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1.
Am J Hum Genet ; 108(2): 337-345, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33434492

RESUMO

Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) is associated with congenital absence of the uterus, cervix, and the upper part of the vagina; it is a sex-limited trait. Disrupted development of the Müllerian ducts (MD)/Wölffian ducts (WD) through multifactorial mechanisms has been proposed to underlie MRKHS. In this study, exome sequencing (ES) was performed on a Chinese discovery cohort (442 affected subjects and 941 female control subjects) and a replication MRKHS cohort (150 affected subjects of mixed ethnicity from North America, South America, and Europe). Phenotypic follow-up of the female reproductive system was performed on an additional cohort of PAX8-associated congenital hypothyroidism (CH) (n = 5, Chinese). By analyzing 19 candidate genes essential for MD/WD development, we identified 12 likely gene-disrupting (LGD) variants in 7 genes: PAX8 (n = 4), BMP4 (n = 2), BMP7 (n = 2), TBX6 (n = 1), HOXA10 (n = 1), EMX2 (n = 1), and WNT9B (n = 1), while LGD variants in these genes were not detected in control samples (p = 1.27E-06). Interestingly, a sex-limited penetrance with paternal inheritance was observed in multiple families. One additional PAX8 LGD variant from the replication cohort and two missense variants from both cohorts were revealed to cause loss-of-function of the protein. From the PAX8-associated CH cohort, we identified one individual presenting a syndromic condition characterized by CH and MRKHS (CH-MRKHS). Our study demonstrates the comprehensive utilization of knowledge from developmental biology toward elucidating genetic perturbations, i.e., rare pathogenic alleles involving the same loci, contributing to human birth defects.


Assuntos
Transtornos 46, XX do Desenvolvimento Sexual/genética , Anormalidades Congênitas/genética , Ductos Paramesonéfricos/anormalidades , Ductos Paramesonéfricos/crescimento & desenvolvimento , Mutação , Ductos Mesonéfricos/crescimento & desenvolvimento , Adulto , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 7/genética , Códon sem Sentido , Feminino , Estudos de Associação Genética , Pleiotropia Genética , Proteínas Homeobox A10/genética , Proteínas de Homeodomínio/genética , Humanos , Fator de Transcrição PAX8/genética , Herança Paterna , Penetrância , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Proteínas Wnt/genética , Ductos Mesonéfricos/anormalidades
3.
Curr Opin Genet Dev ; 66: 10-19, 2020 Dec 28.
Artigo em Inglês | MEDLINE | ID: mdl-33383480

RESUMO

Allele-specific gene expression can influence disease traits. Non-coding germline genetic variants that alter regulatory elements can cause allele-specific gene expression and contribute to cancer susceptibility. In tumors, both somatic copy number alterations and somatic single nucleotide variants have been shown to lead to allele-specific expression of genes, many of which are considered drivers of tumor growth. Here, we review recent studies revealing the pervasive presence of this phenomenon in cancer susceptibility and progression. Furthermore, we underscore the importance of careful experimental design and computational analysis for accurate allelic expression quantification and avoidance of false positives. Finally, we discuss additional methodological challenges encountered in cancer studies and in the burgeoning field of single-cell transcriptomics.

4.
Bioinformatics ; 36(Supplement_2): i919-i927, 2020 Dec 30.
Artigo em Inglês | MEDLINE | ID: mdl-33381818

RESUMO

MOTIVATION: Recent technological advances have led to an increase in the production and availability of single-cell data. The ability to integrate a set of multi-technology measurements would allow the identification of biologically or clinically meaningful observations through the unification of the perspectives afforded by each technology. In most cases, however, profiling technologies consume the used cells and thus pairwise correspondences between datasets are lost. Due to the sheer size single-cell datasets can acquire, scalable algorithms that are able to universally match single-cell measurements carried out in one cell to its corresponding sibling in another technology are needed. RESULTS: We propose Single-Cell data Integration via Matching (SCIM), a scalable approach to recover such correspondences in two or more technologies. SCIM assumes that cells share a common (low-dimensional) underlying structure and that the underlying cell distribution is approximately constant across technologies. It constructs a technology-invariant latent space using an autoencoder framework with an adversarial objective. Multi-modal datasets are integrated by pairing cells across technologies using a bipartite matching scheme that operates on the low-dimensional latent representations. We evaluate SCIM on a simulated cellular branching process and show that the cell-to-cell matches derived by SCIM reflect the same pseudotime on the simulated dataset. Moreover, we apply our method to two real-world scenarios, a melanoma tumor sample and a human bone marrow sample, where we pair cells from a scRNA dataset to their sibling cells in a CyTOF dataset achieving 90% and 78% cell-matching accuracy for each one of the samples, respectively. AVAILABILITY AND IMPLEMENTATION: https://github.com/ratschlab/scim. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

7.
Stem Cells Int ; 2019: 3864948, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31065273

RESUMO

Mesenchymal stem cells (MSC) constitute an important cell population of the bone marrow hematopoietic niche that supports normally hematopoietic stem cells (HSC) but eventually also leukemic cells. The alterations that occur in the MSC under leukemic stress are not well known. To deepen on this topic, we have used an in vitro model of the leukemic niche (LN) by coculturing MSC with an acute lymphocytic leukemia cell line (REH) and proceeded to evaluate MSC characteristics and functions. We found that leukemic cells induced in MSC a significant increase both in senescence-associated ß-galactosidase activity and in p53 gene expression. MSC in the LN also showed a persistent production of cytoplasmic reactive oxygen species (ROS) and a G2/M phase arrest of the cell cycle. Another acute leukemic cell line (SUP-B15) produced almost the same effects on MSC. REH cells adhere strongly to MSC possibly as a result of an increased expression of the adhesion molecules VCAM-1, ICAM-1, and CD49e in MSC and of CD49d in REH cells. Although mesensphere formation was normal or even increased, multipotent differentiation capacity was impaired in MSC from the LN. A REH-conditioned medium was only partially (about 50%) capable of inducing the same changes in MSC, suggesting that cell-to-cell contact is more efficient in inducing these changes. Despite these important effects on MSC in the LN, REH cells increased their cell adhesion, proliferation rate, and directed-migration capacity. In conclusion, in this in vitro LN model, leukemic cells affect importantly the MSC, inducing a senescence process that seems to favour leukemic cell growth.

8.
N Engl J Med ; 378(3): 250-261, 2018 01 18.
Artigo em Inglês | MEDLINE | ID: mdl-29298116

RESUMO

BACKGROUND: Sporadic arteriovenous malformations of the brain, which are morphologically abnormal connections between arteries and veins in the brain vasculature, are a leading cause of hemorrhagic stroke in young adults and children. The genetic cause of this rare focal disorder is unknown. METHODS: We analyzed tissue and blood samples from patients with arteriovenous malformations of the brain to detect somatic mutations. We performed exome DNA sequencing of tissue samples of arteriovenous malformations of the brain from 26 patients in the main study group and of paired blood samples from 17 of those patients. To confirm our findings, we performed droplet digital polymerase-chain-reaction (PCR) analysis of tissue samples from 39 patients in the main study group (21 with matching blood samples) and from 33 patients in an independent validation group. We interrogated the downstream signaling pathways, changes in gene expression, and cellular phenotype that were induced by activating KRAS mutations, which we had discovered in tissue samples. RESULTS: We detected somatic activating KRAS mutations in tissue samples from 45 of the 72 patients and in none of the 21 paired blood samples. In endothelial cell-enriched cultures derived from arteriovenous malformations of the brain, we detected KRAS mutations and observed that expression of mutant KRAS (KRASG12V) in endothelial cells in vitro induced increased ERK (extracellular signal-regulated kinase) activity, increased expression of genes related to angiogenesis and Notch signaling, and enhanced migratory behavior. These processes were reversed by inhibition of MAPK (mitogen-activated protein kinase)-ERK signaling. CONCLUSIONS: We identified activating KRAS mutations in the majority of tissue samples of arteriovenous malformations of the brain that we analyzed. We propose that these malformations develop as a result of KRAS-induced activation of the MAPK-ERK signaling pathway in brain endothelial cells. (Funded by the Swiss Cancer League and others.).


Assuntos
Malformações Arteriovenosas Intracranianas/genética , Mutação , Proteínas Proto-Oncogênicas p21(ras)/genética , Adulto , Células Cultivadas , Análise Mutacional de DNA , Exoma , Expressão Gênica , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Malformações Arteriovenosas Intracranianas/etiologia , Malformações Arteriovenosas Intracranianas/patologia , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Fosforilação , Proteínas Proto-Oncogênicas p21(ras)/metabolismo
9.
Int J Mol Sci ; 18(2)2017 Feb 14.
Artigo em Inglês | MEDLINE | ID: mdl-28216566

RESUMO

An understanding of the cell interactions occurring in the leukemic microenvironment and their functional consequences for the different cell players has therapeutic relevance. By co-culturing mesenchymal stem cells (MSC) with the REH acute lymphocytic leukemia (ALL) cell line, we have established an in vitro leukemic niche for the functional evaluation of hematopoietic stem/progenitor cells (HSPC, CD34+ cells). We showed that the normal homeostatic control exerted by the MSC over the HSPC is considerably lost in this leukemic microenvironment: HSPC increased their proliferation rate and adhesion to MSC. The adhesion molecules CD54 and CD44 were consequently upregulated in HSPC from the leukemic niche. Consequently, with this adhesive phenotype, HSPC showed less Stromal derived factor-1 (SDF-1)-directed migration. Interestingly, multipotency was severely affected with an important reduction in the absolute count and the percentage of primitive progenitor colonies. It was possible to simulate most of these HSPC alterations by incubation of MSC with a REH-conditioned medium, suggesting that REH soluble factors and their effect on MSC are important for the observed changes. Of note, these HSPC alterations were reproduced when primary leukemic cells from an ALL type B (ALL-B) patient were used to set up the leukemic niche. These results suggest that a general response is induced in the leukemic niche to the detriment of HSPC function and in favor of leukemic cell support. This in vitro leukemic niche could be a valuable tool for the understanding of the molecular events responsible for HSPC functional failure and a useful scenario for therapeutic evaluation.


Assuntos
Células-Tronco Hematopoéticas/metabolismo , Leucemia/metabolismo , Leucemia/patologia , Fenótipo , Microambiente Tumoral , Antígenos CD34/metabolismo , Biomarcadores , Adesão Celular , Comunicação Celular , Diferenciação Celular , Movimento Celular , Proliferação de Células , Técnicas de Cocultura , Citocinas/metabolismo , Células-Tronco Hematopoéticas/citologia , Humanos , Linfonodos/metabolismo , Células-Tronco Mesenquimais , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Nicho de Células-Tronco
10.
Plant Dis ; 101(6): 916-923, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30682941

RESUMO

Rhizoctonia foliar blight, caused by Rhizoctonia solani, is an important disease of Brachiaria spp. in tropical America. Host-plant resistance is an attractive option for disease management. In this study, we evaluated three inoculum types (mycelium-infected agar disc, microdiscs suspensions, and microencapsulated-mycelium suspensions) in order to identify a rapid and accurate method for mass screening of Brachiaria genotypes for resistance to Rhizoctonia spp. in greenhouse trials. Visual damage score, area under the disease progress curve, and percent chlorophyll loss were estimated to determine the most accurate and precise method for evaluating Rhizoctonia resistance. The microencapsulated-mycelium solution (0.75 g/ml in potato dextrose broth sprayed on plants 30 days after planting) caused greater foliar damage than the other inoculum types and allowed effective discrimination between resistant and susceptible genotypes. The effectiveness of spray-applied, microencapsulated-mycelium was further corroborated by the evaluation of 350 genotypes not previously selected for resistance to Rhizoctonia spp., which varied significantly in their reaction to R. solani. The microencapsulated-mycelium methodology has several advantages over existing methods, including its high-throughput capacity, efficient use of time and space, ease of quantification of inoculum, and consistent results over replicate trials. This methodology could be applied to assess resistance to Rhizoctonia spp. in other crops.

11.
Nat Genet ; 48(4): 398-406, 2016 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-26950094

RESUMO

Basal cell carcinoma (BCC) of the skin is the most common malignant neoplasm in humans. BCC is primarily driven by the Sonic Hedgehog (Hh) pathway. However, its phenotypic variation remains unexplained. Our genetic profiling of 293 BCCs found the highest mutation rate in cancer (65 mutations/Mb). Eighty-five percent of the BCCs harbored mutations in Hh pathway genes (PTCH1, 73% or SMO, 20% (P = 6.6 × 10(-8)) and SUFU, 8%) and in TP53 (61%). However, 85% of the BCCs also harbored additional driver mutations in other cancer-related genes. We observed recurrent mutations in MYCN (30%), PPP6C (15%), STK19 (10%), LATS1 (8%), ERBB2 (4%), PIK3CA (2%), and NRAS, KRAS or HRAS (2%), and loss-of-function and deleterious missense mutations were present in PTPN14 (23%), RB1 (8%) and FBXW7 (5%). Consistent with the mutational profiles, N-Myc and Hippo-YAP pathway target genes were upregulated. Functional analysis of the mutations in MYCN, PTPN14 and LATS1 suggested their potential relevance in BCC tumorigenesis.


Assuntos
Carcinoma Basocelular/genética , Transdução de Sinais/efeitos da radiação , Neoplasias Cutâneas/genética , Anilidas/uso terapêutico , Antineoplásicos/uso terapêutico , Carcinogênese/genética , Carcinoma Basocelular/tratamento farmacológico , Carcinoma Basocelular/patologia , Análise Mutacional de DNA , Progressão da Doença , Exoma , Estudos de Associação Genética , Predisposição Genética para Doença , Células HEK293 , Humanos , Mutação , Piridinas/uso terapêutico , Neoplasias Cutâneas/tratamento farmacológico , Neoplasias Cutâneas/patologia , Transcriptoma
13.
PLoS One ; 10(8): e0135555, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-26317209

RESUMO

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.


Assuntos
Metilação de DNA , Síndrome de Down/genética , Gêmeos Monozigóticos , Ilhas de CpG , Síndrome de Down/metabolismo , Epigênese Genética , Fibroblastos , Regulação da Expressão Gênica , Biblioteca Gênica , Histonas/metabolismo , Humanos , Fenótipo , Regiões Promotoras Genéticas
14.
Oncotarget ; 6(16): 14596-613, 2015 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-25999349

RESUMO

Ewing's sarcoma (ES) is the second most common bone cancer in children and young people. Edelfosine (1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine) is the prototype of a family of synthetic antitumor compounds, collectively known as alkylphospholipid analogs (APLs). We have found that APLs ranked edelfosine>perifosine>erucylphosphocholine>miltefosine for their capacity to promote apoptosis in ES cells. Edelfosine accumulated in the endoplasmic reticulum (ER) and triggered an ER stress response that eventually led to caspase-dependent apoptosis in ES cells. This apoptotic response involved mitochondrial-mediated processes, with cytochrome c release, caspase-9 activation and generation of reactive oxygen species. Edelfosine-induced apoptosis was also dependent on sustained c-Jun NH2-terminal kinase activation. Oral administration of edelfosine showed a potent in vivo antitumor activity in an ES xenograft animal model. Histochemical staining gave evidence for ER stress response and apoptosis in the ES tumors isolated from edelfosine-treated mice. Edelfosine showed a preferential action on ES tumor cells as compared to non-transformed osteoblasts, and appeared to be well suited for combination therapy regimens. These results demonstrate in vitro and in vivo antitumor activity of edelfosine against ES cells that is mediated by caspase activation and ER stress, and provide the proof of concept for a putative edelfosine- and ER stress-mediated approach forES treatment.


Assuntos
Antineoplásicos/uso terapêutico , Neoplasias Ósseas/tratamento farmacológico , Retículo Endoplasmático/metabolismo , Éteres Fosfolipídicos/uso terapêutico , Sarcoma de Ewing/tratamento farmacológico , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose , Humanos , Camundongos , Camundongos SCID , Éteres Fosfolipídicos/administração & dosagem , Éteres Fosfolipídicos/farmacologia , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Nature ; 508(7496): 345-50, 2014 Apr 17.
Artigo em Inglês | MEDLINE | ID: mdl-24740065

RESUMO

Trisomy 21 is the most frequent genetic cause of cognitive impairment. To assess the perturbations of gene expression in trisomy 21, and to eliminate the noise of genomic variability, we studied the transcriptome of fetal fibroblasts from a pair of monozygotic twins discordant for trisomy 21. Here we show that the differential expression between the twins is organized in domains along all chromosomes that are either upregulated or downregulated. These gene expression dysregulation domains (GEDDs) can be defined by the expression level of their gene content, and are well conserved in induced pluripotent stem cells derived from the twins' fibroblasts. Comparison of the transcriptome of the Ts65Dn mouse model of Down's syndrome and normal littermate mouse fibroblasts also showed GEDDs along the mouse chromosomes that were syntenic in human. The GEDDs correlate with the lamina-associated (LADs) and replication domains of mammalian cells. The overall position of LADs was not altered in trisomic cells; however, the H3K4me3 profile of the trisomic fibroblasts was modified and accurately followed the GEDD pattern. These results indicate that the nuclear compartments of trisomic cells undergo modifications of the chromatin environment influencing the overall transcriptome, and that GEDDs may therefore contribute to some trisomy 21 phenotypes.


Assuntos
Síndrome de Down/genética , Regulação da Expressão Gênica/genética , Genoma/genética , Transcriptoma/genética , Animais , Células Cultivadas , Cromatina/química , Cromatina/metabolismo , Cromossomos Humanos Par 21/genética , Cromossomos de Mamíferos/genética , Período de Replicação do DNA , Síndrome de Down/patologia , Feminino , Feto/citologia , Fibroblastos , Histonas/química , Histonas/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lisina/metabolismo , Masculino , Metilação , Camundongos , Gêmeos Monozigóticos/genética
16.
Plant Dis ; 98(3): 306-310, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30708412

RESUMO

Rhizoctonia foliar blight, caused by Rhizoctonia solani anastomosis group 1, is an economically important fungal disease found throughout the world. The fungus attacks numerous crops, including cereals, roots and tubers, legumes, and cruciferous, horticultural, and ornamental plants. In tropical America, this invasive and destructive disease also attacks most Brachiaria spp. used as forages in the ranching industry, especially in the production of cattle. Research to solve this constraint has been ongoing at the International Center for Tropical Agriculture and has generated new Brachiaria hybrids with excellent agronomic performance, tolerance to poor soils, and, particularly, high resistance to biotic factors such as Rhizoctonia foliar blight. These hybrids belong to lines obtained from Brachiaria humidicola, B. brizantha, and B. decumbens. To identify resistance among Brachiaria hybrid genotypes, the hybrid clones were evaluated for their variability in resistance, and their disease reaction was also determined and characterized. Results led to the identification of hybrids that not only were highly resistant to the blight but also had excellent agronomic characteristics.

17.
Plant Dis ; 97(6): 772-779, 2013 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30722606

RESUMO

Up to 50% of Brachiaria production in the tropics is affected by foliar blight caused by Rhizoctonia spp. Monothallic isolates of Rhizoctonia (n = 147) were cultured from different Brachiaria genotypes in Colombia and morphologically characterized and evaluated in pathogenicity trials in the greenhouse. Based on restriction fragment length polymorphism of the internal transcribed spacer region, 101 of the isolates were identified as Rhizoctonia solani anastomosis group (AG)-1 IA and were multinucleated, with high growth rate, brown mycelium, and high virulence; and 46 isolates were identified as Rhizoctonia sp. AG-D and were binucleated, with low growth rate, white mycelium, and lower virulence on the Brachiaria genotypes tested. The Rhizoctonia isolates also showed variation according to geographic origin, with R. solani AG-1 IA prevalent in warm lowland areas and Rhizoctonia sp. AG-D occurring in cooler areas. Ten Brachiaria genotypes were challenged with different Rhizoctonia isolates and resistant reactions were seen in three of these genotypes, including Brachiaria hybrid (International Center for Tropical Agriculture [CIAT] 36062), Brachiaria brizantha 'Marandú' (CIAT 6294), and Brachiaria hybrid 'Mulato II' (CIAT 36087). These results will contribute to a greater understanding of the interaction of diverse Rhizoctonia isolates on different Brachiaria genotypes, supporting improvement of Brachiaria spp. for disease resistance.

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