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Quick and early detection of rare diseases

Lysosomal storage diseases (LSD) are a group of around 50 rare inherited metabolic disorders. Only 12 LSDs have been described biochemically and microbiologically. Prof. Dr. Michael Przybylski from the Steinbeis Transfer Centre for Biopolymer Analysis and Proteomics at the University of Constance now hopes to change this situation. In a project carried out in cooperation with the biotech company Genzyme CEE Konstanz and the University of Timisoara, Przybylski has developed two highly sensitive molecular methods involving mass spectrometry and fluorimetry and validated them for use in clinical diagnostics. The rapid and simple enzyme tests enable the reliable diagnosis of inherited defects of lysosomal enzymes, including Gaucher’s and Fabry’s disease, already in newborn babies.

Dr. Michael Przybylski is the head of the Steinbeis Transfer Centre for Biopolymer Analysis & Proteomics at the University of Constance © Michael Statnik

Lysosomes are intracellular vesicles that are responsible for breaking up cellular debris as well as high-molecular substances, such as lipids, proteins, glycoproteins, and glycosaminoglycans that have been taken up into the cell. Lysosomal storage diseases such as Gaucher's disease, Niemann-Pick disease, and Fabry's disease are caused by lysosomal dysfunction, usually as a consequence of the deficiency of an enzyme required for the metabolism of lipids etc. Some of the disease symptoms are life-threatening and affect the heart, liver and central nervous system. They can lead to bone deformation, organ enlargement and failure as well as stroke. LSDs occur with an incidence of around 1 out of 75001 newborns. Specific enzyme replacement therapies have been available for quite some years for the treatment of Gaucher's disease, Fabry's disease and mucopolysaccharidose type I. These therapies were developed by the biotechnology company Genzyme. In general, it is important to diagnose LSDs at a very early stage, since enzyme replacement therapy is of no effect when the diseases are already in an advanced stage.

Two methods, one result

The collection of blood samples on paper, known as dried blood spots ('dry-blood-spot' procedure) © Laboratory of Anal. Chemistry/M. Przybylski

Prof. Michael Przybylski has developed diagnostic methods at the Steinbeis Transfer Centre for Biopolymer Analysis & Proteomics at the University of Constance. In cooperation with Genzyme CEE and the University of Timisoara (Romania) the methods will be introduced in the long-term at Central and Eastern European hospitals and laboratories for the screening of Fabry's disease. "The test is a simple and quick blood test. A few microlitres of blood are adsorbed by a special membrane filter and the enzyme values are then determined," said Przybylski explaining the ‘dry-blood-spot' (DBS) method. Sample extraction involves the use of DBS sample aliquots with a diameter of three millimetres. These aliquots can be used for repeated measurements.

Scientists at the Steinbeis Transfer Centre and its partners are developing two complementary methods for use in diagnostics. The tests, which are both reliable and unambiguous, are based on fluorimetric determination and tandem mass spectrometry. "Within a few minutes, we can find out whether the enzyme concentration is in the standard range of healthy people or whether it is basically zero, which means that the patient is severely ill," said Prof. Michael Przybylski explaining that the tests involve two specific procedures that have been tested by the German and Romanian scientists on around 350 healthy volunteers and they delivered identical results of high accuracy.

The method, involving a tandem mass spectrometer, analyses the products that are produced to a lesser degree in patients lacking a lysosomal enzyme. The quantitative determination of the product of a structural analogue of the natural enzyme substrate, for example galactosylceramide in the case of Fabry's disease (α-galactosidase), is done with an isotope-labelled internal standard. Fabry's disease is caused by the deficiency or dysfunction of the enzyme alpha-galactosidase. The enzymatic reaction is monitored using the ‘multiple reaction monitoring' (MRM) procedure involving tandem mass spectrometry, which is based on the fragmentation of ions and used for measuring the enzymatic conversion of substrates to products. "We compare the signal intensities of a product ion with those of an isotope-labelled standard ion (e.g., 13C; 15N), which are chemically identical."

Since this procedure requires detailed analytical knowledge as well as expensive and comprehensive technical equipment, which Eastern European hospitals often do not have, Przybylski and his partners have developed a second method, which is based on fluorimetry (fluorescence spectrometry). This method determines the enzyme activity using a fluorescent substrate analogue. “4-methylumbelliferone is produced by the enzymatic cleavage of galactosyl substrate. We can determine the activity of alpha-galactosidase from the fluorescent product following the addition of substrate,” explained Prof. Michael Przybylski. The fluorimetry-based method can thus be carried out by a laboratory assistant with the simpler devices available in Eastern Europe and is suitable for rapid clinical screening diagnostics.

To date, lysosomal storage diseases are mainly analysed using time-consuming genetic methods, followed by the characterisation of proteins. “We use specific analytical methods with which we can isolate and identify proteins that are the cause of certain lysosomal storage diseases right from the start,” highlights Prof. Michael Przybylski.

The method has already been clinically tested on 300 blood samples of healthy and Fabry’s disease patients for its suitability to diagnose Fabry’s disease, and enabled the partners to optimise and validate the two methods.

Protein misfolding and aggregation is still unknown

The major goals of the cooperative project, which was started at the end of 2009, also focus on the identification of target proteins of known lysosomal storage diseases on the molecular level and the structural characterisation of lysosomal protein aggregations found in human glycosylation diseases. "We are focusing on the protein beta-glucocerebrosidase (GCase), which is found in the lysosome. The mutated form of the protein causes Gaucher's disease." Gaucher's disease is the most common lysosomal glycosphingolipidosis. It is characterised by the accumulation of glucocerebroside in the macrophages. The team of researchers has plans to elucidate the basic mechanisms that lead to the instability/reactivity of the aggregation products and clarify the reaction pathways in detail. For doing so, the researchers use ion mobility mass spectrometry for the analysis of the proteins. The method is relatively new and can be used to characterise the spatial arrangement of proteins. "The defective folding of GCase is also associated with neurodegenerative diseases such as Parkinson's disease," said Przybylski. The structure and reactivity/aggregation of more than 50 lysosomal enzymes that are potentially involved in defective protein folding and aggregation are not yet known.

The findings provide a basis for diagnostic methods that can be used to determine not only the deficiency of one enzyme, but also the lack of several enzymes simultaneously. The large number of symptoms associated with lysosomal storage diseases and the fact that the sugar-metabolising enzymes affected are found in almost all organs, makes the diagnosis of such diseases still very difficult.

1  E. Paschke: Untersuchungen zur frühzeitigen Diagnose und Prognose des Phänotyps bei lysosomalen Speichererkrankungen. Project description as of 1 January 2009

Further information:
Prof. Dr. Dr.h.c. Michael Przybylski
University of Konstanz
Department of Chemistry
Laboratory of Analytical Chemistry
and Biopolymer Structure Analysis
D-78457 Konstanz
Tel.: +49- 7531 882249
Fax: +49- 7531 883097
E-mail: Michael.Przybylski(at)uni-konstanz.de

Website address: https://www.gesundheitsindustrie-bw.de/en/article/news/quick-and-early-detection-of-rare-diseases