LYSOPLEX: An Efficient Toolkit to Detect Sequence Variations in Genes Involved in the Lysosomal-Autophagic Pathway and Neurodegenerative Diseases

Aims: The lysosomal-autophagic pathway (LAP) regulates cellular homeostasis and plays a crucial role in human diseases, such as lysosomal storage disorders (LSDs) and common neurodegenerative diseases. Therefore, the identification of sequence variations in genes involved in the LAP and their association with human diseases would have a significant impact on human health. To this aim, we devised a next generation sequencing–based workflow (Lysoplex) that allowed us to obtain a uniform and accurate sequence coverage of a comprehensive set of 891 lysosomal, endocytic, and autophagic genes.

Methods: To initially validate Lysoplex performance, we tested a training set of 14 patients affected by different genetic forms of LSDs for which the underlying mutations were already known. We then applied Lysoplex to analyze the DNA from 46 mutations unknown patients with a clinical phenotype of neuronal ceroid lipofuscinosis (NCL), most of which had been unsuccessfully analyzed by capillary sequencing.

Results: We identified mutations in both the alleles of a known NCL gene in 63% of patients. In three patients, we found potential disease-causing variants in novel NCL candidate genes. We then compared the variant detection power of Lysoplex with data derived from public ongoing whole-exome sequencing efforts. On an average, 50% higher number of validated amino acid changes and truncating variations per gene were identified. This catalog of previously missed variants may reflect interindividual differences of the LAP pathway in the human population.

Conclusion: Lysoplex is the first workflow that can provide a comprehensive assessment of sequence variability in LAP genes, thus potentially shedding further light on its contribution to human disease.