DNA methylation analysis

Epigenetic processes regulate chromatin conformation and gene expression without altering the nucleotide sequence of the DNA. DNA methylation is a heritable epigenetic mark playing an important role in cell differentiation. In general, hypermethylation of the promoter region of genes is associated with gene silencing.

Environmental factors, e.g. diet, exposure to toxic substances and stressful life events have been shown to alter gene expression via changes in DNA methylation. Aberrant DNA methylation patterns have been detected in a variety of complex diseases, including neurodegenerative and autoimmune disorders and cancer. Silencing of tumor suppressor genes by promoter hypermethylation is considered as one of the earliest events during carcinogenesis.

Our group deals with the development and validation of analytical methods based on methylation-sensitive high resolution melting (MS-HRM) analysis and pyrosequencing for determining the DNA methylation status in the promoter region of candidate genes.

The methods are applied to the following topics:

• We investigate if the promoter methylation status of tumor suppressor genes (e.g. APC, BRCA1, HIN-1, RASSF1A, GSTP1 and MGMT) in tumor tissues of breast cancer patients differs from that in adjacent and histologically normal breast tissues. Alterations in the DNA methylation pattern may explain why histologically normal tissues are at risk for local recurrence. (in co-operation with Georg Pfeiler, Medical University of Vienna, Department of Obstetrics and Gynaecology).

• In co-operation with Bernhard Keppler (University of Vienna, Institute of Inorganic Chemistry) and Walter Berger (Medical University of Vienna, Institute of Cancer Research) we investigate if drugs with antitumor activity influence the DNA methylation status of candidate genes, e.g. ABCB1, in multidrug resistant cancer cells.

• Statins and bisphosphonates, frequently prescribed for lowering cholesterol levels and treating osteoporosis, respectively, have been found to show anticancer activity by influencing apoptosis, cell proliferation, angiogenesis and metastasis. In co-operation with Franz Varga (Ludwig Boltzmann Institute of Osteology, Vienna) we investigate the influence of statins and bisphosphonates on DNA methylation of candidate genes in order to elucidate their exact mode of action.

• Recent evidence suggests that epigenetic mechanisms, including DNA methylation, are essential regulators of neuronal plasticity. In co-operation with Gert Lubec (Medical University of Vienna, The Gert Lubec Proteomics Laboratory), we investigate if spatial learning of rats (eg. in the Morris water maze) leads to DNA methylation changes of candidate genes, e.g. AMPA receptors, BDNF and reelin, in the hippocampus.

Food allergen analysis

Food allergens may elicit severe allergic reactions in sensitized persons, including life-threatening anaphylaxis. The only option for allergic patients is to strictly avoid the consumption of the food they are allergic to. In order to make diet planning easier, 14 allergenic foods and products thereof have to be declared in the European Union according to the Directive 2007/68/EC.

Our group deals with the development and validation of analytical methods allowing to control the implementation of the legal regulations. In order to be applicable, the analytical methods have to be selective, sensitive and suitable for both raw and highly processed foodstuffs. In particular, we aim at the development and validation of enzyme linked immunosorbent assays (ELISAs) and methods based on PCR. Recently, competitive and sandwich ELISAs have been developed for the determination of sesame and lupine in food. Currently, we are developing a real-time PCR assay for potentially allergenic poppy seeds.

Real-time PCR assays for mustard and celery have been developed and validated in co-operation with Rupert Hochegger (Austrian Agency for Health and Food Safety (AGES), Institute for Food Safety, Department of Molecularbiology and Microbiology, Vienna)). Our highlight is a triplex real-time PCR assay allowing the simultaneous detection of white, black and brown mustard and celery in one and the same PCR tube.

Detection of game meat adulteration

Food manufacturers not only have to ensure the safety but also the authenticity of their food products. Game meat is particularly susceptible for fraudulent labelling since it is deemed to be more valuable than meat of domestic animals. In addition, it is especially appreciated due to its distinctive flavour and its low fat and cholesterol content.

According to the Codex Alimentarius Austriacus, in sausages that are declared as “game sausages” the proportion of game meat has to be ≥ 38% of the total meat content. Analytical methods applicable for the detection of game meat adulteration must therefore allow not only the identification of meat species but also the quantitative determination of the meat content in foods. Quantification of meat species is, however, known to be a difficult task.

In co-operation with Rupert Hochegger (AGES) we have recently developed and validated a real-time PCR method for the quantification of roe deer as well as a single real-time PCR method to detect and quantify adulteration of fallow deer, red deer and sika deer.

Determination of midazolam and its main metabolites in biological samples

Midazolam is a sedative routinely used in intensive care patients. Due to its pharmacological properties, i.e. a half-life of about 3 hours and only minimal influence on hemodynamics, its use in sedation is widely accepted, even for long periods. In patients with renal and liver failure, metabolites of midazolam may, however, accumulate, leading to unwarranted prolongation of sedation.

In co-operation with Michael Hiesmayr (Medical University of Vienna) we are currently developing a HPLC method allowing the separation and quantification of midazolam and its metabolites 1-hydroxymidazolam, 4-hydroxymidazolam, 1-hydroxymidazolam-β-D-glucuronide and 4- hydroxymidazolam-β-D-glucuronide in serum and urine samples. The HPLC method will then be applied to investigate the pharmacokinetic of midazolam in patients with renal and liver failure.