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1.
Cell ; 156(1-2): 343-58, 2014 Jan 16.
Artigo em Inglês | MEDLINE | ID: mdl-24439387

RESUMO

Genome-wide association studies have revealed numerous risk loci associated with diverse diseases. However, identification of disease-causing variants within association loci remains a major challenge. Divergence in gene expression due to cis-regulatory variants in noncoding regions is central to disease susceptibility. We show that integrative computational analysis of phylogenetic conservation with a complexity assessment of co-occurring transcription factor binding sites (TFBS) can identify cis-regulatory variants and elucidate their mechanistic role in disease. Analysis of established type 2 diabetes risk loci revealed a striking clustering of distinct homeobox TFBS. We identified the PRRX1 homeobox factor as a repressor of PPARG2 expression in adipose cells and demonstrate its adverse effect on lipid metabolism and systemic insulin sensitivity, dependent on the rs4684847 risk allele that triggers PRRX1 binding. Thus, cross-species conservation analysis at the level of co-occurring TFBS provides a valuable contribution to the translation of genetic association signals to disease-related molecular mechanisms.


Assuntos
Diabetes Mellitus Tipo 2/genética , Polimorfismo de Nucleotídeo Único , Animais , Linhagem Celular , Células Cultivadas , Sequência Conservada , Regulação da Expressão Gênica , Estudo de Associação Genômica Ampla , Proteínas de Homeodomínio/metabolismo , Humanos , Resistência à Insulina , PPAR gama/genética , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
2.
BMC Bioinformatics ; 21(1): 169, 2020 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-32357829

RESUMO

BACKGROUND: Analysing whole genome bisulfite sequencing datasets is a data-intensive task that requires comprehensive and reproducible workflows to generate valid results. While many algorithms have been developed for tasks such as alignment, comprehensive end-to-end pipelines are still sparse. Furthermore, previous pipelines lack features or show technical deficiencies, thus impeding analyses. RESULTS: We developed wg-blimp (whole genome bisulfite sequencing methylation analysis pipeline) as an end-to-end pipeline to ease whole genome bisulfite sequencing data analysis. It integrates established algorithms for alignment, quality control, methylation calling, detection of differentially methylated regions, and methylome segmentation, requiring only a reference genome and raw sequencing data as input. Comparing wg-blimp to previous end-to-end pipelines reveals similar setups for common sequence processing tasks, but shows differences for post-alignment analyses. We improve on previous pipelines by providing a more comprehensive analysis workflow as well as an interactive user interface. To demonstrate wg-blimp's ability to produce correct results we used it to call differentially methylated regions for two publicly available datasets. We were able to replicate 112 of 114 previously published regions, and found results to be consistent with previous findings. We further applied wg-blimp to a publicly available sample of embryonic stem cells to showcase methylome segmentation. As expected, unmethylated regions were in close proximity of transcription start sites. Segmentation results were consistent with previous analyses, despite different reference genomes and sequencing techniques. CONCLUSIONS: wg-blimp provides a comprehensive analysis pipeline for whole genome bisulfite sequencing data as well as a user interface for simplified result inspection. We demonstrated its applicability by analysing multiple publicly available datasets. Thus, wg-blimp is a relevant alternative to previous analysis pipelines and may facilitate future epigenetic research.


Assuntos
Análise de Sequência de DNA , Software , Sulfitos/química , Sequenciamento Completo do Genoma , Metilação de DNA , Bases de Dados Genéticas , Humanos , Interface Usuário-Computador
3.
Cytogenet Genome Res ; 159(1): 1-11, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31658463

RESUMO

The switch/sucrose non-fermenting (SWI/SNF) complex is an ATP-dependent chromatin remodeller that regulates the spacing of nucleosomes and thereby controls gene expression. Heterozygous mutations in genes encoding subunits of the SWI/SNF complex have been reported in individuals with Coffin-Siris syndrome (CSS), with the majority of the mutations in ARID1B. CSS is a rare congenital disorder characterized by facial dysmorphisms, digital anomalies, and variable intellectual disability. We hypothesized that mutations in genes encoding subunits of the ubiquitously expressed SWI/SNF complex may lead to alterations of the nucleosome profiles in different cell types. We performed the first study on CSS-patient samples and investigated the nucleosome landscapes of cell-free DNA (cfDNA) isolated from blood plasma by whole-genome sequencing. In addition, we studied the nucleosome landscapes of CD14+ monocytes from CSS-affected individuals by nucleosome occupancy and methylome-sequencing (NOMe-seq) as well as their expression profiles. In cfDNA of CSS-affected individuals with heterozygous ARID1B mutations, we did not observe major changes in the nucleosome profile around transcription start sites. In CD14+ monocytes, we found few genomic regions with different nucleosome occupancy when compared to controls. RNA-seq analysis of CD14+ monocytes of these individuals detected only few differentially expressed genes, which were not in proximity to any of the identified differential nucleosome-depleted regions. In conclusion, we show that heterozygous mutations in the human SWI/SNF subunit ARID1B do not have a major impact on the nucleosome landscape or gene expression in blood cells. This might be due to functional redundancy, cell-type specificity, or alternative functions of ARID1B.


Assuntos
Anormalidades Múltiplas/genética , Proteínas de Ligação a DNA/genética , Face/anormalidades , Deformidades Congênitas da Mão/genética , Deficiência Intelectual/genética , Micrognatismo/genética , Pescoço/anormalidades , Proteínas Nucleares/genética , Nucleossomos/genética , Fatores de Transcrição/genética , Adolescente , Ácidos Nucleicos Livres/sangue , Ácidos Nucleicos Livres/genética , Criança , Pré-Escolar , Feminino , Genoma Humano/genética , Estudo de Associação Genômica Ampla , Humanos , Masculino , Monócitos/citologia , Adulto Jovem
4.
Cytogenet Genome Res ; 156(1): 9-13, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30016768

RESUMO

Angelman syndrome (AS) is a neurodevelopmental disorder caused by deletion of the maternally inherited 15q11q13 region, paternal uniparental disomy 15 [upd(15)pat], an imprinting defect of the maternal chromosome region 15q11q13, or a pathogenic mutation of the maternal UBE3A allele. Predisposing factors for upd(15)pat, such as nonhomologous robertsonian translocation involving chromosome 15, have been discussed, but no evidence for this predisposition has been published. In the present study, chromosomal analysis was performed in a child with AS, both parents, and the maternal grandparents. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) was employed on DNA of the index individual, and microsatellite analysis was carried out on DNA of the index individual and his parents. The cytogenetic analysis showed that the mother and maternal grandfather are carriers of a rob(14;15). The index individual has a numerically normal karyotype, but MS-MLPA and microsatellite analyses confirmed the clinical diagnosis of AS and revealed a pattern highly suggestive of isodisomic upd(15)pat. This is the first report of an AS-affected individual with isodisomic upd(15)pat and a numerically normal karyotype that most likely results from a rob(14;15)-associated meiotic error in the maternal germline followed by monosomy 15 rescue in the early embryo.

5.
Orv Hetil ; 159(2): 64-69, 2018 Jan.
Artigo em Húngaro | MEDLINE | ID: mdl-29307221

RESUMO

INTRODUCTION: According to the international literature, DNA methylation analysis of the promoter region of SNRPN locus is the most efficient way to start genetic investigation in patients with suspected Prader-Willi syndrome. AIM: Our aim was to develop a simple, reliable first-tier diagnosis to confirm Prader-Willi syndrome, therefore to compare our self-designed simple, cost-efficient high-resolution melting analysis and the most commonly used methylation-specific multiplex ligation-dependent probe amplification to confirm Prader-Willi syndrome. METHOD: We studied 17 clinically suspected Prader-Willi syndrome children and their DNA samples. With self-designed primers, bisulfite-sensitive polymerase chain reaction, high-resolution melting analysis and, as a control, methylation-specific multiplex ligation-dependent probe amplification were performed. RESULTS: Prader-Willi syndrome was genetically confirmed in 6 out of 17 clinically suspected Prader-Willi syndrome patients. The results of high-resolution melting analysis and methylation-specific multiplex ligation-dependent probe amplification were equivalent in each case. CONCLUSION: Using our self-designed primers and altered bisulfite-specific PCR conditions, high-resolution melting analysis appears to be a simple, fast, reliable and effective method for primarily proving or excluding clinically suspected Prade-Willi syndrome cases. Orv Hetil. 2018; 159(2): 64-69.


Assuntos
Técnicas de Amplificação de Ácido Nucleico/métodos , Reação em Cadeia da Polimerase/métodos , Síndrome de Prader-Willi/diagnóstico , Criança , Pré-Escolar , Cromossomos Humanos Par 15/genética , Feminino , Genótipo , Humanos , Masculino , Síndrome de Prader-Willi/genética
6.
Hum Mol Genet ; 24(5): 1295-304, 2015 Mar 01.
Artigo em Inglês | MEDLINE | ID: mdl-25336341

RESUMO

Imprinted genes are expressed either from the paternal or the maternal allele, because the other allele has been silenced in the mother's or father's germline. Imprints are characterized by DNA methylation at cytosine phosphate guanine sites. Recently, abnormal sperm parameters and male infertility have been linked to aberrant methylation patterns of imprinted genes in sperm DNA. However, these studies did not account for possible epigenetic heterogeneity in sperm. We have investigated whether spermatozoa are a homogeneous cell population regarding DNA methylation of imprinted genes. Swim-up sperm was obtained from 45 men with normal (n = 19) and abnormal (n = 26) sperm parameters. DNA methylation of the imprinted gene KCNQ1OT1 was measured in multiple pools of 10 spermatozoa by a highly sensitive pyrosequencing-based oligo-sperm methylation assay (OSMA). DNA methylation of four imprinted genes (KCNQ1OT1, MEST, H19 and MEG3) was further analysed by deep bisulfite sequencing, which allows analysis at the single-cell level. Using OSMA, we found a significantly increased variation in the DNA methylation values of the maternally methylated gene KCNQ1OT1 in samples with abnormal sperm parameters. DBS showed that normozoospermic samples had a homogenous pattern of DNA methylation, whereas oligoasthenozoospermic samples contained discrete populations of spermatozoa with either normal or abnormal methylation patterns. Aberrant methylation of H19 appears to occur preferentially on the maternally inherited allele. Our results demonstrate the presence of epigenetic mosaicism in the semen of oligoasthenozoospermic men, which probably results from errors in imprint erasure.


Assuntos
Epigênese Genética , Mutação em Linhagem Germinativa , Infertilidade Masculina/genética , Mosaicismo , Espermatozoides/patologia , Adulto , Alelos , Citosina/metabolismo , Metilação de DNA , Epigenômica , Impressão Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genética , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Proteínas/genética , Proteínas/metabolismo , Sulfitos/metabolismo
7.
Am J Hum Genet ; 95(6): 698-707, 2014 Dec 04.
Artigo em Inglês | MEDLINE | ID: mdl-25434003

RESUMO

Mutations in components of the major spliceosome have been described in disorders with craniofacial anomalies, e.g., Nager syndrome and mandibulofacial dysostosis type Guion-Almeida. The U5 spliceosomal complex of eight highly conserved proteins is critical for pre-mRNA splicing. We identified biallelic mutations in TXNL4A, a member of this complex, in individuals with Burn-McKeown syndrome (BMKS). This rare condition is characterized by bilateral choanal atresia, hearing loss, cleft lip and/or palate, and other craniofacial dysmorphisms. Mutations were found in 9 of 11 affected families. In 8 families, affected individuals carried a rare loss-of-function mutation (nonsense, frameshift, or microdeletion) on one allele and a low-frequency 34 bp deletion (allele frequency 0.76%) in the core promoter region on the other allele. In a single highly consanguineous family, formerly diagnosed as oculo-oto-facial dysplasia, the four affected individuals were homozygous for a 34 bp promoter deletion, which differed from the promoter deletion in the other families. Reporter gene and in vivo assays showed that the promoter deletions led to reduced expression of TXNL4A. Depletion of TXNL4A (Dib1) in yeast demonstrated reduced assembly of the tri-snRNP complex. Our results indicate that BMKS is an autosomal-recessive condition, which is frequently caused by compound heterozygosity of low-frequency promoter deletions in combination with very rare loss-of-function mutations.


Assuntos
Atresia das Cóanas/genética , Surdez/congênito , Deleção de Genes , Cardiopatias Congênitas/genética , Regiões Promotoras Genéticas/genética , Ribonucleoproteína Nuclear Pequena U5/genética , Spliceossomos/genética , Alelos , Pré-Escolar , Atresia das Cóanas/diagnóstico , Surdez/diagnóstico , Surdez/genética , Exossomos/genética , Fácies , Feminino , Perfilação da Expressão Gênica , Frequência do Gene , Genes Reporter , Cardiopatias Congênitas/diagnóstico , Heterozigoto , Homozigoto , Humanos , Masculino , Mutação , Análise de Sequência com Séries de Oligonucleotídeos , Linhagem , Fenótipo , Ribonucleoproteína Nuclear Pequena U5/metabolismo , Análise de Sequência de DNA , Spliceossomos/metabolismo
8.
Hum Mol Genet ; 22(3): 544-57, 2013 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-23118352

RESUMO

At chromosome 11p15.5, the imprinting centre 1 (IC1) controls the parent of origin-specific expression of the IGF2 and H19 genes. The 5 kb IC1 region contains multiple target sites (CTS) for the zinc-finger protein CTCF, whose binding on the maternal chromosome prevents the activation of IGF2 and allows that of H19 by common enhancers. CTCF binding helps maintaining the maternal IC1 methylation-free, whereas on the paternal chromosome gamete-inherited DNA methylation inhibits CTCF interaction and enhancer-blocking activity resulting in IGF2 activation and H19 silencing. Maternally inherited 1.4-2.2 kb deletions are associated with methylation of the residual CTSs and Beckwith-Wiedemann syndrome, although with different penetrance and expressivity. We explored the relationship between IC1 microdeletions and phenotype by analysing a number of previously described and novel mutant alleles. We used a highly quantitative assay based on next generation sequencing to measure DNA methylation in affected families and analysed enhancer-blocking activity and CTCF binding in cultured cells. We demonstrate that the microdeletions mostly affect IC1 function and CTCF binding by changing CTS spacing. Thus, the extent of IC1 inactivation and the clinical phenotype are influenced by the arrangement of the residual CTSs. A CTS spacing similar to the wild-type allele results in moderate IC1 inactivation and is associated with stochastic DNA methylation of the maternal IC1 and incomplete penetrance. Microdeletions with different CTS spacing display severe IC1 inactivation and are associated with IC1 hypermethylation and complete penetrance. Careful characterization of the IC1 microdeletions is therefore needed to predict recurrence risks and phenotypical outcomes.


Assuntos
Deleção de Genes , Impressão Genômica , Fator de Crescimento Insulin-Like II/genética , Fenótipo , RNA Longo não Codificante/genética , Proteínas Repressoras/genética , Alelos , Sítios de Ligação/genética , Fator de Ligação a CCCTC , Células Cultivadas , Imunoprecipitação da Cromatina , Cromossomos Humanos Par 11/genética , Metilação de DNA , Regulação da Expressão Gênica , Inativação Gênica , Loci Gênicos , Humanos , Fator de Crescimento Insulin-Like II/metabolismo , Linhagem , RNA Longo não Codificante/metabolismo , Proteínas Repressoras/metabolismo , Análise de Sequência de DNA
9.
J Pathol ; 232(5): 485-7, 2014 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-24395592

RESUMO

Genomic imprinting is an epigenetic process by which the male and the female germline confer different DNA methylation marks and histone modifications onto specific gene regions, so that one allele of an imprinted gene is active and the other one is silent. Since the dosage of imprinted genes is important for normal development, growth and behaviour, the loss or duplication of the active allele can cause disease.


Assuntos
Doenças Genéticas Inatas/genética , Impressão Genômica , Animais , Montagem e Desmontagem da Cromatina , Deleção Cromossômica , Duplicação Cromossômica , Metilação de DNA , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Inativação Gênica , Predisposição Genética para Doença , Humanos , Masculino , Mutação , Fatores de Risco
10.
J Med Genet ; 51(6): 407-12, 2014 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-24721835

RESUMO

BACKGROUND: In a subset of imprinting disorders caused by epimutations, multiple imprinted loci are affected. Familial occurrence of multilocus imprinting disorders is rare. PURPOSE/OBJECTIVE: We have investigated the clinical and molecular features of a familial DNA-methylation disorder. METHODS: Tissues of affected individuals and blood samples of family members were investigated by conventional and molecular karyotyping. Sanger sequencing and RT-PCR of imprinting-associated genes (NLRP2, NLRP7, ZFP57, KHDC3L, DNMT1o), exome sequencing and locus-specific, array-based and genome-wide technologies to determine DNA-methylation were performed. RESULTS: In three offspring of a healthy couple, we observed prenatal onset of severe growth retardation and dysmorphism associated with altered DNA-methylation at paternally and maternally imprinted loci. Array-based analyses in various tissues of the offspring identified the DNA-methylation of 2.1% of the genes in the genome to be recurrently altered. Despite significant enrichment of imprinted genes (OR 9.49), altered DNA-methylation predominately (90.2%) affected genes not known to be imprinted. Sequencing of genes known to cause comparable conditions and exome sequencing in affected individuals and their ancestors did not unambiguously point to a causative gene. CONCLUSIONS: The family presented herein suggests the existence of a familial disorder of DNA-methylation affecting imprinted but also not imprinted gene loci potentially caused by a maternal effect mutation in a hitherto not identified gene.


Assuntos
Metilação de DNA/genética , Doenças Genéticas Inatas/genética , Alelos , Análise Mutacional de DNA , Epigenômica , Feminino , Humanos , Recém-Nascido , Masculino , Linhagem
11.
PLoS Genet ; 8(3): e1002543, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22438814

RESUMO

The individual risk for common diseases not only depends on genetic but also on epigenetic polymorphisms. To assess the role of epigenetic variations in the individual risk for obesity, we have determined the methylation status of two CpG islands at the POMC locus in obese and normal-weight children. We found a hypermethylation variant targeting individual CpGs at the intron 2-exon 3 boundary of the POMC gene by bisulphite sequencing that was significantly associated with obesity. POMC exon 3 hypermethylation interferes with binding of the transcription enhancer P300 and reduces expression of the POMC transcript. Since intron 2 contains Alu elements that are known to influence methylation in their genomic vicinity, the exon 3 methylation variant seems to result from an Alu element-triggered default state of methylation boundary definition. Exon 3 hypermethylation in the POMC locus represents the first identified DNA methylation variant that is associated with the individual risk for obesity.


Assuntos
Elementos Alu , Ilhas de CpG , Metilação de DNA , Epigênese Genética , Obesidade/genética , Pró-Opiomelanocortina/genética , Adolescente , Elementos Alu/genética , Animais , Sítios de Ligação , Criança , Pré-Escolar , Ilhas de CpG/genética , Éxons , Regulação da Expressão Gênica , Humanos , Lactente , Recém-Nascido , Íntrons , Camundongos , Camundongos Endogâmicos C57BL , Polimorfismo Genético , Primatas/genética , Pró-Opiomelanocortina/metabolismo , Fatores de Risco
12.
Hum Mol Genet ; 21(18): 4038-48, 2012 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-22694955

RESUMO

The Prader-Willi syndrome (PWS) region in 15q11q13 harbours a cluster of imprinted genes expressed from the paternal chromosome only. Whereas loss of function of the SNORD116 genes appears to be responsible for the major features of PWS, the role of the other genes is less clear. One of these genes is C15orf2, which has no orthologues in rodents, but appears to be under strong positive selection in primates. C15orf2 encodes a 1156 amino acid protein with six nuclear localisation sequences. By protein BLAST analysis and InterProScan signature recognition search, we found sequence similarity of C15orf2 to the nuclear pore complex (NPC) protein POM121. To determine whether C15orf2 is located at nuclear pores, we generated a stable cell line that inducibly expresses FLAG-tagged C15orf2 and performed immunocytochemical studies. We found that C15orf2 is present at the nuclear periphery, where it colocalizes with NPCs and nuclear lamins. At very high expression levels, we observed invaginations of the nuclear envelope. Extending these observations to three-dimensional structured illumination microscopy, which achieves an 8-fold improved volumetric resolution over conventional imaging, we saw that C15orf2 is located at the inner face of the nuclear envelope where it strongly associates with the NPC. In nuclear envelope isolation and fractionation experiments, we detected C15orf2 in the NPC and lamina fractions. These experiments for the first time demonstrate that C15orf2 is part of the NPC or its associated molecular networks. Based on our findings, we propose 'Nuclear pore associated protein 1' as the new name for C15orf2.


Assuntos
Impressão Genômica , Proteínas do Tecido Nervoso/genética , Síndrome de Prader-Willi/genética , Sequência de Aminoácidos , Células HEK293 , Humanos , Glicoproteínas de Membrana/química , Anotação de Sequência Molecular , Proteínas do Tecido Nervoso/química , Proteínas do Tecido Nervoso/metabolismo , Membrana Nuclear/metabolismo , Membrana Nuclear/ultraestrutura , Poro Nuclear/metabolismo , Poro Nuclear/ultraestrutura , Complexo de Proteínas Formadoras de Poros Nucleares , Análise de Sequência com Séries de Oligonucleotídeos , Estrutura Quaternária de Proteína , Homologia de Sequência de Aminoácidos , Transcrição Gênica , Transcriptoma
13.
Med Genet ; 36(2): 111-120, 2024 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-38854642

RESUMO

Epigenetic control systems are based on chromatin modifications (DNA methylation, histone modifications and nucleosome positioning), which affect the local kinetics of gene expression. They play an important role in maintaining cell fate decisions, X inactivation and genomic imprinting. Aberrant chromatin states that are associated with a deleterious change in gene expression are called epimutations. An epimutation can be a primary epimutation that has occurred in the absence of any genetic change or a secondary epimutation that results from a mutation of a cis-acting regulatory element or trans-acting factor. Epimutations may play a causative role in disease, for example in imprinting disorders, or may be part of the pathogenetic mechanism as in the fragile X syndrome and in syndromes caused by a mutation affecting a chromatin modifier. For several diseases, DNA methylation testing is an important tool in the diagnostic work-up of patients.

14.
Clin Epigenetics ; 16(1): 13, 2024 01 16.
Artigo em Inglês | MEDLINE | ID: mdl-38229153

RESUMO

BACKGROUND: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive cancer with poor prognosis. It is marked by extraordinary resistance to conventional therapies including chemotherapy and radiation, as well as to essentially all targeted therapies evaluated so far. More than 90% of PDAC cases harbor an activating KRAS mutation. As the most common KRAS variants in PDAC remain undruggable so far, it seemed promising to inhibit a downstream target in the MAPK pathway such as MEK1/2, but up to now preclinical and clinical evaluation of MEK inhibitors (MEKi) failed due to inherent and acquired resistance mechanisms. To gain insights into molecular changes during the formation of resistance to oncogenic MAPK pathway inhibition, we utilized short-term passaged primary tumor cells from ten PDACs of genetically engineered mice. We followed gain and loss of resistance upon MEKi exposure and withdrawal by longitudinal integrative analysis of whole genome sequencing, whole genome bisulfite sequencing, RNA-sequencing and mass spectrometry data. RESULTS: We found that resistant cell populations under increasing MEKi treatment evolved by the expansion of a single clone but were not a direct consequence of known resistance-conferring mutations. Rather, resistant cells showed adaptive DNA hypermethylation of 209 and hypomethylation of 8 genomic sites, most of which overlap with regulatory elements known to be active in murine PDAC cells. Both DNA methylation changes and MEKi resistance were transient and reversible upon drug withdrawal. Furthermore, MEKi resistance could be reversed by DNA methyltransferase inhibition with remarkable sensitivity exclusively in the resistant cells. CONCLUSION: Overall, the concept of acquired therapy resistance as a result of the expansion of a single cell clone with epigenetic plasticity sheds light on genetic, epigenetic and phenotypic patterns during evolvement of treatment resistance in a tumor with high adaptive capabilities and provides potential for reversion through epigenetic targeting.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Animais , Camundongos , Metilação de DNA , Proteínas Proto-Oncogênicas p21(ras)/genética , Neoplasias Pancreáticas/tratamento farmacológico , Neoplasias Pancreáticas/genética , Carcinoma Ductal Pancreático/tratamento farmacológico , Carcinoma Ductal Pancreático/genética , DNA/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/genética , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Quinases de Proteína Quinase Ativadas por Mitógeno/uso terapêutico , Linhagem Celular Tumoral , Mutação
15.
Epigenetics Chromatin ; 16(1): 17, 2023 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-37170330

RESUMO

The inheritance of acquired traits in mammals is a highly controversial topic in biology. Recently, Takahashi et al. (Cell 186:715-731, 2023) have reported that insertion of CpG-free DNA into a CpG island (CGI) can induce DNA methylation of the CGI and that this aberrant methylation pattern can be transmitted across generations, even after removal of the foreign DNA. These results were interpreted as evidence for transgenerational inheritance of acquired DNA methylation patterns. Here, we discuss several interpretational issues raised by this study and consider alternative explanations.


Assuntos
Metilação de DNA , DNA , Animais , Ilhas de CpG , Mutação , Mamíferos/genética
16.
Sci Transl Med ; 15(705): eadg1659, 2023 07 19.
Artigo em Inglês | MEDLINE | ID: mdl-37467315

RESUMO

Increasing evidence points toward epigenetic variants as a risk factor for developing obesity. We analyzed DNA methylation of the POMC (pro-opiomelanocortin) gene, which is pivotal for satiety regulation. We identified sex-specific and nongenetically determined POMC hypermethylation associated with a 1.4-fold (confidence interval, 1.03 to 2.04) increased individual risk of developing obesity. To investigate the early embryonic establishment of POMC methylation states, we established a human embryonic stem cell (hESC) model. Here, hESCs (WA01) were transferred into a naïve state, which was associated with a reduction of DNA methylation. Naïve hESCs were differentiated via a formative state into POMC-expressing hypothalamic neurons, which was accompanied by re-establishment of DNA methylation patterning. We observed that reduced POMC gene expression was associated with increased POMC methylation in POMC-expressing neurons. On the basis of these findings, we treated POMC-hypermethylated obese individuals (n = 5) with an MC4R agonist and observed a body weight reduction of 4.66 ± 2.16% (means ± SD) over a mean treatment duration of 38.4 ± 26.0 weeks. In summary, we identified an epigenetic obesity risk variant at the POMC gene fulfilling the criteria for a metastable epiallele established in early embryonic development that may be addressable by MC4R agonist treatment to reduce body weight.


Assuntos
Obesidade , Pró-Opiomelanocortina , Masculino , Gravidez , Feminino , Humanos , Pró-Opiomelanocortina/genética , Pró-Opiomelanocortina/metabolismo , Obesidade/genética , Obesidade/metabolismo , Peso Corporal/fisiologia , Metilação de DNA/genética , Fatores de Risco , Receptor Tipo 4 de Melanocortina/genética , Receptor Tipo 4 de Melanocortina/metabolismo
17.
Blood ; 115(12): 2462-72, 2010 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-20075157

RESUMO

The GFI1 gene encodes a transcriptional repressor, which regulates myeloid differentiation. In the mouse, Gfi1 deficiency causes neutropenia and an accumulation of granulomonocytic precursor cells that is reminiscent of a myelodysplastic syndrome. We report here that a variant allele of GFI1 (GFI1(36N)) is associated with acute myeloid leukemia (AML) in white subjects with an odds ratio of 1.6 (P < 8 x 10(-5)). The GFI1(36N) variant occurred in 1806 AML patients with an allele frequency of 0.055 compared with 0.035 in 1691 healthy control patients in 2 independent cohorts. We observed that both GFI1 variants maintain the same activity as transcriptional repressors but differ in their regulation by the AML1/ETO (RUNX1/RUNX1T1) fusion protein produced in AML patients with a t(8;21) translocation. AML1/ETO interacts and colocalizes with the more common GFI1(36S) form in the nucleus and inhibits its repressor activity. However, the variant GFI1(36N) protein has a different subnuclear localization than GFI1(36S). As a consequence, AML1/ETO does not colocalize with GFI1(36N) and is unable to inhibit its repressor activity. We conclude that both variants of GFI1 differ in their ability to be regulated by interacting proteins and that the GFI1(36N) variant form exhibits distinct biochemical features that may confer a predisposition to AML.


Assuntos
Proteínas de Ligação a DNA/genética , Predisposição Genética para Doença , Leucemia Mieloide Aguda/genética , Polimorfismo de Nucleotídeo Único , Fatores de Transcrição/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Animais , Células COS , Núcleo Celular/metabolismo , Chlorocebus aethiops , Subunidade alfa 2 de Fator de Ligação ao Core/metabolismo , Proteínas de Ligação a DNA/metabolismo , Feminino , Frequência do Gene , Variação Genética , Células HeLa , Humanos , Leucemia Mieloide Aguda/metabolismo , Desequilíbrio de Ligação , Masculino , Camundongos , Pessoa de Meia-Idade , Células NIH 3T3 , Proteínas de Fusão Oncogênica/metabolismo , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/metabolismo , Translocação Genética , Adulto Jovem
18.
PLoS Genet ; 5(12): e1000790, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-20041224

RESUMO

Genomic imprinting is an epigenetic process leading to parent-of-origin-specific DNA methylation and gene expression. To date, approximately 60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5'-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2'-deoxycytidine-treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.


Assuntos
Impressão Genômica/genética , Proteína do Retinoblastoma/genética , Desequilíbrio Alélico/efeitos dos fármacos , Desequilíbrio Alélico/genética , Animais , Azacitidina/farmacologia , Ilhas de CpG/genética , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Loci Gênicos/genética , Impressão Genômica/efeitos dos fármacos , Humanos , Linfócitos/efeitos dos fármacos , Linfócitos/metabolismo , Macaca mulatta/genética , Pan troglodytes/genética , Pseudogenes/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
19.
Clin Epigenetics ; 14(1): 95, 2022 07 28.
Artigo em Inglês | MEDLINE | ID: mdl-35902960

RESUMO

Modern epigenetics emerged about 40 years ago. Since then, the field has rapidly grown. Unfortunately, this development has been accompanied by certain misconceptions and methodological shortcomings. A profound misconception is that chromatin modifications are a distinct layer of gene regulation that is directly responsive to the environment and potentially heritable between generations. This view ignores the fact that environmental factors affect gene expression mainly through signaling cascades and the activation or repression of transcription factors, which recruit chromatin regulators. The epigenome is mainly shaped by the DNA sequence and by transcription. Methodological shortcomings include the insufficient consideration of genetic variation and cell mixture distribution. Mis- and overinterpretation of epigenetic data foster genetic denialism ("We can control our genes") and epigenetic determinism ("You are what your parents ate"). These erroneous beliefs can be overcome by using precise definitions, by raising the awareness about methodological pitfalls and by returning to the basic facts in molecular and cellular biology.


Assuntos
Metilação de DNA , Epigênese Genética , Cromatina/genética , Epigenoma , Epigenômica , Humanos
20.
Sci Rep ; 12(1): 17347, 2022 10 17.
Artigo em Inglês | MEDLINE | ID: mdl-36253434

RESUMO

DNA methylation patterns can be responsive to environmental influences. This observation has sparked interest in the potential for psychological interventions to influence epigenetic processes. Recent studies have observed correlations between DNA methylation changes and therapy outcome. However, most did not control for changes in cell composition. This study had two aims: first, we sought to replicate therapy-associated changes in DNA methylation of commonly assessed candidate genes in isolated monocytes from 60 female patients with post-traumatic stress disorder (PTSD). Our second, exploratory goal was to identify novel genomic regions with substantial pre-to-post intervention DNA methylation changes by performing whole-genome bisulfite sequencing (WGBS) in two patients with PTSD. Equivalence testing and Bayesian analyses provided evidence against physiologically meaningful intervention-associated DNA methylation changes in monocytes of PTSD patients in commonly investigated target genes (NR3C1, FKBP5, SLC6A4, OXTR). Furthermore, WGBS yielded only a limited set of candidate regions with suggestive evidence of differential DNA methylation pre- to post-therapy. These differential DNA methylation patterns did not prove replicable when investigated in the entire cohort. We conclude that there is no evidence for major, recurrent intervention-associated DNA methylation changes in the investigated genes in monocytes of patients with PTSD.


Assuntos
Metilação de DNA , Transtornos de Estresse Pós-Traumáticos , Teorema de Bayes , Epigênese Genética , Feminino , Humanos , Monócitos , Proteínas da Membrana Plasmática de Transporte de Serotonina/genética , Transtornos de Estresse Pós-Traumáticos/genética , Transtornos de Estresse Pós-Traumáticos/psicologia
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