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1.
Blood ; 132(21): 2280-2285, 2018 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-30282799

RESUMO

The WHO Classification of Tumours of Haematopoietic and Lymphoid Tissue notes instances of Burkitt lymphoma/leukemia (BL) with IG-MYC rearrangement displaying a B-cell precursor immunophenotype (termed herein "preBLL"). To characterize the molecular pathogenesis of preBLL, we investigated 13 preBLL cases (including 1 cell line), of which 12 were analyzable using genome, exome, and targeted sequencing, imbalance mapping, and DNA methylation profiling. In 5 patients with reads across the IG-MYC breakpoint junctions, we found evidence that the translocation derived from an aberrant VDJ recombination, as is typical for IG translocations arising in B-cell precursors. Genomic changes like biallelic IGH translocations or VDJ rearrangements combined with translocation into the VDJ region on the second allele, potentially preventing expression of a productive immunoglobulin, were detected in 6 of 13 cases. We did not detect mutations in genes frequently altered in BL, but instead found activating NRAS and/or KRAS mutations in 7 of 12 preBLLs. Gains on 1q, recurrent in BL and preB lymphoblastic leukemia/lymphoma (pB-ALL/LBL), were detected in 7 of 12 preBLLs. DNA methylation profiling showed preBLL to cluster with precursor B cells and pB-ALL/LBL, but apart from BL. We conclude that preBLL genetically and epigenetically resembles pB-ALL/LBL rather than BL. Therefore, we propose that preBLL be considered as a pB-ALL/LBL with recurrent genetic abnormalities.

2.
BMC Cancer ; 18(1): 796, 2018 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-30081852

RESUMO

BACKGROUND: ADAMs (a disintegrin and metalloproteinase) have long been associated with tumor progression. Recent findings indicate that members of the closely related ADAMTS (ADAMs with thrombospondin motifs) family are also critically involved in carcinogenesis. Gene silencing through DNA methylation at CpG loci around e.g. transcription start or enhancer sites is a major mechanism in cancer development. Here, we aimed at identifying genes of the ADAM and ADAMTS family showing altered DNA methylation in the development or colorectal cancer (CRC) and other epithelial tumors. METHODS: We investigated potential changes of DNA methylation affecting ADAM and ADAMTS genes in 117 CRC, 40 lung cancer (LC) and 15 oral squamous-cell carcinoma (SCC) samples. Tumor tissue was analyzed in comparison to adjacent non-malignant tissue of the same patients. The methylation status of 1145 CpGs in 51 ADAM and ADAMTS genes was measured with the HumanMethylation450 BeadChip Array. ADAMTS16 protein expression was analyzed in CRC samples by immunohistochemistry. RESULTS: In CRC, we identified 72 CpGs in 18 genes which were significantly affected by hyper- or hypomethylation in the tumor tissue compared to the adjacent non-malignant tissue. While notable/frequent alterations in methylation patterns within ADAM genes were not observed, conspicuous changes were found in ADAMTS16 and ADAMTS2. To figure out whether these differences would be CRC specific, additional LC and SCC tissue samples were analyzed. Overall, 78 differentially methylated CpGs were found in LC and 29 in SCC. Strikingly, 8 CpGs located in the ADAMTS16 gene were commonly differentially methylated in all three cancer entities. Six CpGs in the promoter region were hypermethylated, whereas 2 CpGs in the gene body were hypomethylated indicative of gene silencing. In line with these findings, ADAMTS16 protein was strongly expressed in globlet cells and colonocytes in control tissue but not in CRC samples. Functional in vitro studies using the colorectal carcinoma cell line HT29 revealed that ADAMTS16 expression restrained tumor cell proliferation. CONCLUSIONS: We identified ADAMTS16 as novel gene with cancer-specific promoter hypermethylation in CRC, LC and SCC patients implicating ADAMTS16 as potential biomarker for these tumors. Moreover, our results provide evidence that ADAMTS16 may have tumor suppressor properties.

3.
J Med Genet ; 55(7): 497-504, 2018 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-29574422

RESUMO

BACKGROUND: Genomic imprinting results from the resistance of germline epigenetic marks to reprogramming in the early embryo for a small number of mammalian genes. Genetic, epigenetic or environmental insults that prevent imprints from evading reprogramming may result in imprinting disorders, which impact growth, development, behaviour and metabolism. We aimed to identify genetic defects causing imprinting disorders by whole-exome sequencing in families with one or more members affected by multilocus imprinting disturbance. METHODS: Whole-exome sequencing was performed in 38 pedigrees where probands had multilocus imprinting disturbance, in five of whom maternal variants in NLRP5 have previously been found. RESULTS: We now report 15 further pedigrees in which offspring had disturbance of imprinting, while their mothers had rare, predicted-deleterious variants in maternal effect genes, including NLRP2, NLRP7 and PADI6. As well as clinical features of well-recognised imprinting disorders, some offspring had additional features including developmental delay, behavioural problems and discordant monozygotic twinning, while some mothers had reproductive problems including pregnancy loss. CONCLUSION: The identification of 20 putative maternal effect variants in 38 families affected by multilocus imprinting disorders adds to the evidence that maternal genetic factors affect oocyte fitness and thus offspring development. Testing for maternal-effect genetic variants should be considered in families affected by atypical imprinting disorders.

4.
Clin Epigenetics ; 9: 111, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-29046733

RESUMO

BACKGROUND: Uniparental disomy of certain chromosomes are associated with a group of well-known genetic syndromes referred to as imprinting disorders. However, the extreme form of uniparental disomy affecting the whole genome is usually not compatible with life, with the exception of very rare cases of patients with mosaic genome-wide uniparental disomy reported in the literature. RESULTS: We here report on a fetus with intrauterine growth retardation and malformations observed on prenatal ultrasound leading to invasive prenatal testing. By cytogenetic (conventional karyotyping), molecular cytogenetic (QF-PCR, FISH, array), and methylation (MS-MLPA) analyses of amniotic fluid, we detected mosaicism for one cell line with genome-wide maternal uniparental disomy and a second diploid cell line of biparental inheritance with trisomy X due to paternal isodisomy X. As expected for this constellation, we observed DNA methylation changes at all imprinted loci investigated. CONCLUSIONS: This report adds new information on phenotypic outcome of mosaic genome-wide maternal uniparental disomy leading to an extreme form of multilocus imprinting disturbance. Moreover, the findings highlight the technical challenges of detecting these rare chromosome disorders prenatally.


Assuntos
Impressão Genômica , Ultrassonografia Pré-Natal/métodos , Dissomia Uniparental/genética , Adulto , Metilação de DNA , Feminino , Estudo de Associação Genômica Ampla , Humanos , Mosaicismo , Gravidez
5.
Pediatr Blood Cancer ; 64(3)2017 03.
Artigo em Inglês | MEDLINE | ID: mdl-27786413

RESUMO

Deregulation of the epigenome is an important pathogenetic mechanism in acute lymphoblastic leukemia (ALL) with lysine (K)-specific methyltransferase 2A rearrangement (KMT2Ar). We performed array-based DNA methylation profiling of KMT2Ar ALL cells from 26 children in comparison to normal B-cell precursors. Significant changes in DNA methylation in KMT2Ar ALL were identified in 2,545 CpG loci, influenced by age and the translocation partners AFF1 and MLLT1. In KMT2Ar ALL, DNA methylation loss was enriched at enhancers and for certain transcription factor binding sites such as BCL11A, EBF, and MEF2A. In summary, DNA methylation changes in KMT2Ar ALL target enhancers, genes involved in leukemogenesis and normal hematopoiesis, as well as transcription factor networks.


Assuntos
Biomarcadores Tumorais/genética , Metilação de DNA , Perfilação da Expressão Gênica , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras B/genética , Linfócitos B/metabolismo , Estudos de Casos e Controles , Hibridização Genômica Comparativa , Feminino , Seguimentos , Humanos , Lactente , Masculino , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patologia , Prognóstico , Regiões Promotoras Genéticas/genética
6.
Epigenomics ; 8(6): 801-16, 2016 06.
Artigo em Inglês | MEDLINE | ID: mdl-27323310

RESUMO

AIM: To characterize the genotypic and phenotypic extent of multilocus imprinting disturbances (MLID). MATERIALS & METHODS: We analyzed 37 patients with imprinting disorders (explorative cohort) for DNA methylation changes using the Infinium HumanMethylation450 BeadChip. For validation, three independent cohorts with imprinting disorders or cardinal features thereof were analyzed (84 patients with imprinting disorders, 52 with growth disorder, 81 with developmental delay). RESULTS: In the explorative cohort 21 individuals showed array-based MLID with each one displaying an Angelman or Temple syndrome phenotype, respectively. Epimutations in ZDBF2 and FAM50B were associated with severe MLID regarding number of affected regions. By targeted analysis we identified methylation changes of ZDBF2 and FAM50B also in the three validation cohorts. CONCLUSION: We corroborate epimutations in ZDBF2 and FAM50B as frequent changes in MLID whereas these rarely occur in other patients with cardinal features of imprinting disorders. Moreover, we show cell lineage specific differences in the genomic extent of FAM50B epimutation.


Assuntos
Metilação de DNA , Deficiências do Desenvolvimento/genética , Impressão Genômica , Estudos de Casos e Controles , Proteínas de Ligação a DNA/genética , Feminino , Estudos de Associação Genética , Humanos , Masculino , Fenótipo , Proteínas/genética , Análise de Sequência de DNA
7.
Birth Defects Res A Clin Mol Teratol ; 106(8): 724-8, 2016 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-27223093

RESUMO

BACKGROUND: The bladder exstrophy-epispadias complex (BEEC) is characterized by a spectrum of genitourinary malformations. Both classical bladder exstrophy and the most severe phenotype, exstrophy of the cloaca, display omphaloceles, a cardinal anomaly of some disorders caused by altered imprinting. Therefore, we hypothesized that BEEC in some patients could occur on the basis of an undiagnosed imprinting disorder. Such altered imprinting is associated with changes in the parent-of-origin-specific DNA methylation. METHODS: We analyzed the DNA methylation of 54 imprinted loci in 23 selected patients with different BEEC subtypes (epispadias n = 1, classical bladder exstrophy n = 10, exstrophy of the cloaca n = 12) using the Infinium HumanMethylation450 BeadChip. A total of 471,722 not imprinted autosomal CpG loci and 891 imprinted CpG loci were investigated. Findings were corroborated by methylation-specific-multiplex ligation-dependent probe amplification (MS-MLPA) and microsatellite analysis. RESULTS: No significant differences in the DNA methylation of the not imprinted and imprinted CpG were observed depending on subtype of BEEC. Nevertheless, in 1 of the 23 patients who displayed a classical bladder exstrophy, we detected hypomethylation of the imprinted PLAGL1 locus in chromosome 6q24. We verified this hypomethylation by MS-MLPA and showed further the methylation loss to be caused most likely by a mosaic epimutation. CONCLUSION: Considering that it is highly unlikely to detect a PLAGL1 epimutation among 23 individuals given the low incidence of this alteration in the population, our observations further support a link between BEEC and imprinting disorders. Birth Defects Research (Part A) 106:724-728, 2016. © 2016 Wiley Periodicals, Inc.


Assuntos
Extrofia Vesical/genética , Proteínas de Ciclo Celular/genética , Cromossomos Humanos Par 6/química , Metilação de DNA , Epispadia/genética , Impressão Genômica , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor/genética , Adolescente , Extrofia Vesical/diagnóstico , Extrofia Vesical/patologia , Criança , Pré-Escolar , Estudos de Coortes , Ilhas de CpG , Epispadia/diagnóstico , Epispadia/patologia , Feminino , Expressão Gênica , Loci Gênicos , Humanos , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase Multiplex
8.
Epigenetics ; 11(3): 216-26, 2016 03 03.
Artigo em Inglês | MEDLINE | ID: mdl-26890210

RESUMO

Gene duplication by retrotransposition, i.e., the reverse transcription of an mRNA and integration of the cDNA into the genome, is an important mechanism in evolution. Based on whole-genome bisulfite sequencing of monocyte DNA, we have investigated the methylation state of all CpG islands (CGIs) associated with a retrocopy (n = 1,319), their genomic environment, as well as the CGIs associated with the ancestral genes. Approximately 10% of retrocopies are associated with a CGI. Whereas almost all CGIs of the human genome are unmethylated, 68% of the CGIs associated with a retrocopy are methylated. In retrocopies resulting from multiple retrotranspositions of the same ancestral gene, the methylation state of the CGI often differs. There is a strong positive correlation between the methylation state of the CGI/retrocopy and their genomic environment, suggesting that the methylation state of the integration site determined the methylation state of the CGI/retrocopy, or that methylation of the retrocopy by a host defense mechanism has spread into the adjacent regions. Only a minor fraction of CGI/retrocopies (n = 195) has intermediate methylation levels. Among these, the previously reported CGI/retrocopy in intron 2 of the RB1 gene (PPP1R26P1) as well as the CGI associated with the retrocopy RPS2P32 identified in this study carry a maternal methylation imprint. In conclusion, these findings shed light on the evolutionary dynamics and constraints of DNA methylation.


Assuntos
Metilação de DNA/genética , Genoma Humano , Impressão Genômica/genética , Proteínas de Ligação a Retinoblastoma/genética , Ubiquitina-Proteína Ligases/genética , Ilhas de CpG/genética , Humanos , Íntrons/genética , Herança Materna/genética , Monócitos/metabolismo , Regiões Promotoras Genéticas , Retroelementos/genética , Análise de Sequência de DNA
9.
Eur J Hum Genet ; 24(9): 1280-6, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-26839037

RESUMO

Beckwith-Wiedemann syndrome (BWS; OMIM #130650) is an overgrowth syndrome caused by different genetic or epigenetic alterations affecting imprinted regions on chromosome 11p15.5. Here we report a family with multiple offspring affected with BWS including giant omphalocoeles in which maternal transmission of a chromosomal rearrangement including an inversion and two deletions leads to hypomethylation of the imprint control region 2 (ICR2). As the deletion includes the promoter and 5' part of the KCNQ1 gene, we suggest that transcription of this gene may be involved in establishing the maternal methylation imprint of the ICR2, which is located in intron 10 of KCNQ1.


Assuntos
Síndrome de Beckwith-Wiedemann/genética , Deleção Cromossômica , Inversão Cromossômica , Cromossomos Humanos Par 11/genética , Metilação de DNA , Impressão Genômica , Herança Paterna , Adulto , Síndrome de Beckwith-Wiedemann/diagnóstico , Feminino , Humanos , Canal de Potássio KCNQ1/genética , Masculino , Linhagem
10.
Methods Mol Biol ; 1381: 75-92, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-26667456

RESUMO

Epigenetic mechanisms including DNA methylation are fundamental for the regulation of gene expression. Epigenetic alterations can lead to the development and the evolution of malignant tumors as well as the emergence of phenotypically different cancer cells or metastasis from one single tumor cell. Here we describe bisulfite pyrosequencing, a technology to perform quantitative DNA methylation analyses, to detect aberrant DNA methylation in malignant tumors.


Assuntos
Impressões Digitais de DNA/métodos , Metilação de DNA , DNA/genética , Sequência de Bases , DNA/análise , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Humanos , Neoplasias/genética , Sulfitos/química
11.
Epigenomics ; 7(7): 1089-97, 2015 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-26541061

RESUMO

AIM: To investigate the DNA-methylation levels in the newly described MEG8 differentially methylated region (DMR) in the imprinted cluster in 14q32 in patients with Temple syndrome. PATIENTS & METHODS: We included three patients with Temple syndrome which were studied by Infinium HumanMethylation450 BeadChips, locus-specific bisulfite-pyrosequencing, methylation-specific-MLPA and microsatellite analyses. The tag-CpG of the MEG8-DMR was investigated using the Infinium HumanMethylation450 BeadChip. RESULTS: In all three patients, the identical pattern of DNA-hypermethylation of the MEG8-DMR was observed along with DNA-hypomethylation of the IG-DMR and MEG3-DMR. CONCLUSION: Based on the observed MEG8-DMR DNA-hypermethylation and previously published data, we conclude that DNA-methylation of the MEG3- and MEG8-DMR is functionally dependent on the DNA-methylation pattern of the IG-DMR. The observed combination of epimutations is predicted to be associated with bi-allelic MEG3 and MEG8 expression in individuals with Temple syndrome.


Assuntos
Cromossomos Humanos Par 14 , Deficiências do Desenvolvimento/genética , Epigênese Genética , Isoformas de RNA/genética , RNA Longo não Codificante/genética , Dissomia Uniparental/genética , Adolescente , Criança , Pré-Escolar , Ilhas de CpG , Metilação de DNA , Deficiências do Desenvolvimento/patologia , Feminino , Impressão Genômica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Repetições de Microssatélites , Reação em Cadeia da Polimerase , Dissomia Uniparental/patologia
12.
Nat Genet ; 47(11): 1316-1325, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26437030

RESUMO

Although Burkitt lymphomas and follicular lymphomas both have features of germinal center B cells, they are biologically and clinically quite distinct. Here we performed whole-genome bisulfite, genome and transcriptome sequencing in 13 IG-MYC translocation-positive Burkitt lymphoma, nine BCL2 translocation-positive follicular lymphoma and four normal germinal center B cell samples. Comparison of Burkitt and follicular lymphoma samples showed differential methylation of intragenic regions that strongly correlated with expression of associated genes, for example, genes active in germinal center dark-zone and light-zone B cells. Integrative pathway analyses of regions differentially methylated in Burkitt and follicular lymphomas implicated DNA methylation as cooperating with somatic mutation of sphingosine phosphate signaling, as well as the TCF3-ID3 and SWI/SNF complexes, in a large fraction of Burkitt lymphomas. Taken together, our results demonstrate a tight connection between somatic mutation, DNA methylation and transcriptional control in key B cell pathways deregulated differentially in Burkitt lymphoma and other germinal center B cell lymphomas.


Assuntos
Linfoma de Burkitt/genética , Metilação de DNA , Linfoma Folicular/genética , Mutação , Transcriptoma/genética , Adolescente , Adulto , Idoso , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Criança , Pré-Escolar , Feminino , Genoma Humano/genética , Centro Germinativo/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas de Fusão Oncogênica/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-myc/genética , Transdução de Sinais/genética , Translocação Genética , Adulto Jovem
13.
Eur J Med Genet ; 58(8): 419-25, 2015 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-26003415

RESUMO

Despite recent progress in molecular karyotyping and clinical sequencing the cause of intellectual disability in a considerable subset of individuals affected by this phenotype remains elusive. As intellectual disability is also a feature of various imprinting disorders and some monogenic forms of intellectual disability are caused by epigenetic modifiers we hypothesized that changes in DNA methylation might be associated with or even causative in some cases of intellectual disability. Therefore, we performed a DNA methylation analysis of peripheral blood samples from 82 patients with intellectual disability and additional features using the HumanMethylation450 BeadChip. The findings were compared to that of 19 normal controls. Differentially methylated loci were validated by bisulfite pyrosequencing. On a global level, we failed to detect a robust DNA methylation signature segregating individuals with intellectual disability from controls. Using an individual approach, we identified 157 regions showing individual DNA methylation changes in at least one patient. These correlated to 107 genes including genes linked to conditions associated with intellectual disability, namely COLEC11, SHANK2, GLI2 and KCNQ2, as well as imprinted genes like FAM50B and MEG3. The latter was suggestive of an undiagnosed Temple syndrome which could be confirmed by diagnostic tests. Subsequent in-depth analysis of imprinted loci revealed DNA methylation changes at additional imprinted loci, i.e. PPIEL, IGF2R, MEG8 and MCTS2/HM13, in up to five patients. Our findings indicate that imprinting disorders are rare but probably under-diagnosed in patients with intellectual disability and moreover point to DNA methylation changes as potential alternative means to identify deregulated genes involved in the pathogenesis of intellectual disability.


Assuntos
Metilação de DNA , Deficiências do Desenvolvimento/genética , Loci Gênicos , Impressão Genômica , Deficiência Intelectual/genética , Adolescente , Estudos de Casos e Controles , Criança , Pré-Escolar , Deficiências do Desenvolvimento/diagnóstico , Deficiências do Desenvolvimento/patologia , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Lactente , Deficiência Intelectual/diagnóstico , Deficiência Intelectual/patologia , Cariótipo , Masculino , Análise em Microsséries , Fenótipo
14.
PLoS One ; 10(3): e0120463, 2015.
Artigo em Inglês | MEDLINE | ID: mdl-25785847

RESUMO

Treatment with recombinant human growth hormone (rhGH) has been consistently reported to induce transcriptional changes in various human tissues including peripheral blood. For other hormones it has been shown that the induction of such transcriptional effects is conferred or at least accompanied by DNA-methylation changes. To analyse effects of short term rhGH treatment on the DNA-methylome we investigated a total of 24 patients at baseline and after 4-day rhGH stimulation. We performed array-based DNA-methylation profiling of paired peripheral blood mononuclear cell samples followed by targeted validation using bisulfite pyrosequencing. Unsupervised analysis of DNA-methylation in this short-term treated cohort revealed clustering according to individuals rather than treatment. Supervised analysis identified 239 CpGs as significantly differentially methylated between baseline and rhGH-stimulated samples (p<0.0001, unadjusted paired t-test), which nevertheless did not retain significance after adjustment for multiple testing. An individualized evaluation strategy led to the identification of 2350 CpG and 3 CpH sites showing methylation differences of at least 10% in more than 2 of the 24 analyzed sample pairs. To investigate the long term effects of rhGH treatment on the DNA-methylome, we analyzed peripheral blood cells from an independent cohort of 36 rhGH treated children born small for gestational age (SGA) as compared to 18 untreated controls. Median treatment interval was 33 months. In line with the groupwise comparison in the short-term treated cohort no differentially methylated targets reached the level of significance in the long-term treated cohort. We identified marked intra-individual responses of DNA-methylation to short-term rhGH treatment. These responses seem to be predominately associated with immunologic functions and show considerable inter-individual heterogeneity. The latter is likely the cause for the lack of a rhGH induced homogeneous DNA-methylation signature after short- and long-term treatment, which nevertheless is well in line with generally assumed safety of rhGH treatment.


Assuntos
Metilação de DNA/efeitos dos fármacos , Hormônio do Crescimento Humano/farmacologia , Adolescente , Criança , Estudos de Coortes , Feminino , Humanos , Leucócitos Mononucleares/efeitos dos fármacos , Leucócitos Mononucleares/metabolismo , Masculino , Fatores de Tempo
15.
Lab Invest ; 94(8): 927-33, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24933424

RESUMO

Alterations in the DNA methylome are characteristic for numerous diseases and a typical hallmark of cancer. Therefore, DNA methylation is currently under investigation in research labs and has also entered diagnostics. Recently, protocols like the BeadChip technology have become commercially available to study DNA methylation in an array format and semiquantitative fashion. However, it is known that fixation of the sample material with formalin prior to BeadChip analysis can affect the results. In this study we compared the influence of fixation on the outcome of BeadChip analysis. From six patients each a lung cancer tissue sample and a corresponding tumor-free lung tissue sample were collected. The samples were separated into three pieces. One piece of each sample was fixed with formalin, another one by the non-cross-linking HOPE technique (Hepes-glutamic acid buffer mediated Organic solvent Protection Effect). Subsequently, both became paraffin embedded. As a reference, the remaining third piece was cryopreserved. In addition we used three adenocarcinoma cell lines (H838, A549, and H1650) to validate the results from patient tissues. We show that using the HOPE technique instead of formalin largely prevents the introduction of formalin-fixation related artifacts. An ANOVA analysis significantly separated HOPE- and cryopreserved from formalin-fixed samples (FDR<0.05), while differences in the methylation data obtained from HOPE-fixed and cryopreserved material were minor. Consequently, HOPE fixation is superior to formalin fixation if a subsequent BeadChip analysis of paraffin-embedded sample material is intended.


Assuntos
Adenocarcinoma/metabolismo , Carcinoma de Células Escamosas/metabolismo , Metilação de DNA , DNA de Neoplasias/metabolismo , Neoplasias Pulmonares/metabolismo , Pulmão/metabolismo , Preservação de Tecido , Adenocarcinoma/patologia , Idoso , Artefatos , Tampões (Química) , Carcinoma Adenoescamoso/metabolismo , Carcinoma Adenoescamoso/patologia , Carcinoma de Células Grandes/metabolismo , Carcinoma de Células Grandes/prevenção & controle , Carcinoma de Células Escamosas/patologia , Linhagem Celular Tumoral , Criopreservação , DNA de Neoplasias/isolamento & purificação , Feminino , Ácido Glutâmico/química , HEPES/química , Humanos , Pulmão/patologia , Neoplasias Pulmonares/patologia , Masculino , Pessoa de Meia-Idade , Análise Serial de Tecidos , Fixação de Tecidos
16.
Cell Metab ; 18(2): 296-302, 2013 Aug 06.
Artigo em Inglês | MEDLINE | ID: mdl-23931760

RESUMO

Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disorder in industrialized countries. Liver samples from morbidly obese patients (n = 45) with all stages of NAFLD and controls (n = 18) were analyzed by array-based DNA methylation and mRNA expression profiling. NAFLD-specific expression and methylation differences were seen for nine genes coding for key enzymes in intermediate metabolism (including PC, ACLY, and PLCG1) and insulin/insulin-like signaling (including IGF1, IGFBP2, and PRKCE) and replicated by bisulfite pyrosequening (independent n = 39). Transcription factor binding sites at NAFLD-specific CpG sites were >1,000-fold enriched for ZNF274, PGC1A, and SREBP2. Intraindividual comparison of liver biopsies before and after bariatric surgery showed NAFLD-associated methylation changes to be partially reversible. Postbariatric and NAFLD-specific methylation signatures were clearly distinct both in gene ontology and transcription factor binding site analyses, with >400-fold enrichment of NRF1, HSF1, and ESRRA sites. Our findings provide an example of treatment-induced epigenetic organ remodeling in humans.


Assuntos
Cirurgia Bariátrica/efeitos adversos , Metilação de DNA/genética , Fígado Gorduroso/genética , Fígado/metabolismo , Obesidade Mórbida/cirurgia , Adulto , Idoso , Feminino , Regulação da Expressão Gênica , Humanos , Insulina/metabolismo , Masculino , Pessoa de Meia-Idade , Hepatopatia Gordurosa não Alcoólica , Obesidade Mórbida/genética , Obesidade Mórbida/metabolismo , Transdução de Sinais/genética
17.
Eur J Hum Genet ; 21(8): 838-43, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23232699

RESUMO

Various genes located at imprinted loci and regulated by epigenetic mechanisms are involved in the control of growth and differentiation. The broad phenotypic variability of imprinting disorders suggests that individuals with inborn errors of imprinting might remain undetected among patients born small for gestational age (SGA). We evaluated quantitative DNA methylation analysis at differentially methylated regions (DMRs) of 10 imprinted loci (PLAGL1, IGF2R DMR2, GRB10, H19 DMR, IGF2, MEG3, NDN, SNRPN, NESP, NESPAS) by bisulphite pyrosequencing in 98 patients born SGA and 50 controls. For IGF2R DMR2, methylation patterns of additional 47 parent pairs and one mother (95 individuals) of patients included in the SGA cohort were analyzed. In six out of 98 patients born SGA, we detected DNA methylation changes at single loci. In one child, the diagnosis of upd(14)mat syndrome owing to an epimutation of the MEG3 locus in 14q32 could be established. The remaining five patients showed hypomethylation at GRB10 (n=2), hypomethylation at the H19 3CTCF-binding site (n=1), hypermethylation at NDN (n=1) and hypermethylation at IGF2 (n=1). IGF2R DMR2 hypermethylation was detected in five patients, six parents of patients in the SGA cohort and two controls. We conclude that aberrant methylation at imprinted loci in children born SGA exists but seems to be rare if known imprinting syndromes are excluded. Further investigations on the physiological variations and the functional consequences of the detected aberrant methylation are necessary before final conclusions on the clinical impact can be drawn.


Assuntos
Metilação de DNA , Loci Gênicos/genética , Impressão Genômica/genética , Recém-Nascido Pequeno para a Idade Gestacional , Anormalidades Múltiplas/genética , Anormalidades Múltiplas/patologia , Adolescente , Criança , Pré-Escolar , Estudos de Coortes , Saúde da Família , Feminino , Humanos , Lactente , Recém-Nascido , Masculino , Linhagem , Fenótipo , Análise de Sequência de DNA , Síndrome
18.
PLoS One ; 7(6): e39014, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22723920

RESUMO

Epigenetic mechanisms including DNA methylation are supposed to play a key role in fetal development. Here we have investigated fetal DNA-methylation levels of 27,578 CpG loci in 47 chorionic villi (CVS) and 16 amniotic cell (AC) samples. Methylation levels differed significantly between karyotypically normal AC and CVS for 2,014 genes. AC showed more extreme DNA-methylation levels of these genes than CVS and the differentially methylated genes are significantly enriched for processes characteristic for the different cell types sampled. Furthermore, we identified 404 genes differentially methylated in CVS with trisomy 21. These genes were significantly enriched for high CG dinucleotid (CpG) content and developmental processes associated with Down syndrome. Our study points to major tissue-specific differences of fetal DNA-methylation and gives rise to the hypothesis that part of the Down syndrome phenotype is epigenetically programmed in the first trimester of pregnancy.


Assuntos
Âmnio/metabolismo , Vilosidades Coriônicas/metabolismo , Metilação de DNA , Epigênese Genética , Epigenômica , Feto/metabolismo , Aberrações Cromossômicas , Análise por Conglomerados , Ilhas de CpG , Síndrome de Down/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Especificidade de Órgãos/genética , Gravidez
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