Objective: To sequence the GBA1 gene in PD patients with reduced GCase activity using nanopore amplicon-targeted sequencing.

To characterize benign, VUS, and pathogenic GBA1 variants.

Background: Mutations in the GBA1 gene are a risk factor for Parkinson’s disease (PD) and are associated with decreased glucocerebrosidase (GCase) activity, earlier disease onset, and heterogeneous clinical progression. Avoiding pseudogene variants however can be a difficult task with conventional sequencing. We used enzyme analysis of GCase activity followed by Nanopore amplicon-targeted Long read sequencing to resolve GBA1 variants while avoiding interference from the nearby pseudogene GBAP1.

Method: We measured GCase activity in 110 PD patients and 10 age-matched controls using the fluorometric substrate 4-MUG and classified into normal, heterozygous-deficient, or homozygous-deficient categories. Thirty PD samples with low GCase activity were selected for sequencing. GBA1-specific amplicons were generated to avoid GBAP1, followed by native barcoding and sequencing on the MinION MK1B platform. Data were processed using Dorado SUP, wf-alignment, and wf-human-variation workflows in EPI2ME. Variant interpretation was performed using ClinVar and Franklin (Genoox).

Results: Patients were segregated into normal (N=72), heterozygous (N=30), and homozygous-deficient (N=8) activity groups. Early-onset PD (<50 years) demonstrated a higher frequency of heterozygous carriers. Nanopore sequencing identified common benign polymorphisms (c.455-206A>G, c.1225-34C>A), intronic VUS, and pathogenic variants including c.492C>G and c.1448T>C (L444P). These pathogenic variants were detected in RT-QuIC–positive early-onset cases. A promoter-region VUS (c.-68-82A>G) was also noted.

Conclusion: Nanopore amplicon sequencing reliably resolves GBA1 variation and, when combined with enzymatic and RT-QuIC analyses, offers a robust multimodal approach for genetic risk assessment in Parkinson’s disease

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MDS Abstracts - https://www.mdsabstracts.org/abstract/integrative-analysis-of-gba1-mutations-and-gcase-deficiency-in-parkinsons-disease-using-long-read-nanopore-sequencing/