RESUMEN
OBJECTIVE: To study whether gynecologic or reproductive disorders show association with trisomic conceptions. METHODS: This nationwide cohort study utilized the Registry of Congenital Malformations to identify women who had a trisomic pregnancy (n = 5784), either with trisomy 13 (T13; n = 351), trisomy 18 (T18; n = 1065) or trisomy 21 (T21; n = 4369) from 1987 to 2018. We used the Finnish Maternity cohort to match the cases to population controls (n = 34 422) on the age, residence, and timing of pregnancy. These data were cross-linked to the ICD-10 diagnoses of the national Care Registry for Health Care data on specialized health care in Finland during 1996 to 2019. Both inflammatory (ICD-10 diagnoses: N70-N77) and noninflammatory disorders of the genital tract (N80-N98) were studied. Crude odds ratios (ORs) with 95% CIs were calculated for associations between diagnoses and trisomic conceptions. RESULTS: The diagnosis of female infertility (N97) at any time was associated with trisomic conceptions (OR: 1.19, 95% CI: 1.08-1.32). In the subgroup analysis, this association was found for T18 (OR: 1.29, 95% CI: 1.03-1.61) and T21 (OR: 1.17, 95% CI: 1.04-1.32), but not for T13 (OR: 1.15, 95% CI: 0.75-1.72). When restricting the timing of the diagnosis of female infertility, an elevated OR was found only after the index pregnancy (OR: 1.81, 95% CI: 1.56-2.09). These increased odds for infertility after trisomic conceptions were observed both in women <35 years (T18 OR: 1.91, 95% CI: 1.21-3.00; T21 OR: 1.68, 95% CI: 1.31-2.14) and in women ≥35 years (T18 OR: 2.17, 95% CI: 1.40-3.33; T21 OR: 1.87; 95% CI: 1.47-2.39), but not after T13 conceptions. CONCLUSION: Our observational data suggest a link between trisomic conceptions and subsequent diagnoses of infertility but do not demonstrate causality. These data implicate that partially similar mechanisms might predispose to trisomy and infertility, regardless of maternal age.
RESUMEN
Transcribed enhancer maps can reveal nuclear interactions underpinning each cell type and connect specific cell types to diseases. Using a 5' single-cell RNA sequencing approach, we defined transcription start sites of enhancer RNAs and other classes of coding and noncoding RNAs in human CD4+ T cells, revealing cellular heterogeneity and differentiation trajectories. Integration of these datasets with single-cell chromatin profiles showed that active enhancers with bidirectional RNA transcription are highly cell type-specific and that disease heritability is strongly enriched in these enhancers. The resulting cell type-resolved multimodal atlas of bidirectionally transcribed enhancers, which we linked with promoters using fine-scale chromatin contact maps, enabled us to systematically interpret genetic variants associated with a range of immune-mediated diseases.
Asunto(s)
Linfocitos T CD4-Positivos , Elementos de Facilitación Genéticos , Predisposición Genética a la Enfermedad , Sitio de Iniciación de la Transcripción , Transcripción Genética , Humanos , Linfocitos T CD4-Positivos/inmunología , Diferenciación Celular , Cromatina/metabolismo , Cromatina/genética , Regiones Promotoras Genéticas , Linfocitos T Colaboradores-Inductores/inmunología , Análisis de Expresión Génica de una Sola Célula , Atlas como AsuntoRESUMEN
The HLA region, especially HLA class I and II genes, which encode molecules for antigen presentation to T cells, plays a major role in the predisposition to autoimmune disorders. To clarify the mechanisms behind this association, we examined genome-wide DNA methylation by microarrays to cover over 850,000 CpG sites in the CD4+ T cells and CD19+ B cells of healthy subjects homozygous either for DRB1*15-DQA1*01-DQB1*06:02 (DR2-DQ6, n = 14), associated with a strongly decreased T1D risk, DRB1*03-DQA1*05-DQB1*02 (DR3-DQ2, n = 19), or DRB1*04:01-DQA1*03-DQB1*03:02 (DR4-DQ8, n = 17), associated with a moderately increased T1D risk. In total, we discovered 14 differentially methylated CpG probes, of which 10 were located in the HLA region and six in the HLA-DRB1 locus. The main differences were between the protective genotype DR2-DQ6 and the risk genotypes DR3-DQ2 and DR4-DQ8, where the DR2-DQ6 group was hypomethylated compared to the other groups in all but four of the differentially methylated probes. The differences between the risk genotypes DR3-DQ2 and DR4-DQ8 were small. Our results indicate that HLA variants have few systemic effects on methylation and that their effect on autoimmunity is conveyed directly by HLA molecules, possibly by differences in expression levels or function.
Asunto(s)
Islas de CpG , Metilación de ADN , Diabetes Mellitus Tipo 1 , Predisposición Genética a la Enfermedad , Genotipo , Humanos , Diabetes Mellitus Tipo 1/genética , Diabetes Mellitus Tipo 1/inmunología , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Linfocitos B/inmunología , Linfocitos B/metabolismo , Femenino , Masculino , Cadenas HLA-DRB1/genética , Cadenas HLA-DRB1/inmunología , AlelosRESUMEN
A previous study suggested that fetal inheritance of chromosomally integrated human herpesvirus 6 (ici-HHV6) is associated with the hypertensive pregnancy disorder preeclampsia (PE). We aimed to study this question utilizing cord plasma samples (n = 1276) of the Finnish Genetics of Preeclampsia Consortium (FINNPEC) cohort: 539 from a pregnancy with PE and 737 without. We studied these samples and 30 placentas from PE pregnancies by a multiplex qPCR for the DNAs of all nine human herpesviruses. To assess the population prevalence of iciHHV-6, we studied whole-genome sequencing data from blood-derived DNA of 3421 biobank subjects. Any herpes viral DNA was detected in only two (0.37%) PE and one (0.14%) control sample (OR 2.74, 95% CI 0.25-30.4). One PE sample contained iciHHV-6B and another HHV-7 DNA. The control's DNA was of iciHHV-6B; the fetus having growth restriction and preterm birth without PE diagnosis. Placentas showed no herpesviruses. In the biobank data, 3 of 3421 subjects (0.08%) had low level HHV-6B but no iciHHV-6. While iciHHV-6 proved extremely rare, both fetuses with iciHHV-6B were growth-restricted, preterm, and from a pregnancy with maternal hypertension. Our findings suggest that human herpesviruses are not a significant cause of PE, whereas iciHHV-6 may pose some fetal risk.
Asunto(s)
Herpesvirus Humano 6 , Preeclampsia , Humanos , Femenino , Embarazo , Preeclampsia/virología , Preeclampsia/epidemiología , Adulto , Herpesvirus Humano 6/genética , Herpesvirus Humano 6/aislamiento & purificación , Estudios de Cohortes , Sangre Fetal/virología , Finlandia/epidemiología , ADN Viral/genética , ADN Viral/sangre , Placenta/virología , Herpesviridae/genéticaRESUMEN
The human genome is pervasively transcribed and produces a wide variety of long non-coding RNAs (lncRNAs), constituting the majority of transcripts across human cell types. Some specific nuclear lncRNAs have been shown to be important regulatory components acting locally. As RNA-chromatin interaction and Hi-C chromatin conformation data showed that chromatin interactions of nuclear lncRNAs are determined by the local chromatin 3D conformation, we used Hi-C data to identify potential target genes of lncRNAs. RNA-protein interaction data suggested that nuclear lncRNAs act as scaffolds to recruit regulatory proteins to target promoters and enhancers. Nuclear lncRNAs may therefore play a role in directing regulatory factors to locations spatially close to the lncRNA gene. We provide the analysis results through an interactive visualization web portal at https://fantom.gsc.riken.jp/zenbu/reports/#F6_3D_lncRNA.
Asunto(s)
Cromatina , ARN Largo no Codificante , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Cromatina/metabolismo , Cromatina/genética , Humanos , Anotación de Secuencia Molecular , Núcleo Celular/metabolismo , Núcleo Celular/genética , Genoma Humano , Regiones Promotoras GenéticasRESUMEN
Reading skills and developmental dyslexia, characterized by difficulties in developing reading skills, have been associated with brain anomalies within the language network. Genetic factors contribute to developmental dyslexia risk, but the mechanisms by which these genes influence reading skills remain unclear. In this preregistered study (https://osf.io/7sehx), we explored if developmental dyslexia susceptibility genes DNAAF4, DCDC2, NRSN1, and KIAA0319 are associated with brain function in fluently reading adolescents and young adults. Functional MRI and task performance data were collected during tasks involving written and spoken sentence processing, and DNA sequence variants of developmental dyslexia susceptibility genes previously associated with brain structure anomalies were genotyped. The results revealed that variation in DNAAF4, DCDC2, and NRSN1 is associated with brain activity in key language regions: the left inferior frontal gyrus, middle temporal gyrus, and intraparietal sulcus. Furthermore, NRSN1 was associated with task performance, but KIAA0319 did not yield any significant associations. Our findings suggest that individuals with a genetic predisposition to developmental dyslexia may partly employ compensatory neural and behavioral mechanisms to maintain typical task performance. Our study highlights the relevance of these developmental dyslexia susceptibility genes in language-related brain function, even in individuals without developmental dyslexia, providing valuable insights into the genetic factors influencing language processing.
Asunto(s)
Dislexia , Fenómenos Fisiológicos del Sistema Nervioso , Adolescente , Humanos , Adulto Joven , Encéfalo/diagnóstico por imagen , Dislexia/diagnóstico por imagen , Dislexia/genética , Genotipo , Proteínas Asociadas a Microtúbulos/genética , LecturaRESUMEN
BACKGROUND: Primary cilia emanate from most human cell types, including neurons. Cilia are important for communicating with the cell's immediate environment: signal reception and transduction to/from the ciliated cell. Deregulation of ciliary signaling can lead to ciliopathies and certain neurodevelopmental disorders. In the developing brain cilia play well-documented roles for the expansion of the neural progenitor cell pool, while information about the roles of cilia during post-mitotic neuron differentiation and maturation is scarce. RESULTS: We employed ciliated Lund Human Mesencephalic (LUHMES) cells in time course experiments to assess the impact of ciliary signaling on neuron differentiation. By comparing ciliated and non-ciliated neuronal precursor cells and neurons in wild type and in RFX2 -/- mutant neurons with altered cilia, we discovered an early-differentiation "ciliary time window" during which transient cilia promote axon outgrowth, branching and arborization. Experiments in neurons with IFT88 and IFT172 ciliary gene knockdowns, leading to shorter cilia, confirm these results. Cilia promote neuron differentiation by tipping WNT signaling toward the non-canonical pathway, in turn activating WNT pathway output genes implicated in cyto-architectural changes. CONCLUSIONS: We provide a mechanistic entry point into when and how ciliary signaling coordinates, promotes and translates into anatomical changes. We hypothesize that ciliary alterations causing neuron differentiation defects may result in "mild" impairments of brain development, possibly underpinning certain aspects of neurodevelopmental disorders.
Asunto(s)
Células-Madre Neurales , Vía de Señalización Wnt , Humanos , Cilios/metabolismo , Neuronas/fisiología , Diferenciación Celular , Células-Madre Neurales/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas Adaptadoras Transductoras de Señales/metabolismoRESUMEN
AIM: Clinical risk scores for coronary artery disease (CAD) are used in clinical practice to select patients for diagnostic testing and therapy. Several studies have proposed that polygenic risk scores (PRSs) can improve the prediction of CAD, but the scores need to be validated in clinical populations with accurately characterized phenotypes. We assessed the predictive power of the three most promising PRSs for the prediction of coronary atherosclerosis and obstructive CAD. METHODS: This study was conducted on 943 symptomatic patients with suspected CAD for whom the phenotype was accurately characterized using anatomic and functional imaging. Previously published genome-wide polygenic scores were generated to compare a genetic model based on PRSs with a model based on clinical data. The test and PRS cohorts were predominantly Caucasian of northern European ancestry. RESULTS: All three PRSs predicted coronary atherosclerosis and obstructive CAD statistically significantly. The predictive accuracy of the models combining clinical data and different PRSs varied between 0.778 and 0.805 in terms of the area under the receiver operating characteristic (AUROC), being close to the model including only clinical variables (AUROC 0.769). The difference between the clinical model and combined clinical + PRS model was not significant for PRS1 (p=0.627) and PRS3 (p=0.061). Only PRS2 slightly improved the predictive power of the model (p=0.04). The likelihood ratios showed the very weak diagnostic power of all PRSs. CONCLUSION: The addition of PRSs to conventional risk factors did not clinically significantly improve the predictive accuracy for either coronary atherosclerosis or obstructive CAD, showing that current PRSs are not justified for routine clinical use in CAD.
Asunto(s)
Enfermedad de la Arteria Coronaria , Puntuación de Riesgo Genético , Anciano , Femenino , Humanos , Masculino , Persona de Mediana Edad , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/diagnóstico , Estudio de Asociación del Genoma Completo , Fenotipo , Pronóstico , Medición de Riesgo/métodos , Curva ROCRESUMEN
Embryonic genome activation (EGA) occurs during preimplantation development and is characterized by the initiation of de novo transcription from the embryonic genome. Despite its importance, the regulation of EGA and the transcription factors involved in this process are poorly understood. Paired-like homeobox (PRDL) family proteins are implicated as potential transcriptional regulators of EGA, yet the PRDL-mediated gene regulatory networks remain uncharacterized. To investigate the function of PRDL proteins, we are identifying the molecular interactions and the functions of a subset family of the Eutherian Totipotent Cell Homeobox (ETCHbox) proteins, seven PRDL family proteins and six other transcription factors (TFs), all suggested to participate in transcriptional regulation during preimplantation. Using mass spectrometry-based interactomics methods, AP-MS and proximity-dependent biotin labeling, and chromatin immunoprecipitation sequencing we derive the comprehensive regulatory networks of these preimplantation TFs. By these interactomics tools we identify more than a thousand high-confidence interactions for the 21 studied bait proteins with more than 300 interacting proteins. We also establish that TPRX2, currently assigned as pseudogene, is a transcriptional activator.
Asunto(s)
Proteínas de Homeodominio , Factores de Transcripción , Humanos , Factores de Transcripción/metabolismo , Proteínas de Homeodominio/genética , Genes Homeobox , GenomaRESUMEN
BACKGROUND: The current knowledge about the molecular mechanisms underlying the health benefits of exercise is still limited, especially in childhood. We set out to investigate the effects of a 20-week exercise intervention on whole-blood transcriptome profile (RNA-seq) in children with overweight/obesity. METHODS: Twenty-four children (10.21 ± 1.33 years, 46% girls) with overweight/obesity, were randomized to either a 20-week exercise program (intervention group; n = 10), or to a no-exercise control group (n = 14). Whole-blood transcriptome profile was analyzed using RNA-seq by STRT technique with GlobinLock technology. RESULTS: Following the 20-week exercise intervention program, 161 genes were differentially expressed between the exercise and the control groups among boys, and 121 genes among girls (p-value <0.05), while after multiple correction, no significant difference between exercise and control groups persisted in gene expression profiles (FDR >0.05). Genes enriched in GO processes and molecular pathways showed different immune response in boys (antigen processing and presentation, infections, and T cell receptor complex) and in girls (Fc epsilon RI signaling pathway) (FDR <0.05). CONCLUSION: These results suggest that 20-week exercise intervention program alters the molecular pathways involved in immune processes in children with overweight/obesity.
Asunto(s)
Sobrepeso , Transcriptoma , Masculino , Niño , Femenino , Humanos , Sobrepeso/genética , Sobrepeso/terapia , Obesidad/genética , Ejercicio Físico/fisiologíaRESUMEN
BACKGROUND: Dilated cardiomyopathy (DCM) is a life-threatening heart disease and a common cause of heart failure due to systolic dysfunction and subsequent left or biventricular dilatation. A significant number of cases have a genetic etiology; however, as a complex disease, the exact genetic risk factors are largely unknown, and many patients remain without a molecular diagnosis. METHODS: We performed GWAS followed by whole-genome, transcriptome, and immunohistochemical analyses in a spontaneously occurring canine model of DCM. Canine gene discovery was followed up in three human DCM cohorts. RESULTS: Our results revealed two independent additive loci associated with the typical DCM phenotype comprising left ventricular systolic dysfunction and dilatation. We highlight two novel candidate genes, RNF207 and PRKAA2, known for their involvement in cardiac action potentials, energy homeostasis, and morphology. We further illustrate the distinct genetic etiologies underlying the typical DCM phenotype and ventricular premature contractions. Finally, we followed up on the canine discoveries in human DCM patients and discovered candidate variants in our two novel genes. CONCLUSIONS: Collectively, our study yields insight into the molecular pathophysiology of DCM and provides a large animal model for preclinical studies.
Asunto(s)
Cardiomiopatía Dilatada , Humanos , Animales , Perros , Cardiomiopatía Dilatada/genética , Cardiomiopatía Dilatada/veterinaria , Homeostasis , Modelos Animales , Fenotipo , Factores de RiesgoRESUMEN
The nuclear receptor subfamily 5 group A member 1 (NR5A1), encoding steroidogenic factor 1 (SF-1), has been identified as a critical factor in gonadal development in animal studies. A previous study of ours suggested that upregulation of NR5A1 during early gonadal differentiation in male (46,XY) human pluripotent stem cells steers the cells into a more mature gonadal cell type. However, the detailed role of NR5A1 in female gonadal differentiation has yet to be determined. In this study, by combining the processes of gonadal differentiation and conditional gene activation, we show that NR5A1 induction predominantly upregulates the female gonadal marker inhibin subunit α (INHA) and steroidogenic markers steroidogenic acute regulatory protein (STAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), cytochrome P450 family 17 subfamily A member 1 (CYP17A1), hydroxy-delta-5-steroid dehydrogenase (HSD3B2) and hydroxysteroid 17-beta dehydrogenase 1 (HSD17B1). In contrast, NR5A1 induction did not seem to affect the bipotential gonadal markers gata binding protein 4 (GATA4) and Wilms' tumour suppressor 1 (WT1) nor the female gonadal markers r-spondin 1 (RSPO1) and wnt family member 4 (WNT4). Differentially expressed genes were highly associated with adrenal and ovarian steroidogenesis pathways. Moreover, time-series analysis revealed different dynamic changes between male and female induced samples, where continuously upregulated genes in female gonadal differentiation were mostly associated with adrenal steroidogenesis. Thus, in contrast to male gonadal differentiation, NR5A1 is necessary but not sufficient to steer human embryonic stem cell (hESC)-derived bipotential gonadal-like cells towards a more mature somatic, female cell fate. Instead, it seems to direct bipotential gonadal-like cells more towards a steroidogenic-like cell population. The information obtained in this study helps in elucidating the role of NR5A1 in gonadal differentiation of a female stem cell line.
Asunto(s)
Células Madre Embrionarias Humanas , Animales , Humanos , Femenino , Masculino , Sistemas CRISPR-Cas , Factor Esteroidogénico 1/genética , Diferenciación Celular/genética , Familia 17 del Citocromo P450RESUMEN
OBJECTIVE: To identify the combination of maternal characteristics in women with hypertensive disorders of pregnancy (HDP) associated with hypertensive and other cardiovascular diseases (CVDs) within ten years following delivery. The aim is to understand who should receive the most intensive primary cardiovascular disease prevention. STUDY DESIGN: A prospective cohort study. MAIN OUTCOME: The population was the FINNPEC cohort (2008-2011), including women with (n = 1837) and without (n = 847) HDP. The main exposures were maternal hypertensive pregnancy complications linked with maternal pregnancy data from hospital records. The outcomes were hypertensive diseases and other CVDs (International Classification of Diseases, Tenth Revision). RESULTS: Women with de novo pre-eclampsia (PE) had an elevated risk for hypertensive diseases within ten years following delivery. The risk of CVD was increased in women with superimposed PE and chronic hypertension (CHT) only. Women with de novo PE and hypertensive diseases were more often primiparous (41.4% vs. 23.0%, p = 0.020), had gestational diabetes (GDM) (31.0% vs. 11.7%, p = 0.002), and higher pre-pregnancy body mass index (BMI) (28.7 ± 5.8 vs. 24.6 ± 4.8 kg/m2, p = 0.001), compared with women who remained normotensive. Women with superimposed PE with CVD had more likely early-onset PE, preterm delivery and were older than women without later CVD. CONCLUSIONS: Healthcare professionals should target early prevention of CVDs in women with chronic hypertension during pregnancy; of those who developed superimposed PE prior to 34th weeks of gestation and who delivered preterm. Women with de novo PE who are overweight/obese, primiparous, and with concurrent GDM need regular blood pressure monitoring.
Asunto(s)
Enfermedades Cardiovasculares , Diabetes Gestacional , Hipertensión Inducida en el Embarazo , Hipertensión , Preeclampsia , Complicaciones del Embarazo , Embarazo , Recién Nacido , Femenino , Humanos , Estudios Prospectivos , Hipertensión/epidemiología , Enfermedades Cardiovasculares/epidemiología , Enfermedades Cardiovasculares/etiología , Hipertensión Inducida en el Embarazo/epidemiologíaRESUMEN
Embryonic genome activation (EGA) is a critical step in embryonic development. However, while EGA has been studied in mice using mouse 2-cell-like cells, human EGA remains incompletely elucidated due to the lack of an in vitro cell model recapitulating the early blastomere stage in humans. Recently, five groups independently reported human 8-cell-like cells (8CLCs, also called induced blastomere-like cells) developed from pluripotent stem cells and used single-cell RNA sequencing (scRNA-seq) to specify their cellular identities. Here we summarize the methods developed to produce the 8CLCs and compare their transcriptomic profiles by integrating them with the scRNA-seq datasets of human embryos. These observations will allow comparison and validation of the models, stimulate further in-depth research to characterize the genes involved in human EGA and pre-implantation development, and facilitate studies on human embryogenesis.
Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Embarazo , Femenino , Humanos , Animales , Ratones , Embrión de Mamíferos , Desarrollo Embrionario/genética , Genoma HumanoRESUMEN
Importance: A genetic contribution to preeclampsia susceptibility has been established but is still incompletely understood. Objective: To disentangle the underlying genetic architecture of preeclampsia and preeclampsia or other maternal hypertension during pregnancy with a genome-wide association study (GWAS) of hypertensive disorders of pregnancy. Design, Setting, and Participants: This GWAS included meta-analyses in maternal preeclampsia and a combination phenotype encompassing maternal preeclampsia and preeclampsia or other maternal hypertensive disorders. Two overlapping phenotype groups were selected for examination, namely, preeclampsia and preeclampsia or other maternal hypertension during pregnancy. Data from the Finnish Genetics of Pre-eclampsia Consortium (FINNPEC, 1990-2011), Finnish FinnGen project (1964-2019), Estonian Biobank (1997-2019), and the previously published InterPregGen consortium GWAS were combined. Individuals with preeclampsia or other maternal hypertension during pregnancy and control individuals were selected from the cohorts based on relevant International Classification of Diseases codes. Data were analyzed from July 2020 to February 2023. Exposures: The association of a genome-wide set of genetic variants and clinical risk factors was analyzed for the 2 phenotypes. Results: A total of 16â¯743 women with prior preeclampsia and 15â¯200 with preeclampsia or other maternal hypertension during pregnancy were obtained from FINNPEC, FinnGen, Estonian Biobank, and the InterPregGen consortium study (respective mean [SD] ages at diagnosis: 30.3 [5.5], 28.7 [5.6], 29.7 [7.0], and 28 [not available] years). The analysis found 19 genome-wide significant associations, 13 of which were novel. Seven of the novel loci harbor genes previously associated with blood pressure traits (NPPA, NPR3, PLCE1, TNS2, FURIN, RGL3, and PREX1). In line with this, the 2 study phenotypes showed genetic correlation with blood pressure traits. In addition, novel risk loci were identified in the proximity of genes involved in the development of placenta (PGR, TRPC6, ACTN4, and PZP), remodeling of uterine spiral arteries (NPPA, NPPB, NPR3, and ACTN4), kidney function (PLCE1, TNS2, ACTN4, and TRPC6), and maintenance of proteostasis in pregnancy serum (PZP). Conclusions and Relevance: The findings indicate that genes related to blood pressure traits are associated with preeclampsia, but many of these genes have additional pleiotropic effects on cardiometabolic, endothelial, and placental function. Furthermore, several of the associated loci have no known connection with cardiovascular disease but instead harbor genes contributing to maintenance of successful pregnancy, with dysfunctions leading to preeclampsialike symptoms.
Asunto(s)
Hipertensión Inducida en el Embarazo , Preeclampsia , Humanos , Femenino , Embarazo , Preeclampsia/epidemiología , Preeclampsia/genética , Preeclampsia/diagnóstico , Estudio de Asociación del Genoma Completo , Canal Catiónico TRPC6/genética , Placenta , Factores de RiesgoRESUMEN
Here, we present a modification of single-cell tagged reverse transcription protocol to study gene expression on a single-cell level or with limited RNA input. We describe different enzymes for reverse transcription and cDNA amplification, modified lysis buffer, and additional clean-up steps before cDNA amplification. We also detail an optimized single-cell RNA sequencing method for handpicked single cells, or tens to hundreds of cells, as input material to study mammalian preimplantation development. For complete details on the use and execution of this protocol, please refer to Ezer et al.1.
Asunto(s)
Mamíferos , Tetranitrato de Pentaeritritol , Animales , ADN Complementario/genética , ARN/genética , Análisis de Secuencia de ARNRESUMEN
BACKGROUND: DYX1C1 (DNAAF4) and DCDC2 are two of the most replicated dyslexia candidate genes in genetic studies. They both have demonstrated roles in neuronal migration, in cilia growth and function and they both are cytoskeletal interactors. In addition, they both have been characterized as ciliopathy genes. However, their exact molecular functions are still incompletely described. Based on these known roles, we asked whether DYX1C1 and DCDC2 interact on the genetic and the protein level. RESULTS: Here, we report the physical protein-protein interaction of DYX1C1 and DCDC2 as well as their respective interactions with the centrosomal protein CPAP (CENPJ) on exogenous and endogenous levels in different cell models including brain organoids. In addition, we show a synergistic genetic interaction between dyx1c1 and dcdc2b in zebrafish exacerbating the ciliary phenotype. Finally, we show a mutual effect on transcriptional regulation among DYX1C1 and DCDC2 in a cellular model. CONCLUSIONS: In summary, we describe the physical and functional interaction between the two genes DYX1C1 and DCDC2. These results contribute to the growing understanding of the molecular roles of DYX1C1 and DCDC2 and set the stage for future functional studies.
Asunto(s)
Cilios , Chaperonas Moleculares , Proteínas de Pez Cebra , Pez Cebra , Animales , Movimiento Celular/genética , Regulación de la Expresión Génica , Fenotipo , Pez Cebra/genética , Chaperonas Moleculares/genética , Proteínas de Pez Cebra/genética , Cilios/patologíaRESUMEN
Primary cilia are antenna-like organelles emanating from the cell surface. They are involved in cell-to-cell communication and bidirectional signal transduction to/from the extracellular environment. During brain formation, cilia critically aid in neurogenesis and maturation of neuronal structures such as axons, dendrites and synapses. Aberrations in cilia function can induce neuron differentiation defects and pathological consequences of varying severity, resulting in ciliopathies and likely a number of neurodevelopmental disorders. Despite the documented relevance of cilia for proper brain development, human neuronal models to recognize and study cilia biology are still scarce. We have established two types of cell models, Lund Human Mesencephalic (LUHMES) cells and neuroepithelial stem (NES) cells derived from induced pluripotent stem cells (iPSC), to investigate cilia biology in both proliferating neuronal progenitors/precursors and during the entire neuron differentiation and maturation process. We employ improved immunocytochemistry assays able to specifically detect cilia by confocal and super-resolution microscopy. We provide straightforward and robust methods to easily maintain cells in culture, for immunostaining and characterization of cilia orientation, anatomy and shape in human neurons across all stages of differentiation.
Asunto(s)
Cilios , Células Madre Pluripotentes Inducidas , Humanos , Cilios/fisiología , Neuronas/metabolismo , Diferenciación Celular , Neurogénesis/fisiologíaRESUMEN
Inorganic polyphosphates are evolutionarily conserved bioactive phosphate polymers found as various chain lengths in all living organisms. In mammals, polyphosphates play a vital role in the regulation of cellular metabolism, coagulation, and inflammation. Long-chain polyphosphates are found along with endotoxins in pathogenic gram-negative bacteria and can participate in bacterial virulence. We aimed to investigate whether exogenously administered polyphosphates modulate human leukocyte function in vitro by treating the cells with 3 different chain lengths of polyphosphates (P14, P100, and P700). The long-chain polyphosphates, P700, had a remarkable capacity to downregulate type I interferon signaling dose dependently in THP1-Dual cells while only a slight elevation could be observed in the NF-κB pathway with the highest dose of P700. P700 treatment decreased lipopolysaccharide-induced IFNß transcription and secretion, reduced STAT1 phosphorylation, and downregulated subsequent interferon-stimulated gene expression in primary human peripheral blood mononuclear cells. P700 also augmented lipopolysaccharide-induced secretion of IL-1α, IL-1ß, IL-4, IL-5, IL-10, and IFNγ. Furthermore, P700 has previously been reported to increase the phosphorylation of several intracellular signaling mediators, such as AKT, mTOR, ERK, p38, GSK3α/ß, HSP27, and JNK pathway components, which was supported by our findings. Taken together, these observations demonstrate the extensive modulatory effects P700 has on cytokine signaling and the inhibitory effects specifically targeted to type I interferon signaling in human leukocytes.
Asunto(s)
Interferón Tipo I , Lipopolisacáridos , Animales , Humanos , Lipopolisacáridos/farmacología , Lipopolisacáridos/metabolismo , Leucocitos Mononucleares/metabolismo , Macrófagos/metabolismo , Polifosfatos/farmacología , Polifosfatos/metabolismo , FN-kappa B/metabolismo , Expresión Génica , Citocinas/metabolismo , Interferón Tipo I/metabolismo , Mamíferos/genéticaRESUMEN
The paired-like homeobox transcription factor LEUTX is expressed in human preimplantation embryos between the 4- and 8-cell stages, and then silenced in somatic tissues. To characterize the function of LEUTX, we performed a multiomic characterization of LEUTX using two proteomics methods and three genome-wide sequencing approaches. Our results show that LEUTX stably interacts with the EP300 and CBP histone acetyltransferases through its 9 amino acid transactivation domain (9aaTAD), as mutation of this domain abolishes the interactions. LEUTX targets genomic cis-regulatory sequences that overlap with repetitive elements, and through these elements it is suggested to regulate the expression of its downstream genes. We find LEUTX to be a transcriptional activator, upregulating several genes linked to preimplantation development as well as 8-cell-like markers, such as DPPA3 and ZNF280A. Our results support a role for LEUTX in preimplantation development as an enhancer binding protein and as a potent transcriptional activator.