Wang SC, Oelze, B and Schumacher A (2008) Age-Specific Epigenetic Drift in Late-Onset Alzheimer's Disease PLoS ONE Vol.3 (7)
Abstract: Despite an enormous research effort, most cases of late-onset Alzheimer's disease (LOAD) still remain unexplained and the current biomedical science is still a long way from the ultimate goal of revealing clear risk factors that can help in the diagnosis, prevention and treatment of the disease. Current theories about the development of LOAD hinge on the premise that Alzheimer's arises mainly from heritable causes. Yet, the complex, non-Mendelian disease etiology suggests that an epigenetic component could be involved. Using MALDI-TOF mass spectrometry in post-mortem brain samples and lymphocytes, we have performed an analysis of DNA methylation across 12 potential Alzheimer's susceptibility loci. In the LOAD brain samples we identified a notably age-specific epigenetic drift, supporting a potential role of epigenetic effects in the development of the disease. Additionally, we found that some genes that participate in amyloid-? processing (PSEN1, APOE) and methylation homeostasis (MTHFR, DNMT1) show a significant interindividual epigenetic variability, which may contribute to LOAD predisposition. The APOE gene was found to be of bimodal structure, with a hypomethylated CpG-poor promoter and a fully methylated 3?-CpG-island, that contains the sequences for the ?4-haplotype, which is the only undisputed genetic risk factor for LOAD. Aberrant epigenetic control in this CpG-island may contribute to LOAD pathology. We propose that epigenetic drift is likely to be a substantial mechanism predisposing individuals to LOAD and contributing to the course of disease.
Mill J, Tang T, Kaminsky Z, Khare T, Yazdanpanah S, Bouchard L, Jia P, Assadzadeh A, Flanagan J, Schumacher A, Wang SC, Petronis A. (2008) Epigenomic profiling reveals DNA-methylation changes associated with major psychosis. Amer J Hum Genet82: 696-711; (IF: 12.6)
Abstract: Epigenetic misregulation is consistent with various non-Mendelian features of schizophrenia and bipolar disorder. To date, however, few studies have investigated the role of DNA methylation in major psychosis, and none have taken a genome-wide epigenomic approach. In this study we used CpG-island microarrays to identify DNA-methylation changes in the frontal cortex and germline associated with schizophrenia and bipolar disorder. In the frontal cortex we find evidence for psychosis-associated DNA-methylation differences in numerous loci, including several involved in glutamatergic and GABAergic neurotransmission, brain development, and other processes functionally linked to disease etiology. DNA-methylation changes in a significant proportion of these loci correspond to reported changes of steady-state mRNA level associated with psychosis. Gene-ontology analysis highlighted epigenetic disruption to loci involved in mitochondrial function, brain development, and stress response. Methylome network analysis uncovered decreased epigenetic modularity in both the brain and the germline of affected individuals, suggesting that systemic epigenetic dysfunction may be associated with major psychosis. We also report evidence for a strong correlation between DNA methylation in the MEK1 gene promoter region and lifetime antipsychotic use in schizophrenia patients. Finally, we observe that frontal-cortex DNA methylation in the BDNF gene is correlated with genotype at a nearby nonsynonymous SNP that has been previously associated with major psychosis. Our data are consistent with the epigenetic theory of major psychosis and suggest that DNA-methylation changes are important to the etiology of schizophrenia and bipolar disorder.
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This report was covered in several news stories around the world. Here you can have a look at some of them (PDF-Files):
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A. Schumacher,A. Weinhäusl,
& A. Petronis (2008) Application of
microarrays in DNA methylation profiling. Methods Mol Biol, 439:109-29.
Abstract: Comprehensive analyses of the human epigenome may be of critical importance in understanding the molecular mechanisms of complex diseases, development, ageing, tissue specificity, parental origin effects and sex differences, among other systemic aspects of human biology. However, traditional DNA methylation methods allowed for screening of relatively short DNA fragments only. The advent of microarrays has provided new possibilities in DNA methylation analysis because this technology is able to interrogate a very large number of loci in a highly parallel fashion. There are several permutations of the microarray application in DNA methylation profiling, and such include microarray analysis of bisulfite modified DNA and also the enriched unmethylated or hypermethylated DNA fractions using methylation-sensitive restriction enzymes or antibodies against methylated cytosines. The method described in detail below is based on the analysis of the enriched unmethylated DNA fraction, using a series of treatments with methylation-sensitive restriction enzymes, adaptor ligation, PCR amplification, and quantitative mapping of unmethylated DNA sequences using microarrays. The key advantages of this approach are the ability to investigate DNA methylation patterns using very small DNA amounts and also relatively high informativeness in comparison to the other restriction enzyme- based strategies for DNA methylation profiling
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publication is available at www.springerlink.com.
A. Schumacher,
K. Buiting, M. Zeschnigk, W. Doerfler & B.
Horsthemke (1998) Methylation analysis of the PWS/AS region does not support an
enhancer-competition model. Nature Genet.19: 324-325; (IF: 40.4)
Abstract: We have investigated the methylation status of the AS-SRO, which is 1.15 kb in size and contains 16 CpG dinucleotides. As shown by Southern analysis with the methylation-sensitive restriction enzymes HpaII and CfoI and by sequencing of bisulfite-treated genomic DNA, the ASSRO is extensively methylated (83-87%) on both maternal and paternal chromosomes in healthy, PWS and AS individuals. We show that a maternal imprint has developed in the absence of the AS-SRO, although the enhancer-competition model postulates that this region bears the maternal epigenetic mark which induces spreading of DNA methylation along the chromosome.
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A. Schumacher, P. Koetsier,
J. Hertz & W. Doerfler (2000) Epigenetic and genotype-specific effects on the stability of the novo
imposed methylation patterns in transgenic mice. J. Biol. Chem., 275:
37915-37921 (IF: 7.7)
Abstract: The chloramphenicol acetyltransferase gene under the control of the late E2A promoter of adenovirus type 2 (Ad2) was introduced as transgene into the B6D2F1 mouse strain with mixed genetic background and became extensively de novo methylated. The methylation of this pAd2E2AL-CAT (7-1A) transgene was regulated in a strain-specific manner apparently depending on the site of integration. Transmission of the 7-1A transgene into an inbred DBA/2, 129/sv, or FVB/N genetic background led to a significant loss of methylation in the transgene, whereas C57BL/6, CB20, and Balb/c backgrounds favored the de novo methylation in very specific patterns. The newly established patterns of de novo methylation were transmitted to the offspring and remained stable for many generations, regardless of the heterozygosity of strain-specific DNA sequences present in these mouse strains. Segregation analyses showed a non-mendelian transmission of methylation phenotypes and suggested the involvement of dominant modifiers of methylation.
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A. Schumacher
(2001) Mechanisms and brain specific consequences of genomic imprinting in Prader-Willi and Angelman
syndrome. Gene Funct. Dis., 2: No.1, 1-19, (Wiley-VCH
Top 10 downloaded paper 2001 !)
Abstract: Genomic imprinting is a curious manifestation of epigenetic inheritance that defies normal Mendelian genetics. Most vertebrate genes are expressed from both, the paternal and maternal alleles. However, a subset of mammalian genes is monoallelically expressed in a parent-of-origin manner due to imprinting mechanisms that confer a parent-specific memory to individual cells. Epigenetically correct inheritance of imprinted genes requires appropriate germ-line specific chromosomal modifications like histone acetylation or DNA methylation. This review focuses on the role of these imprinted genes on human chromosome 15q11-13, imprinting center elements, and epigenetic mechanisms in the development of specific regions of the mammalian brain.
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A. Schumacher, S. Arnhold, K. Addicks & W. Doerfler(2003) Staurosporine is a potent activator of neuronal, glial
and "CNS stem cell"-like neurosphere
differentiation in murine embryonic stem cells. Mol
Cell Neurosci., Aug;23(4):669-80 (IF:
5.4)
Abstract: Staurosporine (STS), a broad spectrum protein kinase inhibitor, was previously shown to induce neurite outgrowth in several neuroblastoma cell lines. However, data on the neurotrophic potential of this alkaloid in embryonic stem cell systems were not available. Therefore, three mouse ES cell lines, IB10, RW4, and Bruce 4, were induced to enter neurogenesis in culture at low concentrations of STS. These cells differentiated into epidermal growth factor-responsive neural precursor cells, formed neurospheres, and further developed to neurons and astrocytes. The clonally derived neurospheres consisted of multipotent cells which exhibited some of the classical characteristics of early CNS stem cells and could be propagated in vitro. STS was antagonistic in several ways to retinoic acid (RA), a vitamin A metabolite, which promotes neuritogenesis. Results from RT-PCR experiments and inhibition studies with RA provided evidence that staurosporine exerted its neurotrophic effects through the induction of very late levels of the nerve growth factor and protein kinase C neurogenesis pathways.
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A. Schumacher (2003) Calculation of cell densities and
layer-numbers in staurosporine induced ovoid-like neurospheres. Res Telegraph. (Rus),
1: 2-9
Abstract: By culturing neural stem cells in a serum-free, chemically-defined medium, these cells can differentiate into clusters of cells, known as neurospheres. These spheres contain "neural stem cell"-like cells and are able to detach from the tissue culture dish. One of the main characteristics of all described neurospheres is that they contain a substantial number of cells within the their core. However, recently it was demonstrated that the alkaloid Staurosporine (STS) can induce the formation of a different kind of neurosphere in EGF-dependent embryonic stem cell cultures. These neurospheres have an ovoid-like morphology and are significantly bigger (200 um up to >2 mm) than previously described cell aggregates. This study shows that these spheres consist of only a fraction of the cells expected in filled neurospheres. Layer calculations implicate that the ovoid structures possess an outer shell made by a limited amount of cell layers (~1-5).
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A. Schumacher & W. Doerfler(2004) Influence
of in vitro manipulation on the stability of methylation patterns in the Snurf/Snrpn-imprinting region in mouse embryonic stem
cells. Nucl. Acids Res., Volume 32, 5: 1566-1576 (IF: 7.3)
Abstract: Recent work on embryonic stem (ES) cells showed that stem cell-derived tissues and embryos, cloned from ES cell nuclei, often fail to maintain epigenetic states of imprinted genes. This deregulation is frequently associated with in vitro manipulations and culture conditions which might affect the cells potential to develop into normal fetuses. Usually, epigenetic instability is reported in differentially methylated regions of mostly growth-related imprinted genes. However, little is known about the epigenetic stability of genes that function late in organogenesis. Hence, we set out to investigate the epigenetic stability of neuronal genes and analyzed DNA methylation patterns in the Snurf/Snrpn imprinted cluster in several cultured mouse ES cell lines. We also determined the effects of in vitro stress factors such as consecutive passaging, trypsination, mechanical handling, single cell cloning, centrifugation, staurosporine-induced neurogenesis and the insertion of viral (foreign) DNA into the host genome.
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T. Schwarzbraun, J.B. Vincent, A. Schumacher, D.H. Geschwind, J.
Oliveira, Ch. Windpassinger, L. Ofner,
M. Ledinegg, P.M. Kroisel,
K. Wagner & E. Petek (2004) Cloning, genomic structure, and expression profiles of TULIP1 (GARNL1), a
brain-expressed candidate gene for 14q13-linked neurological phenotypes, and
its murine homologue. Genomics, Sep; 84(3):577-86.
(IF: 3.5)
Abstract: Previously, we have described the clinical and molecular characterization of a de novo 14q13.1-q21.1 microdeletion, less than 3.5 Mb in size, in a patient with severe microcephaly, psychomotor retardation, and other clinical anomalies. Here we report the characterization of the genomic structure of the human tuberin-like protein gene 1 (TULIP1; approved gene symbol GARNL1), a CpG island- associated, brain-expressed candidate gene for the neurological findings in our patient, and its murine homologue. The human TULIP1 gene was mapped to chromosome band 14q13.2. TULIP1 spans about 271 kb of human genomic DNA and is divided into 41 exons. An untranscribed, processed pseudogene of TULIP1 was found on human chromosome band 9q31.1. The active locus TULIP1, encoding a predicted protein of 2036 amino acids, is expressed ubiquitously in pre- and postnatal human tissues. The murine homologue Tulip1 spans about 220 kb of mouse genomic DNA and is also divided into 41 exons, encoding a predicted protein of 2035 amino acids. Considering the location, expression profile, and predicted function, TULIP1 is a strong candidate for several neurological features seen in 14q deletion patients.
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A.
Schumacher&
A. Petronis (2006) Epigenetics in
complex diseases: from theory to laboratory praxis. Current Topics in Microbiology and Immunolgy:310: 81-115. (IF: 3.0)
Abstract: Despite significant effort, understanding the causes and mechanisms of complex non-Mendelian diseases remains a key challenge. Although numerous molecular genetic linkage and association studies have been conducted in order to explain the heritable predisposition to complex diseases, the resulting data are quite often inconsistent and even controversial. In this article, a new interpretation of the paradigm of "genes plus environment" is presented in which the emphasis is shifted to epigenetic misregulation as amajor etiopathogenic factor. Epigenetic mechanisms are consistent with various non-Mendelian irregularities of complex diseases, such as the existence of clinically indistinguishable sporadic and familial cases, sexual dimorphism, relatively late age of onset and peaks of susceptibility to some diseases, discordance of monozygotic twins and major fluctuations on the course of disease severity. It is argued that epigenetic strategies may significantly advance the discovery of etiopathogenic mechanisms of complex diseases. The second part of this chapter is dedicated to a review of laboratory methods forDNA methylation analysis, which may be useful in the study of complex diseases. In this context, epigenetic microarray technologies are emphasized, as it is evident that such technologies will significantly advance epigenetic analyses in complex diseases.
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A. Schumacher,P. Kapranov, J. Flanagan, Z. Kaminsky, A. Assadzadeh, P. Yau, C. Virtanen, J. Cheng, N. Winegarden, T. Gingeras, &
A. Petronis (2006) Microarray-based DNA methylation
profiling: technology and applications. Nucl. Acids Res., Vol.34, 2:
528-542 (IF: 7.3; on NAR top ‘hot papers’ list 2006 !)
Abstract: This work is dedicated to the development of a technology for unbiased, high-throughput DNA methylation profiling of large genomic regions. In this method, unmethylated and methylated DNA fractions are enriched using a series of treatments with methylation sensitive restriction enzymes, and interrogated on microarrays. We have investigated various aspects of the technology including its replicability, informativeness, sensitivity and optimal PCR conditions usingmicroarrays containing oligonucleotides representing 100 kb of genomic DNA derived from the chromosome 22 COMT region in addition to 12 192 element CpG island microarrays. Several new aspects of methylation profiling are provided, including the parallel identification of confounding effects of DNA sequence variation, the description of the principles of microarray design for epigenomic studies and the optimal choice of methylation sensitive restriction enzymes. We also demonstrate the advantages of using the unmethylated DNA fraction versus the methylated one, which substantially improve the chances of detecting DNA methylation differences. We applied this methodology for fine-mapping of methylation patterns of chromosomes 21 and 22 in eight individuals using tiling microarrays consisting of over 340.000 oligonucleotide probe pairs. The principles developed in this work will help to make epigenetic profiling of the entire human genome a routine procedure.
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J.
Flanagan, V. Popendikyte, N.
Pozdniakovaite, M. Sobolev,
A. Assadzadeh, A.
Schumacher, M. Zangeneh, L. Lau, C. Virtanen, S-C. Wang & A. Petronis
(2006) Intra-
and Inter- Individual Epigenetic Variation in Human Germ Cells.
American Journal of Human Genetics; 79:67-84 (IF: 12.6; a Nature Reviews Genetics Research Highlight for 2006!)
Abstract: The objective of this study was to perform a comprehensive analysis of DNA methylation variation between and within the germlines of normal males. First, methylated cytosines were mapped using bisulphite modification-based sequencing in the promoter regions of the following disease genes: presenilins (PSEN1 and PSEN2), breast cancer (BRCA1 and BRCA2), myotonic dystrophy (DM1), and Huntington disease (HD). Major epigenetic variation was detected within samples, since the majority of sperm cells of the same individual exhibited unique DNA methylation profiles. In the interindividual analysis, 41 of 61 pairwise comparisons revealed distinct DNA methylation profiles. Second, a microarray based epigenetic profiling of the same sperm samples was performed using a 12,198-feature CpG island microarray. The microarray analysis has identified numerous DNA methylation-variable positions in the germ cell genome. The largest degree of variation was detected within the promoter CpG islands and pericentromeric satellites among the single-copy DNA fragments and repetitive elements, respectively. A number of genes, such as EED, CTNNA2, CALM1, CDH13, and STMN2, exhibited age-related DNA methylation changes. Finally, allele-specific methylation patterns in CDH13 were detected. This study provides evidence for significant epigenetic variability in human germ cells, which warrants further research to determine whether such epigenetic patterns can be efficiently transmitted across generations and what impact inherited epigenetic individuality may have on phenotypic outcomes in health and disease.
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A. Schumacher, P.
Friedrich, J. Schmid, B. Ibach, T. Eisele, S.M. Laws, H. Förstl, A. Kurz
& M. Riemenschneider (2006)
No association of chromatin-modifying protein 2B with sporadic frontotemporal
dementia.
Neurobiology of Aging, Sept 14 Epub(IF:
5.5)
Abstract: Mutations of the chromatin modifying protein 2B gene (CHMP2B) were identified, in a Danish pedigree, to cause familial frontotemporal dementia (FTD). To explore the possible genetic contribution of common CHMP2B variants in sporadic FTD, we analyzed 14 single nucleotide polymorphisms covering the entire genomic region of CHMP2B. After adjustment for multiple testing single marker and haplotype analysis revealed no significant association with sporadic FTD. Thus, we conclude that CHMP2B can be excluded as a susceptibility gene conferring risk to sporadic forms of FTD.
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A. Schumacher,
P. Friedrich, J. Diehl, B. Ibach, A. Schoepfer-Wendels, J.C. Mueller, L. Konta, S.M. Laws, A. Kurz, H. Foerstl & M.
Riemenschneider (2007)
No association of common VCP variants with sporadic fronto-temporal dementia.
Neurobiology of aging Jul 4; [Epub ahead of print] (IF: 5.6)
Abstract: Mutations in the gene for valosin containing protein (VCP) cause autosomal dominant inclusion body myopathy associated with Paget disease and frontotemporal dementia (IBMPFD). To investigate the role of this novel gene in sporadic forms of frontotemporal dementia (FTD), we genotyped 27 single nucleotide polymorphisms covering the entire VCP genomic region in 198 patients with sporadic FTD and 184 matched controls from Germany. No significant association could be demonstrated. There is no evidence, that common variants in VCP confer a strong risk to the development of sporadic FTD.
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A. Schumacher,
P. Friedrich, J. Diehl-Schmid, B. Ibach, R. Perneczky, T. Eisele, R. Vukovich, H. Foerstl & M.
Riemenschneider (2007)
No association of TDP-43 with sporadic frontotemporal dementia.
Neurobiology of aging Jul 3; [Epub ahead of print] (IF: 5.6)
Abstract: A hyperphosphorylated, ubiquitinated form of TDP-43, known as pathologic TDP-43, was shown to be a central component of ubiquitin-positive, tau-negative and alpha-synuclein-negative inclusions in frontotemporal lobar degeneration (FTLD-U) and amytrophic lateral sclerosis (ALS). To investigate the role of the TDP-43 gene in sporadic forms of frontotemporal dementia (FTD), we genotyped 10 single nucleotide polymorphisms covering the entire TDP-43 genomic region, including the MASP2 gene in 173 patients with sporadic FTD (including 7 patients that were diagnosed with FTD and ALS) and 184 matched controls from Germany. Although we could observe a weak trend towards a disease association in the FTD/ALS patients, no significant association with sporadic FTD could be demonstrated. There is no evidence, that common variants in TDP-43 confer a strong risk to the development of sporadic FTD.
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Other publications:
A. Schumacher(1998) Gametic imprinting and neurogenesis: Analysis of the parental imprinted Snurf/Snrpn gene-cluster in humans and in mouse embryonic stem cells. PhD Thesis (in German!); University Cologne
Abstract: Not available.
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Several other publications are either in press, submitted or in preparation.
