Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson's disease cohort
Introduction
Several monogenic forms of Parkinson's disease (PD) and many genetic risk factors, which predispose to development of PD, are now known. One of the strongest genetic risk factors for PD is mutation of the gene known as GBA, which encodes the lysosomal enzyme glucocerebrosidase (GCase) [1]. GCase is an enzyme involved in glycolipid metabolism, and mutations in GBA lead to a marked decrease in GCase activity [2]. Bi-allelic mutations in GBA result in excessive accumulation of the GCase substrate, GlcCer in macrophages, causing the rare lysosomal storage disorder, Gaucher disease (GD) [3,4]. Heterozygous GBA mutations were initially believed to be benign; however, Parkinsonism was frequently reported in GD patients and their non-GD family members [5]. It is now clear that individuals with these GBA mutations associated with GD in the heterozygous state have a substantially elevated risk of developing PD compared with non-carriers [3,6]. Furthermore, PD patients with GBA mutations have a noticeably earlier age of PD onset and a slightly increased risk of cognitive effects such as dementia, as well as a higher incidence of psychosis and delirium [7,8].
Over 300 different GBA variants have been described, including single nucleotide polymorphisms (SNPs) (the majority of which are missense variants), frameshift mutations, splice-site alterations, and a range of structural variants (SVs) (i.e. variations >50bp) [4]. However, analysis of GBA is not straightforward. A nearby pseudogene called GBAP1 shares 96% sequence homology with the coding region of the GBA gene, and care is required to prevent pseudogene contamination when analyzing GBA [9,10].
Although application of a long-PCR step targeted at GBA can effectively solve many (but not all) of the issues arising from structural variants and the presence of a pseudogene [10], challenges remain using short read sequencing with such amplicons, including the inability to precisely assign haplotypes. Long read sequencing approaches such as Oxford Nanopore Sequencing (Oxford Nanopore Technologies; ONT) and PacBio (Pacific Biosciences) offer new opportunities for analyzing complex genes such as GBA. Indeed a recent report by Leija-Salazar et al. described a procedure for carrying out genotyping and haplotyping of GBA on the MinION sequencer (ONT) [11]. We have further refined the method of Leija-Salazar [11], and applied it for the discovery of GBA variants in a New Zealand PD longitudinal cohort. Our refined protocol utilizes updated hardware and software, thereby reducing the computational workload and improving sequencing accuracy.
Section snippets
Cohort description, recruitment and ethics
DNA for GBA genotyping was from a convenience sample (n = 229) of Parkinson's disease patients from the New Zealand Brain Research Institute (NZBRI) PD cohort recruited through our movement disorders clinic for a range of PD-related research studies. We selected this convenience sample as our lab had previously collected blood samples from these patients for another study. Patients met the UK Brain Bank criteria for PD. Exclusion criteria included an indication of an atypical parkinsonian
Nanopore sequencing of GBA in NZBRI cohort
We applied the nanopore sequencing method to examine GBA amplicons derived from 229 PD and 50 healthy control DNA samples from the NZBRI cohort. We sequenced the amplicons over five libraries containing multiplexed samples, on separate flow cells. All of the nanopore runs resulted in similar yields (~10–15 Gb with QC ≥ 7) (Supplementary Fig. 1). The majority of these were the 8.9 kb fragment of interest (Supplementary Fig. 2). For the first run, ~60% of reads mapped to the GBA reference genome,
Nanopore sequencing as a tool for GBA mutation discovery
We successfully validated the protocol of Leija-Salazar et al. [11] as an effective method for genotyping of GBA. Additionally, we have updated the software pipeline utilized in the method of Leija-Salazar et al. [11], improving the accuracy and reducing the computational workload. More specifically our pipeline utilizes Guppy (flip-flop) instead of the now discontinued base-calling tool Albacore. Guppy runs orders of magnitude faster (1,500,000 bp/s vs 120,000 bps/s) due to its use of GPU
Conclusions
We have successfully validated ONT sequencing of GBA as an efficient and accurate method for detecting a wide range of variants in multiple patient samples, as well as resolving the haplotype of these variants. Applying this method to a sizeable New Zealand cohort of PD revealed the presence of multiple known GBA variants, and a range of additional variants of unclear clinical significance.
These findings may contribute to a better understanding of the effects of these variants on disease
Funding
Support for this work came from the Jim and Mary Carney Charitable Trust (Whangarei, New Zealand), The McGee Fellowship (University of Otago, Christchurch), and The Helen Poole and Ian McDonald Memorial Summer Studentship (Canterbury Medical Research Foundation, Christchurch, New Zealand).
Acknowledgements
The Health Research Council and Lotteries Health provided funding for sample collection and storage. We would also like to acknowledge NZBRI Assistant Research Fellows Leslie Livingston, Sophie Grenfell, and Bob Young for cognitive and clinical assessments, as well as NZBRI Research Fellows Dr Kyla Horne for cognitive testing, and Dr Michael MacAskill and Dr Daniel Myall for cohort database management.
References (30)
- et al.
The link between the GBA gene and parkinsonism
Lancet Neurol.
(2012) - et al.
Increased incidence of Parkinson disease among relatives of patients with Gaucher disease
Blood Cells Mol. Dis.
(2006) Impact of glucocerebrosidase mutations on motor and nonmotor complications in Parkinson’s disease
Neurobiol. Aging
(2015)Reciprocal and nonreciprocal recombination at the glucocerebrosidase gene region: implications for complexity in Gaucher disease
Am. J. Hum. Genet.
(2003)Glucocerebrosidase gene variants are accumulated in idiopathic REM sleep behavior disorder
Park. Relat. Disord.
(2018)Glucocerebrosidase mutations in Parkinson disease
J. Parkinson's Dis.
(2017)Glucocerebrosidase activity in Parkinson's disease with and without GBA mutations
Brain : J. Neurol.
(2015)- et al.
GBA, Gaucher disease, and Parkinson's disease: from genetic to clinic to new therapeutic approaches
Cells
(2019) Multicenter analysis of glucocerebrosidase mutations in Parkinson's disease
N. Engl. J. Med.
(2009)GBA-associated Parkinson's disease: reduced survival and more rapid progression in a prospective longitudinal study
Mov. Disord.
(2015)
Identification of a novel recombinant mutation in Korean patients with Gaucher disease using a long-range PCR approach
J. Hum. Genet.
Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION
Mol. Genet. Genom. Med.
Different PD-MCI criteria and risk of dementia in Parkinson's disease: 4-year longitudinal study
Npj Parkinson's Dis.
A simple salting out procedure for extracting DNA from human nucleated cells
Nucleic Acids Res.
DNA Size Selection (>3-4kb) and Purification of DNA Using an Improved Homemade SPRI Beads Solution
Cited by (14)
Lysosomal functions and dysfunctions: Molecular and cellular mechanisms underlying Gaucher disease and its association with Parkinson disease
2022, Advanced Drug Delivery ReviewsEvaluation of nanopore sequencing technology to identify Salmonella enterica Choleraesuis var. Kunzendorf and Orion var. 15<sup>+</sup>, 34<sup>+</sup>
2021, International Journal of Food MicrobiologyCitation Excerpt :It was shown in our previous study that the workflow including the R9 flow cell, rapid library construction kit and guppy with the transducer basecalling model was sufficient for in silico serotyping prediction using ONT sequences (Xu et al., 2020). A high accuracy (HAC) basecalling model (flip-flop), which was trained on a corpus of sequence contexts that cover a variety of pro- and eukaryotic organisms, was recently released and expected to generate data with higher accuracy than using the transducer model (Graham et al., 2020). Therefore, in workflow 1, the combination of the R9 flow cell, rapid kit and guppy with the HAC basecalling model was applied.
Accurate long-read sequencing identified GBA1 as major risk factor in the Luxembourgish Parkinson’s study
2023, npj Parkinson's DiseaseThe New Zealand Parkinson’s progression programme
2023, Journal of the Royal Society of New Zealand