RESUMO
BACKGROUND: The diagnostic yield of whole-exome sequencing (WES) varies from 30%-50% among patients with mild to severe neurodevelopmental delay (NDD)/intellectual disability (ID). Routine retrospective reanalysis of undiagnosed patients has increased the total diagnostic yield by 10-15%. Here, we performed proband-only WES of 1065 patients with NDD/ID and applied a prospective, daily reanalysis automated pipeline to patients without clinically significant variants to facilitate diagnoses. METHODS: The study included 1065 consecutive patients from 1056 nonconsanguineous unrelated families from 10 multimedical centers in South Korea between April 2018 and August 2021. WES data were analyzed daily using automatically updated databases with variant classification and symptom similarity scoring systems. RESULTS: At the initial analysis, 402 patients from 1056 unrelated families (38.0%, 402/1,056 families) had a positive genetic diagnosis. Daily prospective, automated reanalysis resulted in the identification of 34 additional diagnostic variants in 31 patients (3%), which increased our molecular diagnostic yield to 41% (433/1056 families). Among these 31 patients, 26 were diagnosed with 23 different diseases that were newly discovered after 2019. The time interval between the first analysis and the molecular diagnosis by reanalysis was 1.2 ± 0.9 years, which was shorter in the patients enrolled during the latter part of the study period. CONCLUSION: Daily updated databases and reanalysis systems enhance the diagnostic performance in patients with NDD/ID, contributing to the rapid diagnosis of undiagnosed patients by applying the latest molecular genetic information.
Assuntos
Exoma , Testes Genéticos , Exoma/genética , Testes Genéticos/métodos , Humanos , Estudos Prospectivos , Estudos Retrospectivos , Sequenciamento do Exoma/métodosRESUMO
Most infants born with very low birth weight (VLBW, birth weight < 1500 g) show spontaneous catch-up growth in postnatal life. The reasons for the absence of catch-up growth are not entirely understood. We performed a comprehensive investigation of 52 children born with VLBW. Ten children had a history of an external cause that explained the VLBW and five refused genetic evaluation. Twenty-three cases were initially evaluated by a candidate gene approach. Patients with a negative result in the candidate gene approach (n = 14) or without clinical suspicion (n = 14) were assessed by chromosome microarray analysis (CMA) and/or whole-exome sequencing (WES). A genetic condition was identified in 19 of 37 (51.4%) patients without an external cause, nine by candidate gene approach, and 10 by a genomic approach (CMA/WES). Silver-Russell syndrome was the most frequent diagnosis (n = 5) and the remaining patients were diagnosed with other rare monogenic conditions. Almost all patients with a positive genetic diagnosis exhibited syndromic features (94.4%). However, microcephaly, neurodevelopmental disorders, major malformation, or facial dysmorphism were also frequently observed in children with an external cause. In conclusion, a significant proportion of children born with VLBW with persistent short stature have a genetic/epigenetic condition.
Assuntos
Nanismo , Peso ao Nascer , Criança , Nanismo/diagnóstico , Nanismo/epidemiologia , Nanismo/genética , Epigênese Genética , Transtornos do Crescimento/genética , Humanos , Lactente , Recém-Nascido , Recém-Nascido de muito Baixo Peso , Sequenciamento do ExomaRESUMO
OBJECTIVE: Dysfunctional resolution of intestinal inflammation and altered mucosal healing are essential features in the pathogenesis of inflammatory bowel disease (IBD). Intestinal macrophages are vital in the process of inflammation resolution, but the mechanisms underlying their mucosal healing capacity remain elusive. DESIGN: We investigated the role of the prostaglandin E2 (PGE2) receptor PTGER4 on the differentiation of intestinal macrophages in patients with IBD and mouse models of intestinal inflammation. We studied mucosal healing and intestinal epithelial barrier regeneration in Csf1r-iCre Ptger4fl/fl mice during dextran sulfate sodium (DSS)-induced colitis. The effect of PTGER4+ macrophage secreted molecules was investigated on epithelial organoid differentiation. RESULTS: Here, we describe a subset of PTGER4-expressing intestinal macrophages with mucosal healing properties both in humans and mice. Csf1r-iCre Ptger4fl/fl mice showed defective mucosal healing and epithelial barrier regeneration in a model of DSS colitis. Mechanistically, an increased mucosal level of PGE2 triggers chemokine (C-X-C motif) ligand 1 (CXCL1) secretion in monocyte-derived PTGER4+ macrophages via mitogen-activated protein kinases (MAPKs). CXCL1 drives epithelial cell differentiation and proliferation from regenerating crypts during colitis. Specific therapeutic targeting of macrophages with liposomes loaded with an MAPK agonist augmented the production of CXCL1 in vivo in conditional macrophage PTGER4-deficient mice, restoring their defective epithelial regeneration and favouring mucosal healing. CONCLUSION: PTGER4+ intestinal macrophages are essential for supporting the intestinal stem cell niche and regeneration of the injured epithelium. Our results pave the way for the development of a new class of therapeutic targets to promote macrophage healing functions and favour remission in patients with IBD.
Assuntos
Doenças Inflamatórias Intestinais/metabolismo , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Ativação de Macrófagos , Receptores de Prostaglandina E Subtipo EP4/metabolismo , Animais , Diferenciação Celular , Quimiocina CXCL1/metabolismo , Modelos Animais de Doenças , Camundongos , Regeneração , Transdução de SinaisRESUMO
Transcription factors (TFs) are major trans-acting factors in transcriptional regulation. Therefore, elucidating TF-target interactions is a key step toward understanding the regulatory circuitry underlying complex traits such as human diseases. We previously published a reference TF-target interaction database for humans-TRRUST (Transcriptional Regulatory Relationships Unraveled by Sentence-based Text mining)-which was constructed using sentence-based text mining, followed by manual curation. Here, we present TRRUST v2 (www.grnpedia.org/trrust) with a significant improvement from the previous version, including a significantly increased size of the database consisting of 8444 regulatory interactions for 800 TFs in humans. More importantly, TRRUST v2 also contains a database for TF-target interactions in mice, including 6552 TF-target interactions for 828 mouse TFs. TRRUST v2 is also substantially more comprehensive and less biased than other TF-target interaction databases. We also improved the web interface, which now enables prioritization of key TFs for a physiological condition depicted by a set of user-input transcriptional responsive genes. With the significant expansion in the database size and inclusion of the new web tool for TF prioritization, we believe that TRRUST v2 will be a versatile database for the study of the transcriptional regulation involved in human diseases.
Assuntos
Bases de Dados Genéticas , Elementos Reguladores de Transcrição , Fatores de Transcrição/metabolismo , Animais , Regulação da Expressão Gênica , Humanos , Camundongos , Transcrição Gênica , Interface Usuário-ComputadorRESUMO
Spermatogonial stem cells (SSCs) can spontaneously dedifferentiate into embryonic stem cell (ESC)-like cells, which are designated as multipotent SSCs (mSSCs), without ectopic expression of reprogramming factors. Interestingly, SSCs express key pluripotency genes such as Oct4, Sox2, Klf4 and Myc. Therefore, molecular dissection of mSSC reprogramming may provide clues about novel endogenous reprogramming or pluripotency regulatory factors. Our comparative transcriptome analysis of mSSCs and induced pluripotent stem cells (iPSCs) suggests that they have similar pluripotency states but are reprogrammed via different transcriptional pathways. We identified 53 genes as putative pluripotency regulatory factors using an integrated systems biology approach. We demonstrated a selected candidate, Positive cofactor 4 (Pc4), can enhance the efficiency of somatic cell reprogramming by promoting and maintaining transcriptional activity of the key reprograming factors. These results suggest that Pc4 has an important role in inducing spontaneous somatic cell reprogramming via up-regulation of key pluripotency genes.
Assuntos
Reprogramação Celular/genética , Proteínas de Ligação a DNA/genética , Perfilação da Expressão Gênica , Proteínas Nucleares/genética , Fatores de Transcrição/genética , Células-Tronco Adultas/citologia , Células-Tronco Adultas/metabolismo , Animais , Western Blotting , Células Cultivadas , Análise por Conglomerados , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Nucleares/metabolismo , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Biologia de Sistemas/métodos , Fatores de Transcrição/metabolismoRESUMO
The viral vector-mediated overexpression of the defined transcription factors, Brn4/Pou3f4, Sox2, Klf4, and c-Myc (BSKM), could induce the direct conversion of somatic fibroblasts into induced neural stem cells (iNSCs). However, viral vectors may be randomly integrated into the host genome thereby increasing the risk for undesired genotoxicity, mutagenesis, and tumor formation. Here we describe the generation of integration-free iNSCs from mouse fibroblasts by non-viral episomal vectors containing BSKM. The episomal vector-derived iNSCs (e-iNSCs) closely resemble control NSCs, and iNSCs generated by retrovirus (r-iNSCs) in morphology, gene expression profile, epigenetic status, and self-renewal capacity. The e-iNSCs are functionally mature, as they could differentiate into all the neuronal cell types both in vitro and in vivo Our study provides a novel concept for generating functional iNSCs using a non-viral, non-integrating, plasmid-based system that could facilitate their biomedical applicability.
Assuntos
Células-Tronco Neurais/citologia , Animais , Fibroblastos/citologia , Vetores Genéticos , Fator 4 Semelhante a Kruppel , Camundongos , Camundongos Endogâmicos C3H , TransfecçãoRESUMO
BACKGROUND: Tuberous sclerosis complex (TSC) is an autosomal dominant multisystem disorder, caused by a loss-of-function of either TSC1 or TSC2 gene. However, in 10%-15% TSC patients there is no pathogenic variant identified in either TSC1 or TSC2 genes based on standard clinical testing. METHODS: In this study, genome sequencing was performed for families with clinical diagnosis of TSC with negative results from TSC1 and TSC2 single-gene tests. RESULTS: Herein, we report a family presenting a classical TSC phenotype with an unusual, complex structural variant involving the TSC1 gene: an intrachromosomal inverted insertion in the long arm of chromosome 9. We speculate that the inverted 9q33.3q34.13 region was inserted into the q31.2 region with the 3'-end of the breakpoint of the inversion being located within the TSC1 gene, resulting in premature termination of TSC1. CONCLUSIONS: In this study, we demonstrate the utility of genome sequencing for the identification of complex chromosomal rearrangement. Because the breakpoints are located within the deep intronic/intergenic region, this copy-neutral variant was missed by the TSC1 and TSC2 single-gene tests and contributed to an unknown etiology. Together, this finding suggests that complex structural variants may be underestimated causes for the etiology of TSC.
Assuntos
Esclerose Tuberosa , Proteínas Supressoras de Tumor , Humanos , Proteínas Supressoras de Tumor/genética , Proteína 1 do Complexo Esclerose Tuberosa/genética , Proteína 2 do Complexo Esclerose Tuberosa/genética , Mutação , Esclerose Tuberosa/genética , Esclerose Tuberosa/patologia , Cromossomos Humanos Par 9 , República da CoreiaRESUMO
The cortical actin cytoskeleton plays a critical role in maintaining intestinal epithelial integrity, and the loss of this architecture leads to chronic inflammation, as seen in inflammatory bowel disease (IBD). However, the exact mechanisms underlying aberrant actin remodeling in pathological states remain largely unknown. Here, we show that a subset of patients with IBD exhibits substantially higher levels of tripartite motif-containing protein 40 (TRIM40), a gene that is hardly detectable in healthy individuals. TRIM40 is an E3 ligase that directly targets Rho-associated coiled-coil-containing protein kinase 1 (ROCK1), an essential kinase involved in promoting cell-cell junctions, markedly decreasing the phosphorylation of key signaling factors critical for cortical actin formation and stabilization. This causes failure of the epithelial barrier function, thereby promoting a long-lived inflammatory response. A mutant TRIM40 lacking the RING, B-box, or C-terminal domains has impaired ability to accelerate ROCK1 degradation-driven cortical actin disruption. Accordingly, Trim40-deficient male mice are highly resistant to dextran sulfate sodium (DSS)-induced colitis. Our findings highlight that aberrant upregulation of TRIM40, which is epigenetically silenced under healthy conditions, drives IBD by subverting cortical actin formation and exacerbating epithelial barrier dysfunction.
Assuntos
Colite , Doenças Inflamatórias Intestinais , Proteínas com Motivo Tripartido , Animais , Masculino , Camundongos , Actinas/metabolismo , Colite/induzido quimicamente , Colite/genética , Colite/metabolismo , Sulfato de Dextrana/toxicidade , Modelos Animais de Doenças , Doenças Inflamatórias Intestinais/patologia , Mucosa Intestinal/metabolismo , Intestinos , Camundongos Endogâmicos C57BL , Humanos , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismoRESUMO
Inflammatory cytokines are key signaling molecules that can promote an immune response, thus their RNA turnover must be tightly controlled during infection. Most studies investigate the RNA decay pathways in the cytosol or nucleoplasm but never focused on the nucleolus. Although this organelle has well-studied roles in ribosome biogenesis and cellular stress sensing, the mechanism of RNA decay within the nucleolus is not completely understood. Here, we report that the nucleolus is an essential site of inflammatory pre-mRNA instability during infection. RNA-sequencing analysis reveals that not only do inflammatory genes have higher intronic read densities compared with non-inflammatory genes, but their pre-mRNAs are highly enriched in nucleoli during infection. Notably, nucleolin (NCL) acts as a guide factor for recruiting cytosine or uracil (C/U)-rich sequence-containing inflammatory pre-mRNAs and the Rrp6-exosome complex to the nucleolus through a physical interaction, thereby enabling targeted RNA delivery to Rrp6-exosomes and subsequent degradation. Consequently, Ncl depletion causes aberrant hyperinflammation, resulting in a severe lethality in response to LPS. Importantly, the dynamics of NCL post-translational modifications determine its functional activity in phases of LPS. This process represents a nucleolus-dependent pathway for maintaining inflammatory gene expression integrity and immunological homeostasis during infection.
Assuntos
Nucléolo Celular , Lipopolissacarídeos , Nucléolo Celular/metabolismo , Núcleo Celular , Lipopolissacarídeos/metabolismo , RNA/metabolismo , Estabilidade de RNARESUMO
Patients with non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) mutations exhibit an unfavorable response to PD-1 inhibitor through unclear mechanisms. Hypothesizing that EGFR mutations alter tumor-immune interactions, we compare tumor-infiltrating lymphocytes between EGFR mutant (EGFR-MT) and wild type (EGFR-WT) tumors through single-cell transcriptomic analysis. We find that B cells, CXCL13-producing follicular helper CD4+ T (TFH)-like cells, and tissue-resident memory CD8+ T (TRM)-like cells decreased in EGFR-MT tumors. The NOTCH-RBPJ regulatory network, which is vital for persistence of TRM state, is perturbed, and the interactions between TFH and B cells through the CXCL13-CXCR5 axis disappear in EGFR-MT tumors. Notably, the proportion of TRM-like cells is predictive for anti-PD-1 response in NSCLC. Our findings suggest that the impairment of TFH-B-TRM cooperation in tertiary lymphoid structure formation, accompanied by the dysregulation of TRM homeostasis and the loss of TFH-B crosstalk, underlies unfavorable anti-PD-1 response in EGFR-MT lung tumors.
Assuntos
Receptores ErbB/genética , Receptores ErbB/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Cooperação Linfocítica/fisiologia , Linfócitos B , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Quimiocina CXCL13/metabolismo , Feminino , Homeostase , Humanos , Neoplasias Pulmonares/patologia , Linfócitos do Interstício Tumoral , Masculino , Mutação , Receptores CXCR5/metabolismoRESUMO
Identifying genes that are capable of inducing tumorigenesis has been a major challenge in cancer research. In many cases, such genes frequently show somatic mutations in tumor samples; thus various computational methods for predicting cancer genes have been developed based on "significantly mutated genes." However, this approach is intrinsically limited by the fact that there are many cancer genes infrequently mutated in cancer genomes. Therefore, we recently developed MUFFINN (Mutations For Functional Impact on Network Neighbors), a method for cancer gene prediction based not only on mutation occurrences in each gene but also those of neighbors in functional networks. This enables the identification of cancer genes with infrequent mutation occurrence. We demonstrated that MUFFINN could retrieve known cancer genes more efficiently than gene-based methods and predicted cancer genes with low mutation occurrences in tumor samples. Users can freely access a web server ( http://www.inetbio.org/muffinn ) and run predictions with either public or private data of cancer somatic mutations. For given information of mutation occurrence profiles, the MUFFINN server returns lists of candidate cancer genes by four distinct predictions with different combinations between gene networks and scoring algorithms. Stand-alone software is also available, which allows MUFFINN to be run on local machines with a custom gene network. Here, we present an overall guideline for using the MUFFINN web server and stand-alone software for the discovery of novel cancer genes.
Assuntos
Biologia Computacional/métodos , Análise Mutacional de DNA/métodos , Redes Reguladoras de Genes , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mutação , Proteínas de Neoplasias/genética , Neoplasias/genética , Algoritmos , Perfilação da Expressão Gênica , Humanos , Neoplasias/diagnóstico , Transdução de Sinais , SoftwareRESUMO
Gene set enrichment analysis (GSEA) is a popular tool to identify underlying biological processes in clinical samples using their gene expression phenotypes. GSEA measures the enrichment of annotated gene sets that represent biological processes for differentially expressed genes (DEGs) in clinical samples. GSEA may be suboptimal for functional gene sets; however, because DEGs from the expression dataset may not be functional genes per se but dysregulated genes perturbed by bona fide functional genes. To overcome this shortcoming, we developed network-based GSEA (NGSEA), which measures the enrichment score of functional gene sets using the expression difference of not only individual genes but also their neighbors in the functional network. We found that NGSEA outperformed GSEA in identifying pathway gene sets for matched gene expression phenotypes. We also observed that NGSEA substantially improved the ability to retrieve known anti-cancer drugs from patient-derived gene expression data using drug-target gene sets compared with another method, Connectivity Map. We also repurposed FDA-approved drugs using NGSEA and experimentally validated budesonide as a chemical with anti-cancer effects for colorectal cancer. We, therefore, expect that NGSEA will facilitate both pathway interpretation of gene expression phenotypes and anti-cancer drug repositioning. NGSEA is freely available at www.inetbio.org/ngsea.
Assuntos
Regulação da Expressão Gênica , Redes Reguladoras de Genes , Área Sob a Curva , Budesonida/farmacologia , Budesonida/uso terapêutico , Neoplasias Colorretais/tratamento farmacológico , Neoplasias Colorretais/genética , Bases de Dados Genéticas , Sistemas de Liberação de Medicamentos , Regulação da Expressão Gênica/efeitos dos fármacos , Redes Reguladoras de Genes/efeitos dos fármacos , Humanos , Internet , Fenótipo , Curva ROCRESUMO
Disorders of the biliary epithelium, known as cholangiopathies, cause severe and irreversible liver diseases. The limited accessibility of bile duct precludes modeling of several cholangiocyte-mediated diseases. Therefore, novel approaches for obtaining functional cholangiocytes with high purity are needed. Previous work has shown that the combination of Hnf1ß and Foxa3 could directly convert mouse fibroblasts into bipotential hepatic stem cell-like cells, termed iHepSCs. However, the efficiency of converting fibroblasts into iHepSCs is low, and these iHepSCs exhibit extremely low differentiation potential into cholangiocytes, thus hindering the translation of iHepSCs to the clinic. Here, we describe that the expression of Hnf1α and Foxa3 dramatically facilitates the robust generation of iHepSCs. Notably, prolonged in vitro culture of Hnf1α- and Foxa3-derived iHepSCs induces a Notch signaling-mediated secondary conversion into cholangiocyte progenitor-like cells that display dramatically enhanced differentiation capacity into mature cholangiocytes. Our study provides a robust two-step approach for obtaining cholangiocyte progenitor-like cells using defined factors.
Assuntos
Sistema Biliar/citologia , Diferenciação Celular , Fibroblastos/citologia , Células-Tronco/citologia , Animais , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Fibroblastos/metabolismo , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Fator 3-gama Nuclear de Hepatócito/metabolismo , Hepatócitos/citologia , Hepatócitos/metabolismo , Fígado/citologia , Camundongos Endogâmicos C57BL , Receptores Notch/metabolismo , Transdução de Sinais , Células-Tronco/metabolismo , Transcrição GênicaRESUMO
The reconstruction of transcriptional regulatory networks (TRNs) is a long-standing challenge in human genetics. Numerous computational methods have been developed to infer regulatory interactions between human transcriptional factors (TFs) and target genes from high-throughput data, and their performance evaluation requires gold-standard interactions. Here we present a database of literature-curated human TF-target interactions, TRRUST (transcriptional regulatory relationships unravelled by sentence-based text-mining, http://www.grnpedia.org/trrust), which currently contains 8,015 interactions between 748 TF genes and 1,975 non-TF genes. A sentence-based text-mining approach was employed for efficient manual curation of regulatory interactions from approximately 20 million Medline abstracts. To the best of our knowledge, TRRUST is the largest publicly available database of literature-curated human TF-target interactions to date. TRRUST also has several useful features: i) information about the mode-of-regulation; ii) tests for target modularity of a query TF; iii) tests for TF cooperativity of a query target; iv) inferences about cooperating TFs of a query TF; and v) prioritizing associated pathways and diseases with a query TF. We observed high enrichment of TF-target pairs in TRRUST for top-scored interactions inferred from high-throughput data, which suggests that TRRUST provides a reliable benchmark for the computational reconstruction of human TRNs.