Clinical diagnosis of lysosomal storage diseases

Steinbeis and partners analyze inborn errors of metabolism

Lysosomal storage diseases (LSDs) are a group of mostly inherited metabolic disorders caused by the malfunction of lysosomal enzymes. If left untreated, LSDs are terminal – frequently within several years. Since efficient enzyme replacement therapy (ERT) has become amenable for a number of LSDs, such as Gaucher’s Disease and Fabry’s Disease, rapid and unequivocal diagnosis is critically important. In collaboration with Genzyme CEE and the University of Timis¸oara in Romania, the Steinbeis Transfer Center for Biopolymer Analysis, Proteomics and Protein Chemistry at the University of Konstanz has developed highly sensitive molecular diagnostic methods for LSDs using both mass spectrometry and fluorescence spectroscopy, and has validated these methods for use in clinical diagnosis.

Lysosomal storage diseases are a group of mostly inherited metabolic disorders caused by the malfunction or failure of lysosomal enzymes. As a consequence, the corresponding substrates are not metabolized and thus accumulate in lysosomes, leading to severe pathophysiological effects including hepatomegaly and splenomegaly (enlargement of the liver and spleen), cardiac muscle atrophy, respiratory diseases and multiple organ failure. If left untreated, LSDs are frequently terminal. Of the approximately fifty hitherto known LSDs, some are amenable to treatment using enzyme replacement therapy, including Fabrys Disease, Gauchers disease and glycogen storage disease type II. Genzyme has recently developed highly efficient ERTs for treatment of Gauchers Disease and Fabrys Disease, which present high chances for curative success if early and confirmed diagnosis is available.

Until now, rapid and accurate diagnosis of LSDs is frequently a major problem, and children with undetected LSDs may die before treatment is amenable. In the case of positive biochemical diagnosis, genetic diagnosis can be performed for confirmation and can be extended to the patients family. Aim of the present study of the Steinbeis Center for Biopolymer Analysis at the University of Konstanz in collaboration with its partners was (i), the development of efficient molecular tools for the clinical diagnosis of LSDs using specific quantitative determination of enzyme activity in blood, and (ii), to validate and establish these methods for clinical application. Furthermore, diagnostic applications were initiated in collaboration with Clinical Departments in Central and Eastern Europe in which no corresponding methods for diagnostic confirmation had been hitherto amenable.

For accurate and sensitive clinical diagnostics of LSDs in blood, techniques for sample preparation and analysis were developed based on the dried blood spot (DBS) method. Using the DBS method, blood samples of approximately 30 microlitres were absorbed onto a membrane filter (protein saver card), which in dried and air-tight form are stable for long-term storage and transport. For sample extraction, disk aliquots of 3 mm diameter were punched out of the DBS card and used for multiple determinations (see Figure 1). Two different methods were developed and evaluated in complementary manner for diagnosis of LSDs:

  • Specific determination of enzyme activity using tandem mass spectrometry. With the mass spectrometric method, quantitative determination of the product formation of a suitable structural analogue of the natural substrate is performed using an isotopically labelled internal standard (galactosyl ceramide for α-galactosidase determination in the case of Fabrys Disease)
  • Fluorimetric determination of enzyme activity using a fluorescent substrate analogue. In the enzymatic cleavage of the galactosyl-substrate for determination of α-galactodidase, formation of the product 4-methylumbelliferone (4-MU) is measured. The substrate is placed in a microtitre plate, and following addition of the DBS the pH brought to 4.5 using a buffer solution. Fluorimetric determination of 4-MU is performed at 360 nm excitation, and an emission wavelength of 400 nm. Thus the fluorimetric determination is suitable for rapid clinical screening analysis.

HPLC tandem mass spectrometry analyzes specific fragments of the product of substrate conversion of the respective enzyme. This approach, referred to as Multiple Reaction Monitoring (MRM) enables highly specific (absolute) diagnostic determinations; the MRM technique is feasible to determine several target enzymes thus suitable for simultaneous diagnosis of several LSDs. Initial clinical diagnostics of Fabrys Disease (FD) were performed by double-blind procedure on DBS samples from healthy control persons and FD patients from Clinical Departments of several Universities. The sensitivity, accuracy and reproducibility of α-galactosidase determination was validated with approximately 350 control samples, using both the mass spectrometric and fluorimetric methods. In this study two patients with Fabrys Disease were clearly diagnosed; in addition patients were identified as high-risk cases of FD by their low blood levels, and thus could be classified to further detailed diagnostic determination.

In the present study methods of high sensitivity and specificity have been developed and validated for the clinical diagnosis of target enzymes for Lysosomal Storage Diseases. These approaches providing diagnostic determinations for LSDs with known target enzymes will also enable the development and validation of mass spectrometric methods for identifying biomarkers of LSDs for which presently no quantifications are amenable. Corresponding diagnostic methods are particularly relevant for LSDs in which therapeutic modalities by enzyme replacement are already available. Of particular interest are most recent reports that provide initial proof of the connection between LSD target enzymes and the aggregation of key proteins for neurodegenerative diseases, such as Parkinsons disease, in agreement with established neuropathological effects of LSDs. Protein-analytical methods pursued at the Steinbeis Transfer Center for Biopolymer Analysis Konstanz should be suitable to elucidate the structures of aggregation products of LSD target enzymes.


Claudia Cozma | Sebastian Dilly | Marius Iurascu |
Prof. Dr. Michael Przybylski

Steinbeis Transfer Center Biopolymer Analysis/Protein Chemistry and Proteom Analysis Konstanz (Konstanz)

Christina Mosoarka | Mirela Galusca |
Prof. Dr. Alina Zamfir

Laboratory of Biomolecular Mass Spectrometry
University of Timisoara, Rumänien

Dr. Thomas Fritz | Dr. Michael März |
Dr. Sybille Petersohn

Genzyme CEE (Konstanz)

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