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1.
PLoS One ; 19(6): e0304607, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38848383

RESUMEN

BACKGROUND: Hepatocellular carcinoma (HCC) is a highly aggressive liver cancer with significant morbidity and mortality rates. AXIN1 is one of the top-mutated genes in HCC, but the mechanism by which AXIN1 mutations contribute to HCC development remains unclear. METHODS: In this study, we utilized CRISPR/Cas9 genome editing to repair AXIN1-truncated mutations in five HCC cell lines. RESULTS: For each cell line we successfully obtained 2-4 correctly repaired clones, which all show reduced ß-catenin signaling accompanied with reduced cell viability and colony formation. Although exposure of repaired clones to Wnt3A-conditioned medium restored ß-catenin signaling, it did not or only partially recover their growth characteristics, indicating the involvement of additional mechanisms. Through RNA-sequencing analysis, we explored the gene expression patterns associated with repaired AXIN1 clones. Except for some highly-responsive ß-catenin target genes, no consistent alteration in gene/pathway expression was observed. This observation also applies to the Notch and YAP/TAZ-Hippo signaling pathways, which have been associated with AXIN1-mutant HCCs previously. The AXIN1-repaired clones also cannot confirm a recent observation that AXIN1 is directly linked to YAP/TAZ protein stability and signaling. CONCLUSIONS: Our study provides insights into the effects of repairing AXIN1 mutations on ß-catenin signaling, cell viability, and colony formation in HCC cell lines. However, further investigations are necessary to understand the complex mechanisms underlying HCC development associated with AXIN1 mutations.


Asunto(s)
Proteína Axina , Sistemas CRISPR-Cas , Carcinoma Hepatocelular , Neoplasias Hepáticas , Mutación , beta Catenina , Proteína Axina/genética , Proteína Axina/metabolismo , Humanos , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/metabolismo , Línea Celular Tumoral , beta Catenina/metabolismo , beta Catenina/genética , Regulación Neoplásica de la Expresión Génica , Edición Génica , Transducción de Señal/genética
2.
PLoS Negl Trop Dis ; 18(3): e0012074, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38536871

RESUMEN

Seoul orthohantavirus (SEOV) is a rat-borne zoonotic virus that is transmitted via inhalation of aerosolized infectious excreta, and can cause hemorrhagic fever with renal syndrome (HFRS) in humans worldwide. In rats, SEOV predominantly exists as a persistent infection in the absence of overt clinical signs. Lack of disease in rats is attributed to downregulation of pro-inflammatory and upregulation of regulatory host responses. As lung microvascular endothelial cells (LMECs) represent a primary target of infection in both human and rats, infections in these cells provide a unique opportunity to study the central role of LMECs in the dichotomy between pathogenicity in both species. In this study, host responses to SEOV infection in primary human and rat LMECs were directly compared on a transcriptional level. As infection of rat LMECs was more efficient than human LMECs, the majority of anti-viral defense responses were observed earlier in rat LMECs. Most prominently, SEOV-induced processes in both species included responses to cytokine stimulus, negative regulation of innate immune responses, responses to type I and II interferons, regulation of pattern recognition receptor signaling and MHC-I signaling. However, over time, in the rat LMECs, responses shifted from an anti-viral state towards a more immunotolerant state displayed by a PD-L1, B2M-, JAK2-focused interaction network aiding in negative regulation of cytotoxic CD8-positive T cell activation. This suggests a novel mechanism by which species-specific orthohantavirus-induced endothelium and T cell crosstalk may play a crucial role in the development of acute disease in humans and persistence in rodents.


Asunto(s)
Infecciones por Hantavirus , Fiebre Hemorrágica con Síndrome Renal , Virus Seoul , Humanos , Ratas , Animales , Células Endoteliales , Seúl , Virus Seoul/genética , Pulmón , Roedores , Antivirales
3.
Genes (Basel) ; 14(12)2023 Dec 16.
Artículo en Inglés | MEDLINE | ID: mdl-38137048

RESUMEN

Single cell RNAseq has been a big leap in many areas of biology. Rather than investigating gene expression on a whole organism level, this technology enables scientists to get a detailed look at rare single cells or within their cell population of interest. The field is growing, and many new methods appear each year. We compared methods utilized in our core facility: Smart-seq3, PlexWell, FLASH-seq, VASA-seq, SORT-seq, 10X, Evercode, and HIVE. We characterized the equipment requirements for each method. We evaluated the performances of these methods based on detected features, transcriptome diversity, mitochondrial RNA abundance and multiplets, among others and benchmarked them against bulk RNA sequencing. Here, we show that bulk transcriptome detects more unique transcripts than any single cell method. While most methods are comparable in many regards, FLASH-seq and VASA-seq yielded the best metrics, e.g., in number of features. If no equipment for automation is available or many cells are desired, then HIVE or 10X yield good results. In general, more recently developed methods perform better. This also leads to the conclusion that older methods should be phased out, and that the development of single cell RNAseq methods is still progressing considerably.


Asunto(s)
Perfilación de la Expresión Génica , Transcriptoma , Humanos , Transcriptoma/genética , Perfilación de la Expresión Génica/métodos , Análisis de Secuencia de ARN/métodos
4.
Genes (Basel) ; 14(3)2023 03 02.
Artículo en Inglés | MEDLINE | ID: mdl-36980900

RESUMEN

Functional perturbation and action mechanism studies have shown that the transcription factor Zeb2 controls cell fate decisions, differentiation, and/or maturation in multiple cell lineages in embryos and after birth. In cultured embryonic stem cells (ESCs), Zeb2's mRNA/protein upregulation is necessary for the exit from primed pluripotency and for entering general and neural differentiation. We edited mouse ESCs to produce Flag-V5 epitope-tagged Zeb2 protein from one endogenous allele. Using chromatin immunoprecipitation coupled with sequencing (ChIP-seq), we mapped 2432 DNA-binding sites for this tagged Zeb2 in ESC-derived neuroprogenitor cells (NPCs). A new, major binding site maps promoter-proximal to Zeb2 itself. The homozygous deletion of this site demonstrates that autoregulation of Zeb2 is necessary to elicit the appropriate Zeb2-dependent effects in ESC-to-NPC differentiation. We have also cross-referenced all the mapped Zeb2 binding sites with previously obtained transcriptome data from Zeb2 perturbations in ESC-derived NPCs, GABAergic interneurons from the ventral forebrain of mouse embryos, and stem/progenitor cells from the post-natal ventricular-subventricular zone (V-SVZ) in mouse forebrain, respectively. Despite the different characteristics of each of these neurogenic systems, we found interesting target gene overlaps. In addition, our study also contributes to explaining developmental disorders, including Mowat-Wilson syndrome caused by ZEB2 deficiency, and also other monogenic syndromes.


Asunto(s)
Neuronas , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc , Animales , Ratones , Sitios de Unión , ADN/química , ADN/metabolismo , Homocigoto , Neuronas/metabolismo , Neuronas/patología , Eliminación de Secuencia , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/metabolismo , Células Madre Embrionarias de Ratones/metabolismo
5.
Blood Adv ; 5(9): 2339-2349, 2021 05 11.
Artículo en Inglés | MEDLINE | ID: mdl-33938942

RESUMEN

The BCL11A gene encodes a transcriptional repressor with essential functions in multiple tissues during human development. Haploinsufficiency for BCL11A causes Dias-Logan syndrome (OMIM 617101), an intellectual developmental disorder with hereditary persistence of fetal hemoglobin (HPFH). Due to the severe phenotype, disease-causing variants in BCL11A occur de novo. We describe a patient with a de novo heterozygous variant, c.1453G>T, in the BCL11A gene, resulting in truncation of the BCL11A-XL protein (p.Glu485X). The truncated protein lacks the 3 C-terminal DNA-binding zinc fingers and the nuclear localization signal, rendering it inactive. The patient displayed high fetal hemoglobin (HbF) levels (12.1-18.7% of total hemoglobin), in contrast to the parents who had HbF levels of 0.3%. We used cultures of patient-derived erythroid progenitors to determine changes in gene expression and chromatin accessibility. In addition, we investigated DNA methylation of the promoters of the γ-globin genes HBG1 and HBG2. HUDEP1 and HUDEP2 cells were used as models for fetal and adult human erythropoiesis, respectively. Similar to HUDEP1 cells, the patient's cells displayed Assay for Transposase-Accessible Chromatin (ATAC) peaks at the HBG1/2 promoters and significant expression of HBG1/2 genes. In contrast, HBG1/2 promoter methylation and genome-wide gene expression profiling were consistent with normal adult erythropoiesis. We conclude that HPFH is the major erythroid phenotype of constitutive BCL11A haploinsufficiency. Given the essential functions of BCL11A in other hematopoietic lineages and the neuronal system, erythroid-specific targeting of the BCL11A gene has been proposed for reactivation of γ-globin expression in ß-hemoglobinopathy patients. Our data strongly support this approach.


Asunto(s)
Haploinsuficiencia , Proteínas Nucleares , Adulto , Proteínas Portadoras/genética , Humanos , Proteínas Nucleares/genética , Fenotipo , Proteínas Represoras/genética
6.
Hum Mol Genet ; 29(15): 2535-2550, 2020 08 29.
Artículo en Inglés | MEDLINE | ID: mdl-32628253

RESUMEN

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons.


Asunto(s)
Cromatina/ultraestructura , Elementos de Facilitación Genéticos/genética , Enfermedad de Hirschsprung/genética , Discapacidad Intelectual/genética , Microcefalia/genética , Caja Homeótica 2 de Unión a E-Box con Dedos de Zinc/genética , Animales , Diferenciación Celular/genética , Linaje de la Célula/genética , Cromatina/genética , Facies , Regulación de la Expresión Génica/genética , Enfermedad de Hirschsprung/patología , Proteínas de Homeodominio/genética , Humanos , Discapacidad Intelectual/patología , Ratones , Microcefalia/patología , Células-Madre Neurales/metabolismo , Células-Madre Neurales/patología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/ultraestructura , Secuencias Reguladoras de Ácidos Nucleicos
7.
Front Genet ; 11: 337, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32425970

RESUMEN

Telomeres are important for maintaining genomic stability. Telomere length has been associated with aging, disease, and mortality and is highly heritable (∼82%). In this study, we aimed to identify rare genetic variants associated with telomere length using whole-exome sequence data. We studied 1,303 participants of the Erasmus Rucphen Family (ERF) study, 1,259 of the Rotterdam Study (RS), and 674 of the British Heart Foundation Family Heart Study (BHF-FHS). We conducted two analyses, first we analyzed the family-based ERF study and used the RS and BHF-FHS for replication. Second, we combined the summary data of the three studies in a meta-analysis. Telomere length was measured by quantitative polymerase chain reaction in blood. We identified nine rare variants significantly associated with telomere length (p-value < 1.42 × 10-7, minor allele frequency of 0.2-0.5%) in the ERF study. Eight of these variants (in C11orf65, ACAT1, NPAT, ATM, KDELC2, and EXPH5) were located on chromosome 11q22.3 that contains ATM, a gene involved in telomere maintenance. Although we were unable to replicate the variants in the RS and BHF-FHS (p-value ≥ 0.21), segregation analysis showed that all variants segregate with shorter telomere length in a family. In the meta-analysis of all studies, a nominally significant association with LTL was observed with a rare variant in RPL8 (p-value = 1.48 × 10-6), which has previously been associated with age. Additionally, a novel rare variant in the known RTEL1 locus showed suggestive evidence for association (p-value = 1.18 × 10-4) with LTL. To conclude, we identified novel rare variants associated with telomere length. Larger samples size are needed to confirm these findings and to identify additional variants.

8.
Immunogenetics ; 72(1-2): 101-108, 2020 02.
Artículo en Inglés | MEDLINE | ID: mdl-31797007

RESUMEN

The domestic ferret, Mustela putorius furo, is an important mammalian animal model to study human respiratory infection. However, insufficient genomic annotation hampers detailed studies of ferret T cell responses. In this study, we analyzed the published T cell receptor beta (TRB) locus and performed high-throughput sequencing (HTS) of peripheral blood of four healthy adult ferrets to identify expressed V, D, J, and C genes. The HTS data is used as a guide to manually curate the expressed V, D, J, and C genes. The ferret locus appears to be most similar to that of the dog. Like other mammalian TRB loci, the ferret TRB locus contains a library of variable genes located upstream of two D-J-C gene clusters, followed by a (in the ferret non-functional) V gene with an inverted transcriptional orientation. All TRB genes (expressed or not) reported here have been approved by the IMGT/WHO-IUIS nomenclature committee.


Asunto(s)
Regulación de la Expresión Génica , Reordenamiento Génico de la Cadena beta de los Receptores de Antígenos de los Linfocitos T/genética , Receptores de Antígenos de Linfocitos T alfa-beta/genética , Animales , Hurones , Secuenciación de Nucleótidos de Alto Rendimiento
9.
Hum Mutat ; 40(11): 2131-2145, 2019 11.
Artículo en Inglés | MEDLINE | ID: mdl-31322790

RESUMEN

Noncoding RNAs have been widely recognized as essential mediators of gene regulation. However, in contrast to protein-coding genes, much less is known about the influence of noncoding RNAs on human diseases. Here we examined the association of genetic variants located in primary microRNA sequences and long noncoding RNAs (lncRNAs) with Alzheimer disease (AD) by leveraging data from the largest genome-wide association meta-analysis of late-onset AD. Variants annotated to 5 miRNAs and 10 lncRNAs (in seven distinct loci) exceeded the Bonferroni-corrected significance threshold (p < 1.02 × 10-6 ). Among these, a leading variant (rs2526377:A>G) at the 17q22 locus annotated to two noncoding RNAs (MIR142 and BZRAP1-AS) was significantly associated with a reduced risk of AD and fulfilled predefined criteria for being a functional variant. Our functional genomic analyses revealed that rs2526377 affects the promoter activity and decreases the expression of miR-142. Moreover, differential expression analysis by RNA-Seq in human iPSC-derived neural progenitor cells and the hippocampus of miR-142 knockout mice demonstrated multiple target genes of miR-142 in the brain that are likely to be involved in the inflammatory and neurodegenerative manifestations of AD. These include TGFBR1 and PICALM, of which their derepression in the brain due to reduced expression levels of miR-142-3p may reduce the risk of AD.


Asunto(s)
Enfermedad de Alzheimer/genética , Predisposición Genética a la Enfermedad , Variación Genética , MicroARNs/genética , Regiones Promotoras Genéticas , Alelos , Enfermedad de Alzheimer/metabolismo , Animales , Línea Celular , Mapeo Cromosómico , Biología Computacional/métodos , Modelos Animales de Enfermedad , Regulación de la Expresión Génica , Estudios de Asociación Genética , Estudio de Asociación del Genoma Completo , Hipocampo/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Polimorfismo de Nucleótido Simple , Interferencia de ARN , ARN no Traducido
10.
Front Genet ; 9: 420, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-30356672

RESUMEN

Carotid intima-media thickness (cIMT) is an established heritable marker for subclinical atherosclerosis. In this study, we aim to identify rare variants with large effects driving differences in cIMT by performing genome-wide linkage analysis of individuals in the extremes of cIMT trait distribution (>90th percentile) in a large family-based study from a genetically isolated population in the Netherlands. Linked regions were subsequently explored by fine-mapping using exome sequencing. We observed significant evidence of linkage on chromosomes 2p16.3 [rs1017418, heterogeneity LOD (HLOD) = 3.35], 19q13.43 (rs3499, HLOD = 9.09), 20p13 (rs1434789, HLOD = 4.10), and 21q22.12 (rs2834949, HLOD = 3.59). Fine-mapping using exome sequencing data identified a non-coding variant (rs62165235) in PNPT1 gene under the linkage peak at chromosome 2 that is likely to have a regulatory function. The variant was associated with quantitative cIMT in the family-based study population (effect = 0.27, p-value = 0.013). Furthermore, we identified several genes under the linkage peak at chromosome 21 highly expressed in tissues relevant for atherosclerosis. To conclude, our linkage analysis identified four genomic regions significantly linked to cIMT. Further analyses are needed to demonstrate involvement of identified candidate genes in development of atherosclerosis.

11.
PLoS Genet ; 13(12): e1007137, 2017 12.
Artículo en Inglés | MEDLINE | ID: mdl-29261648

RESUMEN

Cohesin is crucial for genome stability, cell division, transcription and chromatin organization. Its functions critically depend on NIPBL, the cohesin-loader protein that is found to be mutated in >60% of the cases of Cornelia de Lange syndrome (CdLS). Other mutations are described in the cohesin subunits SMC1A, RAD21, SMC3 and the HDAC8 protein. In 25-30% of CdLS cases no mutation in the known CdLS genes is detected. Until now, functional elements in the noncoding genome were not characterized in the molecular etiology of CdLS and therefore are excluded from mutation screening, although the impact of such mutations has now been recognized for a wide range of diseases. We have identified different elements of the noncoding genome involved in regulation of the NIPBL gene. NIPBL-AS1 is a long non-coding RNA transcribed upstream and antisense to NIPBL. By knockdown and transcription blocking experiments, we could show that not the NIPBL-AS1 gene product, but its actual transcription is important to regulate NIPBL expression levels. This reveals a possibility to boost the transcriptional activity of the NIPBL gene by interfering with the NIPBL-AS1 lncRNA. Further, we have identified a novel distal enhancer regulating both NIPBL and NIPBL-AS1. Deletion of the enhancer using CRISPR genome editing in HEK293T cells reduces expression of NIPBL, NIPBL-AS1 as well as genes found to be dysregulated in CdLS.


Asunto(s)
Elementos de Facilitación Genéticos , Oligonucleótidos Antisentido/genética , Oligonucleótidos Antisentido/metabolismo , Proteínas/genética , Proteínas/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas Cromosómicas no Histona/genética , Proteínas Cromosómicas no Histona/metabolismo , Segregación Cromosómica , Síndrome de Cornelia de Lange/genética , Regulación de la Expresión Génica , Genoma Humano , Células HEK293 , Humanos , Mutación , Fenotipo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , ARN Largo no Codificante/metabolismo , Análisis de Secuencia de ADN , Cohesinas
12.
Front Genet ; 8: 151, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-29093733

RESUMEN

Obstructive sleep apnea (OSA) is a common sleep breathing disorder associated with an increased risk of cardiovascular and cerebrovascular diseases and mortality. Although OSA is fairly heritable (~40%), there have been only few studies looking into the genetics of OSA. In the present study, we aimed to identify genetic variants associated with symptoms of sleep apnea by performing a whole-exome sequence meta-analysis of symptoms of sleep apnea in 1,475 individuals of European descent. We identified 17 rare genetic variants with at least suggestive evidence of significance. Replication in an independent dataset confirmed the association of a rare genetic variant (rs2229918; minor allele frequency = 0.3%) with symptoms of sleep apnea (p-valuemeta = 6.98 × 10-9, ßmeta = 0.99). Rs2229918 overlaps with the 3' untranslated regions of ERCC1 and CD3EAP genes on chromosome 19q13. Both genes are expressed in tissues in the neck area, such as the tongue, muscles, cartilage and the trachea. Further, CD3EAP is localized in the nucleus and mitochondria and involved in the tumor necrosis factor-alpha/nuclear factor kappa B signaling pathway. Our results and biological functions of CD3EAP/ERCC1 genes suggest that the 19q13 locus is interesting for further OSA research.

13.
Genome Biol ; 18(1): 48, 2017 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-28274275

RESUMEN

BACKGROUND: Hirschsprung disease (HSCR), which is congenital obstruction of the bowel, results from a failure of enteric nervous system (ENS) progenitors to migrate, proliferate, differentiate, or survive within the distal intestine. Previous studies that have searched for genes underlying HSCR have focused on ENS-related pathways and genes not fitting the current knowledge have thus often been ignored. We identify and validate novel HSCR genes using whole exome sequencing (WES), burden tests, in silico prediction, unbiased in vivo analyses of the mutated genes in zebrafish, and expression analyses in zebrafish, mouse, and human. RESULTS: We performed de novo mutation (DNM) screening on 24 HSCR trios. We identify 28 DNMs in 21 different genes. Eight of the DNMs we identified occur in RET, the main HSCR gene, and the remaining 20 DNMs reside in genes not reported in the ENS. Knockdown of all 12 genes with missense or loss-of-function DNMs showed that the orthologs of four genes (DENND3, NCLN, NUP98, and TBATA) are indispensable for ENS development in zebrafish, and these results were confirmed by CRISPR knockout. These genes are also expressed in human and mouse gut and/or ENS progenitors. Importantly, the encoded proteins are linked to neuronal processes shared by the central nervous system and the ENS. CONCLUSIONS: Our data open new fields of investigation into HSCR pathology and provide novel insights into the development of the ENS. Moreover, the study demonstrates that functional analyses of genes carrying DNMs are warranted to delineate the full genetic architecture of rare complex diseases.


Asunto(s)
Exoma , Predisposición Genética a la Enfermedad , Estudio de Asociación del Genoma Completo , Secuenciación de Nucleótidos de Alto Rendimiento , Enfermedad de Hirschsprung/genética , Alelos , Animales , Estudios de Casos y Controles , Biología Computacional/métodos , Análisis Mutacional de ADN , Modelos Animales de Enfermedad , Técnicas de Inactivación de Genes , Genotipo , Humanos , Mutación , Fenotipo , Pez Cebra
14.
Biol Psychiatry ; 81(8): 702-707, 2017 04 15.
Artículo en Inglés | MEDLINE | ID: mdl-27745872

RESUMEN

BACKGROUND: Despite high heritability, little success was achieved in mapping genetic determinants of depression-related traits by means of genome-wide association studies. METHODS: To identify genes associated with depressive symptomology, we performed a gene-based association analysis of nonsynonymous variation captured using exome-sequencing and exome-chip genotyping in a genetically isolated population from the Netherlands (n = 1999). Finally, we reproduced our significant findings in an independent population-based cohort (n = 1604). RESULTS: We detected significant association of depressive symptoms with a gene NKPD1 (p = 3.7 × 10-08). Nonsynonymous variants in the gene explained 0.9% of sex- and age-adjusted variance of depressive symptoms in the discovery study, which is translated into 3.8% of the total estimated heritability (h2 = 0.24). Significant association of depressive symptoms with NKPD1 was also observed (n = 1604; p = 1.5 × 10-03) in the independent replication sample despite little overlap with the discovery cohort in the set of nonsynonymous genetic variants observed in the NKPD1 gene. Meta-analysis of the discovery and replication studies improved the association signal (p = 1.0 × 10-09). CONCLUSIONS: Our study suggests that nonsynonymous variation in the gene NKPD1 affects depressive symptoms in the general population. NKPD1 is predicted to be involved in the de novo synthesis of sphingolipids, which have been implicated in the pathogenesis of depression.


Asunto(s)
Depresión/genética , Trastorno Depresivo Mayor/genética , Nucleósido-Trifosfatasa/genética , Exoma , Femenino , Variación Genética , Estudio de Asociación del Genoma Completo , Humanos , Masculino , Proteínas de la Membrana/genética , Persona de Mediana Edad , Proteínas del Tejido Nervioso/genética , Países Bajos , Polimorfismo de Nucleótido Simple , Población Blanca/genética
15.
Methods Mol Biol ; 1507: 199-220, 2017.
Artículo en Inglés | MEDLINE | ID: mdl-27832542

RESUMEN

The development and widespread implementation of chromosome conformation capture (3C) technology has allowed unprecedented new insight into how chromosomes are folded in three-dimensional (3D) space. 3C and its derivatives have contributed tremendously to the now widely accepted view that genome topology plays an important role in many major cellular processes, at a chromosome-wide scale, but certainly also at the level of individual genetic loci. A particularly popular application of 3C technology is to study transcriptional regulation, allowing researchers to draw maps of gene regulatory connections beyond the linear genome through addition of the third dimension. In this chapter, we provide a highly detailed protocol describing 3C coupled to high-throughput sequencing (referred to as 3C-Seq or more commonly 4C-Seq), allowing the unbiased interrogation of genome-wide chromatin interactions with specific genomic regions of interest. Interactions between spatially clustered DNA fragments are revealed by crosslinking the cells with formaldehyde, digesting the genome with a restriction endonuclease and performing a proximity ligation step to link interacting genomic fragments. Next, interactions with a selected DNA fragment are extracted from the 3C library through a second round of digestion and ligation followed by an inverse PCR. The generated products are immediately compatible with high-throughput sequencing, and amplicons from different PCR reactions can easily be multiplexed to dramatically increase throughput. Finally, we provide suggestions for data analysis and visualization.


Asunto(s)
Cromosomas de los Mamíferos/ultraestructura , ADN/aislamiento & purificación , Secuenciación de Nucleótidos de Alto Rendimiento , Análisis de Secuencia de ADN , Animales , Células Cultivadas , Cromosomas de los Mamíferos/genética , ADN/genética , Genoma , Humanos , Reacción en Cadena de la Polimerasa
16.
Mov Disord ; 31(7): 1041-8, 2016 07.
Artículo en Inglés | MEDLINE | ID: mdl-27090768

RESUMEN

BACKGROUND: ECHS1 encodes a mitochondrial enzyme involved in the degradation of essential amino acids and fatty acids. Recently, ECHS1 mutations were shown to cause a new severe metabolic disorder presenting as Leigh or Leigh-like syndromes. The objective of this study was to describe a family with 2 siblings affected by different dystonic disorders as a resulting phenotype of ECHS1 mutations. METHODS: Clinical evaluation, MRI imaging, genome-wide linkage, exome sequencing, urine metabolite profiling, and protein expression studies were performed. RESULTS: The first sibling is 17 years old and presents with generalized dystonia and severe bilateral pallidal MRI lesions after 1 episode of infantile subacute metabolic encephalopathy (Leigh-like syndrome). In contrast, the younger sibling (15 years old) only suffers from paroxysmal exercise-induced dystonia and has very mild pallidal MRI abnormalities. Both patients carry compound heterozygous ECHS1 mutations: c.232G>T (predicted protein effect: p.Glu78Ter) and c.518C>T (p.Ala173Val). Linkage analysis, exome sequencing, cosegregation, expression studies, and metabolite profiling support the pathogenicity of these mutations. Expression studies in patients' fibroblasts showed mitochondrial localization and severely reduced levels of ECHS1 protein. Increased urinary S-(2-carboxypropyl)cysteine and N-acetyl-S-(2-carboxypropyl)cysteine levels, proposed metabolic markers of this disorder, were documented in both siblings. Sequencing ECHS1 in 30 unrelated patients with paroxysmal dyskinesias revealed no further mutations. CONCLUSIONS: The phenotype associated with ECHS1 mutations might be milder than reported earlier, compatible with prolonged survival, and also includes isolated paroxysmal exercise-induced dystonia. ECHS1 screening should be considered in patients with otherwise unexplained paroxysmal exercise-induced dystonia, in addition to those with Leigh and Leigh-like syndromes. Diet regimens and detoxifying agents represent potential therapeutic strategies. © 2016 International Parkinson and Movement Disorder Society.


Asunto(s)
Trastornos Distónicos/genética , Trastornos Distónicos/fisiopatología , Enoil-CoA Hidratasa/deficiencia , Adolescente , Enoil-CoA Hidratasa/genética , Ejercicio Físico , Humanos , Masculino , Linaje
19.
RNA Biol ; 12(1): 30-42, 2015.
Artículo en Inglés | MEDLINE | ID: mdl-25826412

RESUMEN

Current RNA expression profiling methods rely on enrichment steps for specific RNA classes, thereby not detecting all RNA species in an unperturbed manner. We report strand-specific RNAome sequencing that determines expression of small and large RNAs from rRNA-depleted total RNA in a single sequence run. Since current analysis pipelines cannot reliably analyze small and large RNAs simultaneously, we developed TRAP, Total Rna Analysis Pipeline, a robust interface that is also compatible with existing RNA sequencing protocols. RNAome sequencing quantitatively preserved all RNA classes, allowing cross-class comparisons that facilitates the identification of relationships between different RNA classes. We demonstrate the strength of RNAome sequencing in mouse embryonic stem cells treated with cisplatin. MicroRNA and mRNA expression in RNAome sequencing significantly correlated between replicates and was in concordance with both existing RNA sequencing methods and gene expression arrays generated from the same samples. Moreover, RNAome sequencing also detected additional RNA classes such as enhancer RNAs, anti-sense RNAs, novel RNA species and numerous differentially expressed RNAs undetectable by other methods. At the level of complete RNA classes, RNAome sequencing also identified a specific global repression of the microRNA and microRNA isoform classes after cisplatin treatment whereas all other classes such as mRNAs were unchanged. These characteristics of RNAome sequencing will significantly improve expression analysis as well as studies on RNA biology not covered by existing methods.


Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , ARN/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma , Animales , Humanos , Ratones
20.
Eur J Hum Genet ; 23(9): 1142-50, 2015 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-25626705

RESUMEN

Implementation of next-generation DNA sequencing (NGS) technology into routine diagnostic genome care requires strategic choices. Instead of theoretical discussions on the consequences of such choices, we compared NGS-based diagnostic practices in eight clinical genetic centers in the Netherlands, based on genetic testing of nine pre-selected patients with cardiomyopathy. We highlight critical implementation choices, including the specific contributions of laboratory and medical specialists, bioinformaticians and researchers to diagnostic genome care, and how these affect interpretation and reporting of variants. Reported pathogenic mutations were consistent for all but one patient. Of the two centers that were inconsistent in their diagnosis, one reported to have found 'no causal variant', thereby underdiagnosing this patient. The other provided an alternative diagnosis, identifying another variant as causal than the other centers. Ethical and legal analysis showed that informed consent procedures in all centers were generally adequate for diagnostic NGS applications that target a limited set of genes, but not for exome- and genome-based diagnosis. We propose changes to further improve and align these procedures, taking into account the blurring boundary between diagnostics and research, and specific counseling options for exome- and genome-based diagnostics. We conclude that alternative diagnoses may infer a certain level of 'greediness' to come to a positive diagnosis in interpreting sequencing results. Moreover, there is an increasing interdependence of clinic, diagnostics and research departments for comprehensive diagnostic genome care. Therefore, we invite clinical geneticists, physicians, researchers, bioinformatics experts and patients to reconsider their role and position in future diagnostic genome care.


Asunto(s)
Cardiomiopatías/diagnóstico , Pruebas Genéticas/normas , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento/normas , Mutación , Proteínas de Unión al Calcio/genética , Miosinas Cardíacas/genética , Cardiomiopatías/genética , Proteínas Portadoras/genética , Exoma , Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento/instrumentación , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Consentimiento Informado/legislación & jurisprudencia , Ensayos de Aptitud de Laboratorios/estadística & datos numéricos , Quinasas Quinasa Quinasa PAM/genética , Cadenas Pesadas de Miosina/genética , Países Bajos , Proteínas Serina-Treonina Quinasas
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