Purpose: Glycogen Synthase is a glycosyltransferase and key enzyme in glycogenesis, converting glucose into glycogen for storage and therefore playing a major role in regulating glycogen/glucose levels. Mutations in the GYS1 gene are associated with a variety of metabolic disorders in human, foremost glycogen storage disease type 0, diabetes and hyperglycemia.
Lafora disease (Lafora progressive myoclonic epilepsy; MELF) is a currently incurable, fatal glycogen metabolism disorder and neurodegenerative disease, affecting neurons, liver, skin, heart and muscle cells in human and canines. Symptoms mostly begin in late childhood with epileptic seizures and include muscle spasms (myoclonus), difficulty walking, and dementia. Patients experience rapid cognitive deterioration and the disease is often fatal within 10 years of onset casing death before the age of twenty-five.
Here we present a clinical investigation if Glycogen Synthase can be employed as prognostic biomarker for Lafora disease by bioanalytical sample testing in cerebrospinal fluid (CSF) from healthy and diseased children, including bioanalytical assay development to provide the needed sensitivity at minimized sample volume requirement.
Methods: Human CSF samples were collected by lumbar puncture from children and stored frozen at -80°C. Approx. 10 samples from healthy volunteers and 30 samples from Lafora patients were selected for bioanalysis with a custom developed Immuno-PCR (IPCR; Imperacer®) ligand-binding assay (LBA). The Imperacer® IPCR platform combines the specificity of antibody-target binding with extreme signal generation of DNA analytics by exponential PCR amplification of a double-stranded DNA antibody label. High sensitivity in combination with broad assay quantification range make Imperacer® a promising analytical platform to fulfill sensitivity requirements at the lowest possible sample consumption, for patient safety and welfare in juvenile populations.
Results: Assay development on the Imperacer® IPCR immunoassay platform initially focused on sensitivity to quantify endogenous levels of Glycogen Synthase in CSF reference samples from healthy donors, under the assumption that levels in Lafora disease population would be elevated over healthy population. By careful screening of a variety of approx. 10 antibody candidates for binding of Glycogen Synthase and appropriate optimization of an assay protocol using Chimera’s AnySource® sample dilution technology, the sensitivity requirement was set to be considerable below 100 pg/ml. The resulting Imperacer® assay demonstrated an assay range of better 4 log units from 3.2 pg/mL – 10,000 pg / mL in Chimera’s artificial CSF standard curve matrix, for sample testing from 20 µl human CSF per duplicate run. First Glycogen Synthase concentration results from quantification of human CSF samples suggest an approx. 3-fold elevated level in the Lafora disease population (N = 27) with an average of approx. 900 pg/ml over the healthy endogenous levels (N = 10) of approx. 300 pg/ml. The current assay setup can be further modified, e.g. for use in preclinical species and validated according to bioanalytical method validation guidelines for clinical drug development support.
Conclusion: An Imperacer IPCR immunoassay was developed to quantify Glycogen Synthase at the needed sensitivity (below 100 pg/ml) from minimal volume of human CSF from juvenile healthy donors and Lafora patients. First Glycogen Synthase concentration results suggest an approx. 3-fold higher level in Lafora patients over healthy volunteers.