RESUMO
PURPOSE: To determine the utility of single gene non-invasive prenatal screening (NIPS-SGD) in a high-risk reproductive genetics clinic. METHODS: A clinical pilot for NIPS-SGD was conducted from March 2020 to November 2021. A NIPS-SGD panel assessing pathogenic variants in 30 genes was offered to pregnant individuals for the following indications: (1) advanced sperm age ≥40 years, (2) nuchal translucency (NT) ≥ 3.5 mm, (3) fetal anomaly, or (4) family history of a condition covered by the panel. Diagnostic testing was offered concurrently. RESULTS: NIPS-SGD was ordered for 253 individuals: 88 (34.8%) for fetal anomalies, 96 (37.9%) for advanced sperm age, 37 (14.6%) for increased NT, and 5 (2.0%) for family history. Among 228 (90.1%) completed tests, 8 (3.5%) were positive. Diagnostic testing for 78 individuals revealed no false positive or negative results. Of 41 (25.9%) individuals who received a molecular diagnosis, 34 (82.9%) were outside the scope of NIPS-SGD. Positive NIPS-SGD altered medical management in five cases. CONCLUSIONS: NIPS-SGD in a high-risk population can lead to earlier prenatal diagnosis, enhanced surveillance, and targeted genetic analysis, but should not replace clinically indicated diagnostic testing. Potential incidental findings include parental diagnoses and misattributed parentage.
Assuntos
Diagnóstico Pré-Natal , Sêmen , Gravidez , Feminino , Masculino , Humanos , Adulto , Diagnóstico Pré-Natal/métodos , Medição da Translucência Nucal , AneuploidiaRESUMO
PURPOSE: Copy-number variant (CNV) assessment is recommended for patients undergoing prenatal diagnostic testing. Noninvasive screening tests have not been extensively validated for CNV detection. The objective of this study was to compare the ability of genome-wide noninvasive prenatal screening (NIPS) to chromosomal microarray to detect clinically significant findings. METHODS: We prospectively enrolled 198 subjects at the time of consent for diagnostic prenatal testing. Genome-wide NIPS results were compared with diagnostic testing results to assess NIPS test performance (n = 160, 38 subjects without microarray results excluded). Cohen's kappa statistic was used to assess test agreement. RESULTS: Genome-wide NIPS did not detect clinically significant chromosomal abnormalities at the same rate as diagnostic testing, κ = 0.75 (95% confidence interval [CI], 0.62-0.87). When excluding CNVs <7 Mb and findings outside the limits of genome-wide NIPS, test agreement improved, κ = 0.88 (0.79-0.97) driven by agreement for common aneuploidies (κ = 1.0). However, among patients with an abnormal fetal survey, agreement was only fair, κ = 0.38 (0.08-0.67). CONCLUSION: While NIPS is an excellent screening test for common aneuploidies, genome-wide NIPS misses clinically significant findings detected on routine diagnostic testing. False positive and false negative cases highlight the importance of pretest counseling regarding NIPS limitations, especially in the setting of fetal anomalies.
Assuntos
Transtornos Cromossômicos , Teste Pré-Natal não Invasivo , Aneuploidia , Feminino , Humanos , Gravidez , Diagnóstico Pré-Natal , Estudos ProspectivosRESUMO
OBJECTIVES: To assess the incidence of sex chromosome aneuploidy (SCA) predicted by noninvasive prenatal testing (NIPT), assess test performance, and compare it with nuchal translucency (NT) screening among patients seen in our prenatal diagnosis center. METHODS: We identified suspected cases of SCA by reviewing results from all NIPT samples sent from our center to commercial laboratories offering analysis by cell-free DNA between 1 December 2012 and 31 July 2015. Records of pregnancies positive for SCA were reviewed for ultrasound findings, NIPT indications, and karyotype results on maternal, fetal, and postnatal samples. Other SCA cases presenting during this period regardless of NIPT status were identified from genetic counseling and cytogenetics laboratory logbooks. RESULTS: Noninvasive prenatal testing predicted SCA in 18/2851 patients (0.63%). All had diagnostic testing of fetal or newborn samples. No patients terminated pregnancies on the basis of NIPT. NIPT suggested triple X in five cases, two with elevated NT: all were confirmed on karyotype. Two Klinefelter syndrome cases were also accurately predicted by NIPT. NIPT indicated monosomy X in 11 cases. Only one was a true positive. Ten were false positives, with 46, XX found on fetal or newborn karyotype. Maternal karyotype was mosaic (45, X[4], 46, XX[26]) in one case. Over the same time period, four additional cases of 45, X were confirmed on fetal samples, all with cystic hygromas. One of these had had a false negative NIPT result. The remaining patients pursued only direct testing via CVS or amniocentesis. CONCLUSIONS: Sex chromosome aneuploidy was frequently suspected on NIPT. False positive rate for monosomy X was surprisingly high (91%). Prediction of other SCA was more accurate. Diagnostic fetal chromosome analysis should be offered after abnormal NIPT or in the presence of cystic hygromas despite normal NIPT. NIPT limitations should be explained in pretest counseling. © 2017 John Wiley & Sons, Ltd.
Assuntos
Aneuploidia , Diagnóstico Pré-Natal/efeitos adversos , Diagnóstico Pré-Natal/métodos , Aberrações dos Cromossomos Sexuais , Adolescente , Adulto , Síndrome de Down/diagnóstico , Síndrome de Down/genética , Feminino , Testes Genéticos/métodos , Humanos , Cariotipagem , Medição da Translucência Nucal , Gravidez , Estudos Retrospectivos , Cromossomos SexuaisRESUMO
Histone acetyltransferases (HATs) are a class of enzymes that participate in modulating chromatin structure and gene expression. Altered HAT activity has been implicated in a number of diseases, yet little is known about the regulation of HATs. In this study, we report that glycosaminoglycans (GAGs) are potent inhibitors of p300 and pCAF HAT activities in vitro, with heparin and heparan sulfate proteoglycans (HSPGs) being the most potent inhibitors. The mechanism of inhibition by heparin was investigated. The ability of heparin to inhibit HAT activity was in part dependent upon its size and structure, as small heparin-derived oligosaccharides (>8 sugars) and N-desulfated or O-desulfated heparin showed reduced inhibitory activity. Heparin was shown to bind to pCAF; and enzyme assays indicated that heparin shows the characteristics of a competitive-like inhibitor causing an approximately 50-fold increase in the apparent Km of pCAF for histone H4. HSPGs isolated from corneal and pulmonary fibroblasts inhibited HAT activity with similar effectiveness as heparin. As evidence that endogenous GAGs might be involved in modulating histone acetylation, the direct addition of heparin to pulmonary fibroblasts resulted in an approximately 50% reduction of histone H3 acetylation after 6 h of treatment. In addition, Chinese hamster ovary cells deficient in GAG synthesis showed increased levels of acetylated histone H3 compared to wild-type parent cells. GAGs represent a new class of HAT inhibitors that might participate in modulating cell function by regulating histone acetylation.
Assuntos
Glicosaminoglicanos/farmacologia , Histona Acetiltransferases/antagonistas & inibidores , Histona Acetiltransferases/metabolismo , Acetilação , Animais , Núcleo Celular/metabolismo , Células Cultivadas , Cricetinae , Olho/metabolismo , Fibroblastos , Glicosaminoglicanos/química , Glicosaminoglicanos/metabolismo , Histona Acetiltransferases/genética , Histonas/metabolismo , Humanos , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Especificidade por SubstratoRESUMO
Elastin synthesis is essential for lung development and postnatal maturation as well as for repair following injury. Using human embryonic lung fibroblasts that express undetectable levels of elastin as assessed by Northern analyses, we found that treatment with exogenous transforming growth factor-beta (TGF-beta) induced rapid and transient increases in levels of elastin heterogeneous nuclear RNA (hnRNA) followed by increases of elastin mRNA and protein expression. In fibroblasts derived from transgenic mice, TGF-beta induced increases in the expression of a human elastin gene promoter fragment driving a chloramphenicol acetyl transferase reporter gene. The induction of elastin hnRNA and mRNA expression by TGF-beta was abolished by pretreatments with TGF-beta receptor I inhibitor, global transcription inhibitor actinomycin D, and partially blocked by addition of protein synthesis inhibitor cycloheximide, but was not affected by the p44/42 MAPK inhibitor U0126. Pretreatment with the p38 MAPK inhibitor SB-203580 also partially attenuated the levels of TGF-beta-induced elastin mRNA but not its hnRNA. Western analysis indicated that TGF-beta stimulated Akt phosphorylation. Inhibition of phosphatidylinositol 3-kinase and Akt phosphorylation by LY-294002 abolished TGF-beta-induced increases in elastin hnRNA and mRNA expression. Treatment of lung fibroblasts with interleukin-1beta or the histone deacetylase inhibitor trichostatin A inhibited TGF-beta-induced elastin mRNA and hnRNA expression by a mechanism that involved inhibition of Akt phosphorylation. Downregulation of Akt2 but not Akt1 expression employing small interfering RNA duplexes blocked TGF-beta-induced increases of elastin hnRNA and mRNA levels. Together, our results demonstrated that TGF-beta activates elastin transcription that is dependent on phosphatidylinositol 3-kinase/Akt activity.
Assuntos
Elastina/biossíntese , Pulmão/metabolismo , Transcrição Gênica/efeitos dos fármacos , Fator de Crescimento Transformador beta/fisiologia , Células Cultivadas , Cromonas/farmacologia , Regulação para Baixo , Fibroblastos/metabolismo , Inibidores de Histona Desacetilases , Humanos , Ácidos Hidroxâmicos/farmacologia , Morfolinas/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-akt/biossíntese , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Mensageiro/metabolismoRESUMO
Uncontrolled elastase activity is involved in the development of several types of lung disease. Previous reports demonstrated that growth factors are liberated from pulmonary matrix storage sites by elastase; however, release of these entities in vivo is not well defined. In the present study, we investigated the release of fibroblast growth factor-2 (FGF-2) and transforming growth factor-beta (TGF-beta), after intratracheal instillation of porcine pancreatic elastase into mice. We found that elastase promoted a time-dependent release of FGF-2 and TGF-beta1 from the lung into bronchoalveolar lavage (BAL) fluid. A large fraction of the TGF-beta1 in BAL fluid was in the active form (approximately 60%), suggesting that elastase might participate in the activation of TGF-beta1 from its latent form. Analysis of the levels of FGF-2 and TGF-beta1 in mouse blood indicated that the growth factors in BAL fluid were not entirely derived from blood. Moreover, elastase treatment of pulmonary fibroblasts cultures caused the release of TGF-beta1, suggesting that the TGF-beta1 in BAL fluid could have come from lung cells/matrix. Additional in vitro studies also indicated that TGF-beta1 plays a role in upregulating elastin mRNA levels. These data suggest that elastase releases growth factors from lung that participate in elastolytic injury responses.
Assuntos
Fator 2 de Crescimento de Fibroblastos/metabolismo , Pulmão/enzimologia , Pulmão/metabolismo , Elastase Pancreática/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Proteínas Sanguíneas/metabolismo , Células Cultivadas , Elastina/biossíntese , Elastina/genética , Feminino , Fator 2 de Crescimento de Fibroblastos/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Pulmão/fisiopatologia , Camundongos , Elastase Pancreática/farmacologia , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Tempo de Reação/efeitos dos fármacos , Tempo de Reação/fisiologia , Fator de Crescimento Transformador beta/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologiaRESUMO
Elastase degradation of elastin within alveolar walls is an important event in the development of pulmonary emphysema. In addition to elastolytic activities, elastases release growth factors from extracellular matrices and interstitial cell surfaces that can regulate elastogenesis and other cellular responses. In the present study, we demonstrate that brief treatment of matrix-laden rat pulmonary fibroblast cultures with pancreatic elastase results in the release of soluble heparin-binding epidermal growth factor-like growth factor (HB-EGF) concomitant with a decrease in HB-EGF binding to both heparan sulfate proteoglycan and receptor sites on the cells. In undigested, matrix-laden fibroblasts, HB-EGF significantly downregulates elastin mRNA via activation of epidermal growth factor receptor. Results from nuclear run-on analyses show that HB-EGF downregulates elastin mRNA via transcriptional suppression. HBEGF treatment stimulates MAP or ERK kinase (MEK)-dependent ERK1/2 phosphorylation and leads to nuclear accumulation of Fra-1. Blocking ERK1/2 activation by MEK1/2 inhibitors (PD-98059 or U-0126) diminishes HB-EGF-induced Fra-1 accumulation and subsequent downregulation of elastin mRNA. Coaddition of two elastase-released growth factors, HB-EGF and FGF-2, results in an additive inhibitory effect on elastin mRNA levels. Furthermore, HB-EGF addition to pulmonary fibroblasts increases FGF-2 mRNA and protein levels. These data suggest that HB-EGF and FGF-2 act in concert to regulate the synthesis of elastin in injury/repair situations.
Assuntos
Elastina/genética , Fator de Crescimento Epidérmico/farmacologia , Fator 2 de Crescimento de Fibroblastos/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Pulmão/fisiologia , Animais , Animais Recém-Nascidos , Sítios de Ligação , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Fator de Crescimento Epidérmico/farmacocinética , Fibroblastos/citologia , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Flavonoides/farmacologia , Heparina/farmacologia , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Pulmão/citologia , Pulmão/efeitos dos fármacos , Elastase Pancreática/metabolismo , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/farmacologia , Transcrição Gênica/efeitos dos fármacosRESUMO
Previously we have shown that treatment of confluent, pulmonary fibroblast cultures with elastase results in upregulation of elastin mRNA and protein levels. In the present study we focused on determining the level at which elastin expression is upregulated after elastase exposure. We examined as models for this investigation elastin gene expression in primary pulmonary fibroblast cells during the transition from subconfluent to confluent cultures and in confluent, matrix-laden cultures treated briefly with elastase. In addition, we extended our studies to mice that were given an intratracheal dose of elastase; the effects on lung elastin mRNA and elastin promoter activity levels were measured and compared with results from in vitro cell models. The results demonstrate that upregulation of elastin gene expression during the transition of subconfluent to confluent cultures and after elastase injury is associated with an increase in the level of transcription both in vitro and in vivo. Furthermore, intratracheal administration of elastase to transgenic mice illustrates that the increased levels of elastin mRNA are accompanied by increased activity of the elastin gene promoter in cells spatially positioned near major sites of tissue injury.
Assuntos
Elastina/genética , Regulação da Expressão Gênica , Pulmão/patologia , Elastase Pancreática/toxicidade , Animais , Animais Recém-Nascidos , Células Cultivadas , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Humanos , Cinética , Pulmão/efeitos dos fármacos , Pulmão/metabolismo , Camundongos , Camundongos Transgênicos , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
We investigated the role of sulfated proteoglycans in regulating extracellular matrix (ECM) deposition in pulmonary fibroblast cultures. Fibroblast cultures were subject to pharmacologic and enzymatic interventions to modify sulfated proteoglycan levels. Native and proteoglycan-depleted fibroblasts were treated with porcine pancreatic elastase at 2-4-day intervals and the elastase-mediated release of fibroblast growth factor 2 (FGF-2) and glycosaminoglycans was determined. Elastase treatment released significantly less FGF-2 and glycosaminoglycans (GAG) from PG-depleted fibroblasts with respect to native cells. Equilibrium ligand binding studies indicated that 125I FGF-2 binding at both cell surface receptor and heparan sulfate proteoglycan sites was reduced to different extents based on the method of proteoglycan depletion. Quantitation of elastin protein and message levels indicated that biological sulfation is required for the proper incorporation of tropoelastin into the extracellular matrix. These results suggest that sulfated proteoglycans play a central role in modulating pulmonary fibroblast extracellular matrix composition and are important mediators of elastolytic injury.
Assuntos
Heparitina Sulfato/fisiologia , Pulmão/fisiologia , Animais , Células Cultivadas , Elastina/efeitos dos fármacos , Elastina/fisiologia , Matriz Extracelular/efeitos dos fármacos , Matriz Extracelular/fisiologia , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fibroblastos/efeitos dos fármacos , Fibroblastos/fisiologia , Glicosaminoglicanos/metabolismo , Proteoglicanas de Heparan Sulfato/fisiologia , Pulmão/citologia , Pulmão/efeitos dos fármacos , Elastase Pancreática/farmacologia , Ratos , Receptores Proteína Tirosina Quinases/fisiologia , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Suínos , Cicatrização/fisiologiaRESUMO
We report the solid-state 13C and 15N NMR of insoluble elastin which has been synthesized in vitro with isotopically enriched glycine. Most of the glycines reside in a domain with good cross-polarization (CP) efficiencies, although surprisingly, a portion resides in an environment that is not detectable using CP. Our data indicate that much of the 13C population resides in regions of significant conformational flexibility. To support these conclusions, we present 13C and 15N cross-polarization with magic-angle-spinning (CPMAS) data in conjunction with "direct-polarization", nonspinning CP, and T1 measurements.
Assuntos
Elastina/química , Glicina/química , Animais , Isótopos de Carbono , Células Cultivadas , Elastina/biossíntese , Marcação por Isótopo , Músculo Liso/química , Músculo Liso/metabolismo , Isótopos de Nitrogênio , Ressonância Magnética Nuclear Biomolecular , Ratos , Ratos Sprague-DawleyRESUMO
Interleukin (IL)-1beta released after lung injury regulates the production of extracellular matrix components. We found that IL-1beta treatment reduced the rate of elastin gene transcription by 74% in neonatal rat lung fibroblasts. Deletion analysis of the rat elastin promoter detected a cis-acting element located at -118 to -102 bp that strongly bound Sp1 and Sp3 but not nuclear factor (NF)-kappaB. This element mediated IL-1beta-induced inhibition of the elastin promoter. IL-1beta treatment did not affect the level of Sp1 but did induce translocation of the p65 subunit of NF-kappaB. Overexpression of p65 decreased elastin promoter activity and markedly reduced elastin mRNA. Immunoprecipitation studies indicated an interaction between the p65 subunit and Sp1 protein. Microarray analysis of mRNA isolated after overexpression of p65 or treatment with IL-1beta revealed downregulation of alpha-smooth muscle actin and calponin mRNAs. Expression of these genes is associated with the myofibroblast phenotype. These results indicate that IL-1beta activates the nuclear localization of NF-kappaB that subsequently interacts with Sp1 to downregulate elastin transcription and expression of the myofibroblast phenotype.
Assuntos
Elastina/genética , Fibroblastos/fisiologia , Interleucina-1/farmacologia , Pulmão/fisiologia , Músculo Liso/fisiologia , NF-kappa B/fisiologia , Transcrição Gênica/efeitos dos fármacos , Animais , Células Cultivadas , Pulmão/citologia , Músculo Liso/citologia , Análise de Sequência com Séries de Oligonucleotídeos , Fenótipo , Regiões Promotoras Genéticas , Ratos , Ratos Sprague-Dawley , Fator de Transcrição Sp1/fisiologia , Fator de Transcrição RelARESUMO
The extracellular matrix (ECM) protein elastin plays an essential role in the cardiovascular system by imparting elasticity to blood vessel wall. In this study, we examined the effect of basic fibroblast growth factor (bFGF) on the expression of elastin in aortic smooth muscle cells (SMC) to gain insight into events associated with cardiovascular diseases. The results show that bFGF treatment of SMC causes a significant decrease in elastin mRNA and secreted tropoelastin levels. Nuclear run-on analyses demonstrate that the downregulation is due to a decrease in the level of elastin gene transcription. Transient transfections of SMC with wild-type and mutated elastin gene promoter/chloramphenicol acetyl transferase (CAT) constructs show that a previously identified activator protein-1-cAMP response element (AP1/CRE) (-564 to -558-bp) within the elastin promoter mediates the bFGF-dependent downregulation of elastin gene transcription in SMC. Addition of bFGF to SMC activates the extracellular signal-regulated kinases 1/2 (ERK1/2) resulting in their translocation into the nucleus and subsequent induction of Fra-1. The addition of PD-98059, an inhibitor of ERK1/2 kinase, abrogates the bFGF-dependent decrease of elastin mRNA in SMC. The described inhibitory effect of bFGF on elastin gene expression in SMC may significantly contribute to the inefficient repair of elastin in early stages of vascular wall injury.
Assuntos
Aorta/metabolismo , Elastina/genética , Fator 2 de Crescimento de Fibroblastos/metabolismo , Músculo Liso Vascular/metabolismo , Transcrição Gênica , Animais , Aorta/citologia , Células Cultivadas , AMP Cíclico/metabolismo , Regulação para Baixo/efeitos dos fármacos , Elastina/efeitos dos fármacos , Elastina/metabolismo , Inibidores Enzimáticos/farmacologia , Fator 2 de Crescimento de Fibroblastos/farmacologia , Flavonoides/farmacologia , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Regiões Promotoras Genéticas/fisiologia , Proteínas Proto-Oncogênicas c-fos/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Elementos de Resposta/fisiologia , Transdução de Sinais , Fator de Transcrição AP-1/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
Elastase/anti-elastase imbalance is a hallmark of emphysema, a chronic obstructive pulmonary disease associated with the rupture and inefficient repair of interstitial elastin. We report that neutrophil elastase (NE) at low physiologic concentrations, ranging from 35 nm to 1 microm, invokes transient, peaking at 15 min, activation of extracellular signal-regulated kinases 1 and 2 (ERK) in elastogenic lung fibroblasts. ERK activation is preceded by the release of soluble 25-26-kDa forms of epidermal growth factor (EGF) and transactivation of EGF receptor (EGFR) in NE-exposed cells. The stimulatory effect of NE on ERK is abrogated in the presence of anti-EGF-neutralizing antibodies, EGFR tyrosine kinase inhibitor (AG1478), and ERK kinase inhibitor (PD98059), as well as abolished in both EGFR-desensitized and endocytosis-arrested fibroblasts. Nuclear accumulation of activated ERK is associated with transient, peaking at 30 min, induction of c-Fos and sustained, observed at 24-48 h, decrease of tropoelastin mRNA levels in NE-challenged cells. Pretreatment of fibroblasts with AG1478 or PD98059 abrogates the NE-initiated tropoelastin mRNA suppression. We conclude that proteolytically released EGF signals directly via EGFR and ERK to down-regulate tropoelastin mRNA in NE-challenged lung fibroblasts.