GBA Analysis in Next-Generation Era

Abstract

Mutations in the gene encoding the lysosomal enzyme acid β-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.

Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION

“…We observed minimal alignment to the pseudogene. We also detected intronic SNPs, an understudied area in GBA, and other lysosomal disorders (Zampieri et al, 2017). Finally, the long reads allowed the phasing of mutations, enabling a haplotype-resolved personalized assessment.…”

“…Using long reads has several advantages, despite the lower accuracy at the base level (Goodwin et al, ), some of which were evident here. The challenge of aligning short reads to regions with high homology is often not fully appreciated (Mandelker et al, ), with false negatives in GBA ‐targeted Illumina sequencing when the whole genome was used as a reference (Zampieri et al, ). We observed minimal alignment to the pseudogene.…”

“…Detection of recombinants is a known problem. RecNciI can be missed by targeted Illumina sequencing, unless specifically aligning reads to GBA, rather than the whole genome (Zampieri et al, 2017). Recombinant alleles have been reported in PD, with frequencies of 0.7% (Liu et al, 2016) and 0.25% (Neumann et al, 2009), and may be the fifth commonest GBA variant in non-Ashkenazi PD patients (Zhang et al, 2018).…”

“…Established analysis protocols usually involve PCR of up to three fragments, carefully designed to not amplify GBAP (Neumann et al, 2009), followed by Sanger sequencing of coding exons. Illumina targeted sequencing protocols have also recently been developed (Liu et al, 2016;Zampieri, Cattarossi, Bembi, & Dardis, 2017). In recent years, long reads produced by sequencing DNA molecules in real time have become commercially available and have several advantages over short reads (Goodwin, McPherson, & McCombie, 2016).…”

Detection ofGBAmissense mutations and other variants using the Oxford Nanopore MinION

“…Illumina targeted sequencing protocols have also recently been developed. 9,12 In recent years, long reads produced by Single Molecule Real Time (SMRT) sequencing have become commercially available, and have several advantages over short reads. 18 Oxford Nanopore sequencing technology analyses a single DNA molecule while it passes through a pore, producing characteristic changes in current depending on the sequence.…”