital heart disease, skeletal abnormalities, and failure to thrive. The variant c.734A>G (p.Tyr245Cys) was found to occur de novo or cosegregate with disease in five individuals (families 1-3). Additionally, a de novo c.1066G>A (p.Ala356Thr) variant was identified in a sixth individual (family 4). We overexpressed the mutant constructs in HEK 293T cells and observed increased tyrosine phosphorylation, suggesting increased ABL1 kinase activities associated with both the p.Tyr245Cys and p.Ala356Thr substitutions. Our clinical and experimental findings, together with previously reported teratogenic effects of selective BCR-ABL inhibitors in humans and developmental defects in Abl1 knockout mice, suggest that ABL1 has an important role during organismal development.


Assuntos
Anormalidades Múltiplas/genética , Doenças do Desenvolvimento Ósseo/genética , Transtornos Cromossômicos/genética , Anormalidades Craniofaciais/genética , Transtornos da Alimentação e da Ingestão de Alimentos/genética , Proteínas de Fusão bcr-abl/genética , Mutação em Linhagem Germinativa/genética , Cardiopatias Congênitas/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Deformidades Congênitas dos Membros/genética , Animais , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Knockout , Cromossomo Filadélfia/efeitos dos fármacos , Fosforilação/genética , Transdução de Sinais/genética
7.
Neoplasia ; 18(9): 541-52, 2016 09.
Artigo em Inglês | MEDLINE | ID: mdl-27659014

RESUMO

Preclinical studies have suggested that the pancreatic tumor microenvironment both inhibits and promotes tumor development and growth. Here we establish the role of stromal fibroblasts during acinar-to-ductal metaplasia (ADM), an initiating event in pancreatic cancer formation. The transcription factor V-Ets avian erythroblastosis virus E26 oncogene homolog 2 (ETS2) was elevated in smooth muscle actin-positive fibroblasts in the stroma of pancreatic ductal adenocarcinoma (PDAC) patient tissue samples relative to normal pancreatic controls. LSL-Kras(G12D/+); LSL-Trp53(R172H/+); Pdx-1-Cre (KPC) mice showed that ETS2 expression initially increased in fibroblasts during ADM and remained elevated through progression to PDAC. Conditional ablation of Ets-2 in pancreatic fibroblasts in a Kras(G12D)-driven mouse ADM model decreased the amount of ADM events. ADMs from fibroblast Ets-2-deleted animals had reduced epithelial cell proliferation and increased apoptosis. Surprisingly, fibroblast Ets-2 deletion significantly altered immune cell infiltration into the stroma, with an increased CD8+ T-cell population, and decreased presence of regulatory T cells (Tregs), myeloid-derived suppressor cells, and mature macrophages. The mechanism involved ETS2-dependent chemokine ligand production in fibroblasts. ETS2 directly bound to regulatory sequences for Ccl3, Ccl4, Cxcl4, Cxcl5, and Cxcl10, a group of chemokines that act as potent mediators of immune cell recruitment. These results suggest an unappreciated role for ETS2 in fibroblasts in establishing an immune-suppressive microenvironment in response to oncogenic Kras(G12D) signaling during the initial stages of tumor development.


Assuntos
Células Acinares/metabolismo , Transformação Celular Neoplásica/metabolismo , Quimiocinas/biossíntese , Quimiotaxia de Leucócito , Ductos Pancreáticos/metabolismo , Proteína Proto-Oncogênica c-ets-2/metabolismo , Células Estromais/metabolismo , Células Acinares/patologia , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/imunologia , Quimiotaxia de Leucócito/imunologia , Colágeno/metabolismo , Fibroblastos/metabolismo , Deleção de Genes , Expressão Gênica , Humanos , Imuno-Histoquímica , Metaplasia , Camundongos , Camundongos Knockout , Ductos Pancreáticos/imunologia , Ductos Pancreáticos/patologia , Fenótipo , Proteína Proto-Oncogênica c-ets-2/genética , Proteínas Proto-Oncogênicas p21(ras)/genética , Transdução de Sinais
8.
Cancer Lett ; 380(1): 319-29, 2016 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-26688098

RESUMO

Targeted delivery aims to selectively distribute drugs to targeted tumor tissues but not to healthy tissues. This can address many clinical challenges by maximizing the efficacy but minimizing the toxicity of anti-cancer drugs. However, a complex tumor microenvironment poses various barriers hindering the transport of drugs and drug delivery systems. New tumor models that allow for the systematic study of these complex environments are highly desired to provide reliable test beds to develop drug delivery systems for targeted delivery. Recently, research efforts have yielded new in vitro tumor models, the so called tumor-microenvironment-on-chip, that recapitulate certain characteristics of the tumor microenvironment. These new models show benefits over other conventional tumor models, and have the potential to accelerate drug discovery and enable precision medicine. However, further research is warranted to overcome their limitations and to properly interpret the data obtained from these models. In this article, key features of the in vivo tumor microenvironment that are relevant to drug transport processes for targeted delivery were discussed, and the current status and challenges for developing in vitro transport model systems were reviewed.


Assuntos
Antineoplásicos/administração & dosagem , Carcinoma Ductal Pancreático/tratamento farmacológico , Sistemas de Liberação de Medicamentos/métodos , Dispositivos Lab-On-A-Chip , Procedimentos Analíticos em Microchip , Neoplasias Pancreáticas/tratamento farmacológico , Microambiente Tumoral , Animais , Transporte Biológico , Carcinoma Ductal Pancreático/metabolismo , Carcinoma Ductal Pancreático/patologia , Proliferação de Células/efeitos dos fármacos , Portadores de Fármacos , Descoberta de Drogas/instrumentação , Descoberta de Drogas/métodos , Resistencia a Medicamentos Antineoplásicos , Humanos , Técnicas Analíticas Microfluídicas/instrumentação , Micromanipulação/instrumentação , Terapia de Alvo Molecular , Neoplasias Pancreáticas/metabolismo , Neoplasias Pancreáticas/patologia
9.
Genetics ; 197(4): 1267-83, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24931409

RESUMO

Peptidergic neurons are a group of neuronal cells that synthesize and secrete peptides to regulate a variety of biological processes. To identify genes controlling the development and function of peptidergic neurons, we conducted a screen of 545 splice-trap lines and identified 28 loci that drove expression in peptidergic neurons when crossed to a GFP reporter transgene. Among these lines, an insertion in the alan shepard (shep) gene drove expression specifically in most peptidergic neurons. shep transcripts and SHEP proteins were detected primarily and broadly in the central nervous system (CNS) in embryos, and this expression continued into the adult stage. Loss of shep resulted in late pupal lethality, reduced adult life span, wing expansion defects, uncoordinated adult locomotor activities, rejection of males by virgin females, and reduced neuropil area and reduced levels of multiple presynaptic markers throughout the adult CNS. Examination of the bursicon neurons in shep mutant pharate adults revealed smaller somata and fewer axonal branches and boutons, and all of these cellular phenotypes were fully rescued by expression of the most abundant wild-type shep isoform. In contrast to shep mutant animals at the pharate adult stage, shep mutant larvae displayed normal bursicon neuron morphologies. Similarly, shep mutant adults were uncoordinated and weak, while shep mutant larvae displayed largely, although not entirely, normal locomotor behavior. Thus, shep played an important role in the metamorphic development of many neurons.


Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Metamorfose Biológica/genética , Neurogênese/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Sistema Nervoso Central/embriologia , Proteínas de Drosophila/genética , Feminino , Loci Gênicos , Larva/genética , Masculino , Dados de Sequência Molecular , Neurônios/metabolismo , Fenótipo , Filogenia , Proteínas de Ligação a RNA/genética , Análise de Sequência de RNA
10.
Bio Protoc ; 3(19)2013 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-27453905

RESUMO

Normal pancreatic acinar cells are difficult to maintain on traditional plastic culture surfaces due to their physical properties of housing large quantities of digestive enzymes and the formation of intercellular tight junctions and gap junctions (Apte and Wilson 2005; Rukstalis et al., 2003). However, placing primary acinar cells within a 3-dimensional matrix (3D-culture) maintains the cells for sufficient time so that they can be monitored for physiological changes to different stimuli. We have used a modified collagen 3D-culture system that has been adapted from Means et al. (2005) to model the very early events associated with pancreatic cancer development. In this model, KrasG12D-expressing pancreatic acinar cells, or wildtype acinar cells treated with EGFR-dependent growth factors (i.e., TGFα), convert to ductal cysts that mimic the acinar-to-ductal metaplasia (ADM) stage that precedes formation of Pancreatic Intraepithelial Neoplasia (PanIN) and Pancreatic Ductal Adenocarcinoma (PDAC) (Means et al., 2005; Shi et al., 2013).

11.
J Neurosci ; 26(30): 7860-9, 2006 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-16870731

RESUMO

During differentiation, neuroendocrine cells acquire highly amplified capacities to synthesize neuropeptides to overcome dilution of these signals in the general circulation. Once mature, the normal functioning of integrated physiological systems requires that neuroendocrine cells remain plastic to dramatically alter neuropeptide expression for long periods in response to hormonal and electrical cues. The mechanisms underlying the long-term regulation of neuroendocrine systems are poorly understood. Here we show that the Drosophila basic helix-loop-helix protein DIMM, a critical regulator of neuroendocrine cell differentiation, controls secretory capacity in mature neurons. DIMM expression began embryonically but persisted in adults. Through spatial and temporal manipulation of transgene expression in vivo, we defined two phases of prosecretory DIMM activity. During an embryonic critical window, DIMM controlled the differentiation of amplified expression of the neuropeptide leucokinin. At the onset of metamorphosis, levels of DIMM decreased in the insulin-producing cells (IPCs) in parallel with a marked reduction in levels of Drosophila insulin-like peptide 2 and a key neuropeptide biosynthetic enzyme peptidylglycine alpha-monooxygenase (PHM). Overexpression of DIMM in the IPCs prevented the decrease in PHM levels at this stage. In addition, transient overexpression of DIMM in adults produced a dramatic increase in PHM levels in numerous neurons located throughout the brain. These findings provide insights into the mechanisms controlling the maintenance of differentiated cell states, and they suggest an effective means for dynamically adjusting the strength of hormonal signals in diverse homeostatic systems.


Assuntos
Envelhecimento/fisiologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/fisiologia , Neurônios/citologia , Neurônios/fisiologia , Neuropeptídeos/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Drosophila/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia
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