GBA Analysis in Next-Generation Era Pitfalls, Challenges, and Possible Solutions

Mutations in the gene encoding the lysosomal enzyme acid B-glucosidase (GBA) are responsible for Gaucher disease and represent the main genetic risk factor for developing Parkinson disease. In past years, next-generation sequencing (NGS) technology has been applied for the molecular analysis of the GBA gene, both as a single gene or as part of gene panels. However, the presence of complex gene-pseudogene rearrangements, resulting from the presence of a highly homologous pseudogene (GBAP1) located downstream of the GBA gene, makes NGS analysis of GBA challenging. Therefore, adequate strategies should be adopted to avoid misdetection of GBA recombinant mutations. Here, we validated a strategy for the identification of GBA mutations using parallel massive sequencing and provide an overview of the major drawbacks encountered during GBA analysis by NGS. We implemented a NGS workflow, using a set of 38 patients with Gaucher disease carrying different GBA alleles identified previously by Sanger sequencing. As expected, the presence of the pseudogene significantly affected data output. However, the combination of specific procedures for the library preparation and data analysis resulted in maximal repeatability and reproducibility, and a robust performance with 97% sensitivity and 100% specificity. In conclusion, the pipeline described here represents a useful approach to deal with GBA sequencing using NGS technology.