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In higher eukaryotic organisms epigenetic modifications are crucial for proper chromatin folding and thereby proper regulation of gene expression. In the last years the involvement of aberrant epigenetic modifications in inflammatory and autoimmune diseases has been recognized and attracted significant interest. However, the epigenetic mechanisms underlying the different disease phenotypes are still poorly understood. As autoimmune and inflammatory diseases are at least partly T cell mediated, we will provide in this chapter an introduction to the epigenetics of T cell differentiation followed by a summary of the current knowledge on aberrant epigenetic modifications that dysfunctional T cells display in various diseases such as type 1 diabetes, rheumatoid arthritis, systemic lupus erythematosus, multiple sclerosis, inflammatory bowel disease, and asthma.

Parathyroid hormone-related protein (PTHrP) is upregulated in primary breast cancers and a major candidate for osteoclastic bone resorption present at sites of breast cancer to bone metastases. Using a human model of mammary epithelial cell lines differing in tumorigenicity and PTHrP expression, we investigated the role of epigenetic modifications for PTHrP expression. Quantitative analysis of the DNA methylation patterns at a total of 104 CpGs in the promoter region of PTHrP by pyrosequencing showed the absence of methylation in all analyzed cell lines in the large CpG island upstream of exon 1C. In the second intron of promoter 2 (P2) a region was identified containing 4 CpG nucleotides for which differential methylation correlated with the PTHrP expression level. The functional importance of this control mechanism was confirmed by the ability of the demethylating agent 5'-azacytidine to induce PTHrP mRNA and iPTHrP protein expression in previously non-expressing cell lines and increase their production by metastatic NS2T2A1 cells. In particular, transcription from P2 was activated non-tumoral S1T3 cells upon treatment with 5'-azacytidine. Our findings support the hypothesis that the methylation status of specific CpG dinucleotides is the dominant mechanism involved in silencing of PTHrP expression rather than the overall methylation of the CpG island. Methylation of the PTHrP P2 is a potential marker of breast cancer progression and might be used to evaluate the metastatic potential of breast tumors.

Preeclampsia (PE) and vascular intra-uterine growth restriction (vIUGR) are two pathological obstetrical conditions originating from placental dysfunction. Recently, methylation changes at the placental level have been shown to be indicative of these diseases. The alteration of such epigenetic marks is therefore a novel pathway that might be critical for these pathologies. Here, we identified a region located in the distal promoter of the T-box-containing transcription factor TBX15 that is differentially methylated in pathological placentas. The level of methylation correlated significantly with the weight and stature of the newborn. The promoter was found to be hypomethylated in vIUGR coinciding with the down-regulation of its expression. PDX1, a transcription factor important for the regulation of insulin metabolism regulation was able to repress the TBX15 promoter in a methylation-dependent manner, which might, at least partially, explain the specific mRNA decrease of TBX15 observed in vIUGR placentas. Overall, the data presented herein suggest that TBX15 might be involved in the pathophysiology of placental diseases.

Mass spectrometry-based analysis techniques are widely applied in proteomics. This study presents a novel method for quantitative multiplex candidate protein profiling. It applies immunocapture of differentially labeled protein complements on hydrogel antibody arrays and subsequent quantification by MS. To make this approach quantitative a labeling approach was devised. The impact of labeling on the antibody/antigen interaction was assessed in detail by surface plasmon resonance. Owing to there solution by mass more than two protein samples can be compared simultaneously. Direct labeling of crude samples such as sera was developed and so enables the absolute quantification of target proteins straight from crude samples without a protein purification step. It was used to measure the concentration of apolipoprotein A-1 in serum. This method has been termed A2M2S for Affinity Array sand MALDI Mass Spectrometry.

The O(6)-methylguanine-DNA methyltransferase gene (MGMT) is methylated in several cancers, including gliomas. However, the functional role of cysteine-phosphate-guanine (CpG) island (CGI) methylation in MGMT silencing is still controversial. The aim of this study was to investigate whether MGMT CGI methylation correlates inversely with RNA expression of MGMT in glioblastomas and to determine the CpG region whose methylation best reflects the level of expression. The methylation level of CpG sites that are potentially related to expression was investigated in 54 glioblastomas by pyrosequencing, a highly quantitative method, and analyzed with respect to their MGMT mRNA expression status. Three groups of patients were identified according to the methylation pattern of all 52 analyzed CpG sites. Overall, an 85% rate of concordance was observed between methylation and expression (p < 0.0001). When analyzing each CpG separately, six CpG sites were highly correlated with expression (p < 0.0001), and two CpG regions could be used as surrogate markers for RNA expression in 81.5% of the patients. This study indicates that there is good statistical agreement between MGMT methylation and expression, and that some CpG regions better reflect MGMT expression than do others. However, if transcriptional repression is the key mechanism in explaining the higher chemosensitivity of MGMT-methylated tumors, a substantial rate of discordance should lead clinicians to be cautious when deciding on a therapeutic strategy based on MGMT methylation status alone.

Background: Using array comparative genomic hybridization (aCGH), a large number of deleted genomic regions have been identified in human cancers. However, subsequent efforts to identify target genes selected for inactivation in these regions have often been challenging., Methods: We integrated here genome-wide copy number data with gene expression data and non-sense mediated mRNA decay rates in breast cancer cell lines to prioritize gene candidates that are likely to be tumour suppressor genes inactivated by bi-allelic genetic events. The candidates were sequenced to identify potential mutations., Results: This integrated genomic approach led to the identification of RIC8A at 11p15 as a putative candidate target gene for the genomic deletion in the ZR-75-1 breast cancer cell line. We identified a truncating mutation in this cell line, leading to loss of expression and rapid decay of the transcript. We screened 127 breast cancers for RIC8A mutations, but did not find any pathogenic mutations. No promoter hypermethylation in these tumours was detected either. However, analysis of gene expression data from breast tumours identified a small group of aggressive tumours that displayed low levels of RIC8A transcripts. qRT-PCR analysis of 38 breast tumours showed a strong association between low RIC8A expression and the presence of TP53 mutations (P = 0.006)., Conclusion: We demonstrate a data integration strategy leading to the identification of RIC8A as a gene undergoing a classical double-hit genetic inactivation in a breast cancer cell line, as well as in vivo evidence of loss of RIC8A expression in a subgroup of aggressive TP53 mutant breast cancers.

DNA methylation occurring on the 5 position of the pyrimidine ring of cytosines in the context of the dinucleotide sequence CpG forms one of the multiple layers of epigenetic mechanisms controlling and modulating gene expression through chromatin structure. It closely interacts with histone modifications and chromatin-remodeling complexes to form the genomic chromatin landscape. DNA methylation is essential for proper mammalian development, crucial for imprinting, and plays a role in maintaining genomic stability as well as in dosage compensation. DNA methylation patterns are susceptible to change in response to environmental stimuli such as diet or toxins whereby the epigenome seems to be most vulnerable during early in utero development. Aberrant DNA methylation changes have been detected in several diseases, particularly cancer where genome-wide hypomethylation coincides with gene-specific hypermethylation. DNA methylation patterns can be used to detect cancer at very early stages, to classify tumors as well as predict and monitor the response to antineoplastic treatment. As a stable nucleic acid-based modification with limited dynamic range that is technically easy to handle, DNA methylation is a promising biomarker for many applications.

The global increase in lung cancer burden, together with its poor survival and resistance to classical chemotherapy, underscores the need for identification of critical molecular events involved in lung carcinogenesis. Here, we have applied quantitative profiling of DNA methylation states in a panel of five cancer-associated genes (CDH1, CDKN2A, GSTP1, MTHFR, and RASSF1A) to a large case-control study of lung cancer. Our analyses revealed a high frequency of aberrant hypermethylation of MTHFR, RASSF1A, and CDKN2A in lung tumors as compared with control blood samples, whereas no significant increase in methylation levels of GSTP1 and CDH1 was observed, consistent with the notion that aberrant DNA methylation occurs in a tumor-specific and gene-specific manner. Importantly, we found that tobacco smoking, sex, and alcohol intake had a strong influence on the methylation levels of distinct genes (RASSF1A and MTHFR), whereas folate intake, age, and histologic subtype had no significant influence on methylation states. We observed a strong association between MTHFR hypermethylation in lung cancer and tobacco smoking, whereas methylation levels of CDH1, CDKN2A, GSTP1, and RASSF1A were not associated with smoking, indicating that tobacco smoke targets specific genes for hypermethylation. We also found that methylation levels in RASSF1A, but not the other genes under study, were influenced by sex, with males showing higher levels of methylation. Together, this study identifies aberrant DNA methylation patterns in lung cancer and thus exemplifies the mechanism by which environmental factors may interact with key genes involved in tumor suppression and contribute to lung cancer.

Most available protocols for gene-specific DNA methylation analysis are either labor intensive, not quantitative, or limited to the measurement of the methylation status of only one or very few CpG positions. Pyrosequencing is a real-time sequencing technology that overcomes these limitations. After bisulfite modification of genomic DNA, a region of interest is amplified by polymerase chain reaction (PCR) with one of the two primers being biotinylated. The PCR-generated template is rendered single stranded and a pyrosequencing primer is annealed to analyze quantitatively CpGs within 120 bases. Advantages of the pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain high reproducibility and avoid random amplification. The required DNA amount can be provided by an individual sample or a pool of samples to rapidly investigate the presence of variable DNA methylation patterns. The use of pools and serial pyrosequencing, that is, the successive use of several pyrosequencing primers on the same DNA template, significantly reduces cost, labor, and analysis time as well as saving precious DNA samples for the analysis of gene-specific DNA methylation patterns.

The CpG island spanning the transcription start of the glutathione S-transferase P1 becomes methylated in a variety of human cancers including breast cancer. To study the effect of sequence variation on hypermethylation of the GSTP1 promoter, we analyzed the genetic and epigenetic variability in 90 tumors from patients with locally advanced breast cancer. High-resolution quantitative analysis revealed large variability in the DNA methylation levels. Lack of methylation was more often observed in the basal and normal-like estrogen receptor (ER)-negative tumors, and methylated GSTP1 was associated with better overall survival (P = 0.00063). Studies of the genetic variation identified 14 different haplotypes. The distribution of methylation levels of tumors homozygous for the most frequent haplotype was significantly different from other haplotype combinations (P = 0.011), the difference being more pronounced in ER-positive (P = 0.005) and progesterone receptor-positive (P = 0.008) tumors. Regression modeling identified the ER status and haplotype as the main determinants of DNA methylation variability. We identified a putative c-Myb response element (MRE) that was present in one of two minimal promoter haplotypes. In vitro analysis showed that c-Myb binds to the MRE, but binding was weakened by the two polymorphisms. Transient cotransfections in luminal-type and basal-like breast cancer cell lines confirmed cell-specific differential binding of c-Myb to the polymorphic sites, leading to a change in the expression from the GSTP1 promoter in vivo. GSTP1 expression was moderately but significantly (P = 0.01) reduced after siRNA-mediated knockdown of c-Myb. Our results indicate that haplotype structure of a promoter is important for the extent of DNA methylation.

Aberrant DNA methylation patterns have been identified in a variety of human diseases, particularly cancer. Pyrosequencing has evolved in recent years as a sensitive and accurate method for the analysis and quantification of the degree of DNA methylation in specific target regions. However, the number of candidate genes that can be analyzed in clinical specimens is often restricted by the limited amount of sample available. Here, we present a novel screening approach that enables the rapid identification of differentially methylated regions such as promoters by pyrosequencing of etiologically homogeneous sample pools after bisulfite treatment. We exemplify its use by the analysis of five genes (CDKN2A, GSTP1, MLH1, IGF2, and CTNNB1) involved in the pathogenesis of human hepatocellular carcinoma using pools stratified for different parameters of clinical importance. Results were confirmed by the individual analysis of the samples. The screening identified all genes displaying differential methylation successfully, and no false positives occurred. Quantitative comparison of the pools and the samples in the pool analyzed individually showed a deviation of approximately 1.5%, making the method ideally suited for the identification of diagnostic markers based on DNA methylation while saving precious DNA material.

Pyrosequencing is a sequencing-by-synthesis method that quantitatively monitors the real-time incorporation of nucleotides through the enzymatic conversion of released pyrophosphate into a proportional light signal. Quantitative measures are of special importance for DNA methylation analysis in various developmental and pathological situations. Analysis of DNA methylation patterns by pyrosequencing combines a simple reaction protocol with reproducible and accurate measures of the degree of methylation at several CpGs in close proximity with high quantitative resolution. After bisulfite treatment and PCR, the degree of each methylation at each CpG position in a sequence is determined from the ratio of T and C. The process of purification and sequencing can be repeated for the same template to analyze other CpGs in the same amplification product. Quantitative epigenotypes are obtained using this protocol in approximately 4 h for up to 96 DNA samples when bisulfite-treated DNA is already available as the starting material.

As the sequence of the human genome is now nearly finished, genome research turns to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the pathogenesis of various diseases. The real-time luminometric detection of pyrophosphate release upon nucleotide incorporation in the Pyrosequencing technology is ideally suited for the simultaneous analysis and quantification of the methylation degree of several CpG positions in close proximity. We developed and improved this analysis to obtain reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 80 nt. Advantages of the Pyrosequencing technology are the ease of its implementation, the high quality and the quantitative nature of the results, and its ability to identify differentially methylated positions in close proximity, which may be used as DNA methylation markers.

The analysis of deoxyribose nucleic acid (DNA) by mass spectrometry (MS) has evolved to where it can be used to analyze most known types of DNA and ribose nucleic acid (RNA) situations. It can efficiently deal with the analysis of DNA polymorphisms, sequences, haplotypes, human leukocyte antigen (HLA) typing, DNA methylation and RNA expression. Implementations of MS for these forms of DNA analyses are reviewed. The use of DNA analysis by MS is compared with competing technologies. Finally, an overview is given of worthwhile applications where the know-how gained so far could be used for future developments., (Copyright 2006 John Wiley & Sons, Ltd.)

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.

Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry has become one of the most powerful and widely applied technologies for SNP scoring and determination of allele frequencies in the post-genome sequencing era. Although different strategies for allele discrimination combined with MALDI were devised, in practice only primer extension methods are nowadays routinely used. This combination enables the rapid, quantitative, and direct detection of several genetic markers simultaneously in a broad variety of biological samples. In the field of molecular diagnostics, MALDI has been applied to the discovery of genetic markers, that are associated with a phenotype like a disease susceptibility or drug response, as well as an alternative means for diagnostic testing of a range of diseases for which the responsible mutations are already known. It is one of the first techniques with which whole genome scans based on single nucleotide polymorphisms were carried out. It is equally well suited for pathogen identification and the detection of emerging mutant strains as well as for the characterization of the genetic identity and quantitative trait loci mapping in farm animals. MALDI can also be used as a detection platform for a range of novel applications that are more demanding than standard SNP genotyping such as mutation/polymorphism discovery, molecular haplotyping, analysis of DNA methylation, and expression profiling. This review gives an introduction to the application of mass spectrometry for DNA analysis, and provides an overview of most studies using SNPs as genetic markers and MALDI mass spectrometric detection that are related to clinical applications and molecular diagnostics. Further, it aims to show specialized applications that might lead to diagnostic applications in the future. It does not speculate on whether this methodology will ever reach the diagnostic market.

The Human Epigenome Project aims to identify, catalogue, and interpret genome-wide DNA methylation phenomena. Occurring naturally on cytosine bases at cytosine-guanine dinucleotides, DNA methylation is intimately involved in diverse biological processes and the aetiology of many diseases. Differentially methylated cytosines give rise to distinct profiles, thought to be specific for gene activity, tissue type, and disease state. The identification of such methylation variable positions will significantly improve our understanding of genome biology and our ability to diagnose disease. Here, we report the results of the pilot study for the Human Epigenome Project entailing the methylation analysis of the human major histocompatibility complex. This study involved the development of an integrated pipeline for high-throughput methylation analysis using bisulphite DNA sequencing, discovery of methylation variable positions, epigenotyping by matrix-assisted laser desorption/ionisation mass spectrometry, and development of an integrated public database available at http://www.epigenome.org. Our analysis of DNA methylation levels within the major histocompatibility complex, including regulatory exonic and intronic regions associated with 90 genes in multiple tissues and individuals, reveals a bimodal distribution of methylation profiles (i.e., the vast majority of the analysed regions were either hypo- or hypermethylated), tissue specificity, inter-individual variation, and correlation with independent gene expression data., Competing Interests: JL, KB, TH, TO, and AO are employees of Epigenomics AG. IGG and SB are members of the scientific advisory board of Epigenomics AG, but do not benefit financially from their involvement in this study. Epigenomics AG has filed for patents based on some of the work described here. Epigenomics AG was a scientific collaborator in the study. The work described here was funded by a grant from the European Union Framework 5 Programme.

Current protocols for DNA methylation analysis are either labor intensive or limited to the measurement of only one or two CpG positions. Pyrosequencing is a real-time sequencing technology that can overcome these limitations and be used as an epigenotype-mapping tool. Initial experiments demonstrated reliable quantification of the degree of DNA methylation when 2-6 CpGs were analyzed. We sought to improve the sequencing protocol so as to analyze as many CpGs as possible in a single sequencing run. By using an improved enzyme mix and adding single-stranded DNA-binding protein to the reaction, we obtained reproducible results for as many as 10 successive CpGs in a single sequencing reaction spanning up to 75 nucleotides. A minimum amount of 10 ng of bisulfite-treated DNA is necessary to obtain good reproducibility and avoid preferential amplification. We applied the assay to the analysis of DNA methylation patterns in four CpG islands in the vicinity of IGF2 and H19 genes. This allowed accurate and quantitative de novo sequencing of the methylation state of each CpG, showing reproducible variations of methylation state in contiguous CpGs, and proved to be a useful adjunct to current technologies.

As the DNA sequence of the human genome is now nearly finished, the main task of genome research is to elucidate gene function and regulation. DNA methylation is of particular importance for gene regulation and is strongly implicated in the development of cancer. Even minor changes in the degree of methylation can have severe consequences. An accurate quantification of the methylation status at any given position of the genome is a powerful diagnostic indicator. Here we present the first assay for the analysis and precise quantification of methylation on CpG positions in simplex and multiplex reactions based on matrix-assisted laser desorption/ ionisation mass spectrometry detection. Calibration curves for CpGs in two genes were established and an algorithm was developed to account for systematic fluctuations. Regression analysis gave R(2) >or= 0.99 and standard deviation around 2% for the different positions. The limit of detection was approximately 5% for the minor isomer. Calibrations showed no significant differences when carried out as simplex or multiplex analyses. All variable parameters were thoroughly investigated, several paraffin-embedded tissue biopsies were analysed and results were verified by established methods like analysis of cloned material. Mass spectrometric results were also compared to chip hybridisation.

In the last decade, the demand for high-throughput DNA analysis methods has dramatically increased, mainly due to the advent of the human genome sequencing project that is now nearing completion. Even though mass spectrometry did not contribute to that project, it is clear that it will have an important role in the post-genome sequencing era, in genomics and proteomics. In genomics, mainly matrix-assisted laser desorption/ionization (MALDI) mass spectrometry will contribute to large-scale single nucleotide polymorphism (SNP) genotyping projects. Here, the development and history of DNA analysis by mass spectrometry is reviewed and put into the context with the requirements of genomics. All major contributions to the field and their status and limitations are described in detail., (Copyright 2003 Wiley Periodicals, Inc.)

Reconstruction of haplotypes, or the allelic phase, of single nucleotide polymorphisms (SNPs) is a key component of studies aimed at the identification and dissection of genetic factors involved in complex genetic traits. In humans, this often involves investigation of SNPs in case/control or other cohorts in which the haplotypes can only be partially inferred from genotypes by statistical approaches with resulting loss of power. Moreover, alternative statistical methodologies can lead to different evaluations of the most probable haplotypes present, and different haplotype frequency estimates when data are ambiguous. Given the cost and complexity of SNP studies, a robust and easy-to-use molecular technique that allows haplotypes to be determined directly from individual DNA samples would have wide applicability. Here, we present a reliable, automated and high-throughput method for molecular haplotyping in 2 kb, and potentially longer, sequence segments that is based on the physical determination of the phase of SNP alleles on either of the individual paternal haploids. We demonstrate that molecular haplotyping with this technique is not more complicated than SNP genotyping when implemented by matrix-assisted laser desorption/ionisation mass spectrometry, and we also show that the method can be applied using other DNA variation detection platforms. Molecular haplotyping is illustrated on the well-described beta(2)-adrenergic receptor gene.