Objective: To use nanopore sequencing as a cost-effective strategy to determine the association and prevalence of mutations within the GBA gene in patients with Parkinson’s disease (PD) from the Norwegian population.

Background: GBA mutations are the most common genetic risk factor of PD, although the underlying mechanism of the association between GBA mutations and PD is still unknown. The presence of a highly homologous nearby pseudogene (GBAP1) predisposes to a range of structural variants and complicates genetic testing and analysis. An earlier investigation of 311 Norwegian patients with PD and 474 controls used a specific-genotype method to assess GBA N370S and L444P and identified seven heterozygous patients (2.3%) and eight controls (1.7%).

Method: Our study consisted of 444 Norwegian patients with PD and 376 controls. Forty-three out of 444 patients were probands with a family history of PD. The control subjects (n=376) do not carry pathogenic variants in known PD genes. Mutational screening was performed with Oxford Nanopore sequencing on blood-derived genomic DNA amplified with the LongAmp Taq PCR Kit. We sequenced the full GBA gene including introns on the Oxford Nanopore MinION as an 8.9 kb amplicon and investigated other recombinants. Variant annotation of GBA mutations was performed with Annovar.

Results: Across all samples, we obtained a mean coverage of 105.4X (SD ± 3.4X). The cost to sequence the complete GBA gene with this coverage on the nanopore is less than 10USD per sample as samples were multiplexed on the MinION flow cells. Ten mutations within GBA were found in the Norwegian PD cases, seven of them predicted to be probably or likely pathogenic (p.L444P, p.N370S, p.R72W, p.R319C, p.D92H, p.S59L, p.S223G), three predicted as benign (p.T369M, p.E326K, p.K13R). The total carrier frequency of GBA mutations predicted as pathogenic in the PD cohort is 8.6% (38/444).

Conclusion: In our study, 8.6% of Norwegian PD patients carried a pathogenic GBA mutation. The Oxford Nanopore can detect missense mutations in this complex gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants. Further investigations are warranted and the association of GBA mutations with PD will be examined by including controls in the study.

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MDS Abstracts - https://www.mdsabstracts.org/abstract/long-read-nanopore-sequencing-for-cost-effective-and-comprehensive-analysis-of-gba-mutations-in-norwegian-patients-with-parkinsons-disease/