Most of the lysosomal storage diseases analyzed at CENTOGENE are characterized by mutations (defects) of genes that encode enzymes or transport proteins. Lysosomal enzymes are biological active proteins that play a role in degradation of the complex molecules foreign or indigenous to the cell as part of their metabolism. The genetic defect of the respective gene is translated at the protein level (enzyme level) into an impaired or absent function. Thus, molecules which would normally be further processed or degraded by the enzyme, will accumulate – first in the lysosome, then in the cytosol and in the end in the intracellular space. Biochemical analyses can: (i.) assert the specific enzymatic activity, or (ii.) measure the levels of the substrate accumulated in the cells (biomarkers).
Multidisciplinary, step-wise analyses
For a relevant diagnosis, the levels of the biomarkers in the pathologic cases are correlated always with information from the genetic analyses and the enzymatic tests. This helps in confirming the diagnosis (Gaucher and Fabry disease) or to perform a differential diagnosis for Niemann-Pick disease type A/B or C. For some diseases, analyses are performed stepwise, the enzymatic assay is used as screening methods, and, for the pontential patients, it is followed by biomarker quantification and by genetic confirmation.
Although we established classical fluorimetric and spectrophotometric methods available. The detection method of choice is mass spectrometry, more precisely high-resolution multiple reaction monitoring mass spectrometry (high-resolution MRM-MS). MRM-MS, in combination with liquid chromatography, provides high specificity, selectivity, precise quantification of the enzymatic products or of the biomarkers, and requires a minimal amount of biological sample (mainly dried blood spots).
(i.) Determination of enzymatic activity
To determine the enzymatic activity of a biological sample, a small amount of enzyme is extracted from the biological sample (dried blood spots, EDTA blood, leukocytes, fibroblasts) and incubated with a synthetic substrate in conditions that mimic the biology of the enzymatic reaction. The product of the enzymatic assay is detected by different analytical methods such as mass spectrometry, fluorimetry or photometry.
CENTOGENE adapted existing methods and has developed entire new methods for the determination of the enzyme activity in biological samples from patients suffering from lysosomal storage disease such as: Gaucher, Fabry, Farber, Niemann-Pick A/B, Pompe, mucopolysaccharidosis (MPS) 1, MPS 2, MPS 3b, MPS 4A, MPS 4B, MPS 6, metachromatic leukodystrophy (MLD), Wolman.
Each of the biochemical analyses performed in our laboratory require a specific biological material (e.g. blood, leucocytes, protein extract, plasma), but by using protocols developed in our laboratory and techniques capable to quantify in very low amounts of sample ( e.g. mass spectrometry) we adapted most of our assays for the usage of dried blood spots of filter cards (CentoCard®). This enables our clients to send samples easily, at room temperature and at low cost, from any location in the world.
(ii.) Biomarkers quantification
A biomarker is an endogenous molecule linked to clinical manifestation or outcome, that can be reliably quantified, is present in easily accessible clinical samples, realistically reflects burden of the disease, reflects therapeutic measures and elucidates molecular pathogenesis of the disease. Elevated levels of biomarkers in patients are the (direct or indirect) consequence of impaired enzyme/transport protein function, and the severity of the symptoms is in all known cases directly related to their concentration in the tissues. Using mass spectrometry we are able to detect these biomarkers either in dried blood spots or in plasma.
At CENTOGENE, biomarkers quantification methods were developed for the diagnosis, prognosis and treatment evaluation (e.g. enzyme replacement therapy) for the following diseases: Fabry, Gaucher, Farber and Niemann-Pick disease type A/B and C. Moreover, we are continuously working on developing and validating new biomarkers for the diagnosis of lysosomal storage disease. Currently, we have in our pipeline a new biomarker for metachromatic leukodystrophy.
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