Resolving the GBA gene with Oxford Nanopore Technologies

Mutations in the GBA (glucocerebrosidase)gene on human chromosome 1 are a risk factor for Parkinson’s disease, with an increased risk of ~5 to 10-fold compared to non-carriers. Disease modifying therapies that target GBA mutations are being investigated (e.g. the Ambroxol trial). The importance of this gene in human disease is therefore apparent, but sequencing of the GBA region is challenging, mainly due to a pseudogene located nearby that causes a high frequency of recombination events at the locus.

Marco’s team developed a targeted sequencing method to analyse the entire GBA gene in a single 7.9 kb amplicon (Leija-Salazar et al. 2019; Mol Genet Genomic Med.). However, this method can be hindered by some forms of GBA recombination, specifically reciprocal gene fusion. They therefore developed a new set of primers that specifically amplified these recombination events, which could be used in an additional, separate PCR reaction. By nanopore sequencing the products and alignment of the data to a reference, the sequence and breakpoint location could be identified – which is key for determining the pathogenicity of the fusion.

With the awareness that PCR can introduce artifacts into the data, Marco explained that they have been investigating the use of adaptive sampling combined with the ‘UNCALLED‘ software to perform amplification-free, real-time target enrichment of the GBA locus. Enrichment of the locus has proven successful and they are continuing to develop this approach.

Marco concluded that their method of PCR-based targeted sequencing of the GBA locus can successfully resolve the entire gene and detect complex structural variants. This approach is cost-effective and reliable, with up to 96 samples multiplexed for sequencing on one MinION Flow Cell.