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Evaluation of the Detection of GBA Missense Mutations and Other Variants Using the Oxford Nanopore Minion

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Evaluation of the Detection of GBA Missense Mutations and Other Variants Using the Oxford Nanopore Minion View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by UCL Discovery Received: 19 June 2018 | Revised: 23 November 2018 | Accepted: 13 December 2018 DOI: 10.1002/mgg3.564 METHOD Evaluation of the detection of GBA missense mutations and other variants using the Oxford Nanopore MinION Melissa Leija‐Salazar1 | Fritz J. Sedlazeck2 | Marco Toffoli1 | Stephen Mullin1,3 | Katya Mokretar1 | Maria Athanasopoulou4 | Aimee Donald5 | Reena Sharma6 | Derralynn Hughes7 | Anthony H.V. Schapira1 | Christos Proukakis1 1Department of Clinical and Movement Neurosciences, Royal Free Campus, Abstract Institute of Neurology, University College Background: Mutations in GBA cause Gaucher disease when biallelic and are strong London, London, UK risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated 2 Human Genome Sequencing by the nearby pseudogene. We aimed to design and validate a method for sequencing Center, Baylor College of Medicine, Houston, Texas GBA using long reads. 3Institute of Translational and Stratified Methods: We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb ampli- Medicine, Plymouth University Peninsula con from 102 individuals, including patients with Parkinson's and Gaucher diseases. School of Medicine, Plymouth, UK We used NanoOK for quality metrics, NGMLR to align data (after comparing with 4Department of Molecular Neuroscience, GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing. Institute of Neurology, University College London, London, UK Results: We detected all known missense mutations in these samples, including the 5Department of Paediatrics, Royal common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer Manchester Children’s Hospital, ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demon- Manchester, UK strated the ability to phase mutations, confirm compound heterozygosity, and assign 6The Mark Holland Metabolic Unit, Salford Royal Foundation NHS Trust, Salford, UK haplotypes. We also detected two known risk variants in some Parkinson's patients. 7Institute of Immunity and Rare false positives were easily identified and filtered, with the Nanopolish quality Transplantation, Lysosomal Storage score adjusted for the number of reads a very robust discriminator. In two individuals Disorders Unit, Royal Free Hospital, carrying a recombinant allele, we were able to detect and fully define it in one carrier, London, UK where it included a 55‐base pair deletion, but not in another one, suggesting a limita- Correspondence tion of the PCR enrichment method. Missense mutations were detected at the correct Christos Proukakis, Department of Clinical and Movement Neurosciences, Royal Free zygosity, except for the case where the RecNciI one was missed. Campus, Institute of Neurology, University Conclusion: The Oxford Nanopore MinION can detect missense mutations and an College London, Rowland Hill Street, exonic deletion in this difficult gene, with the added advantages of phasing and in- London NW3 2PF, UK. Email: [email protected] tronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants. Funding information Consejo Nacional de Ciencia y Tecnología; Medical Research Council; Michael J. Fox KEYWORDS Foundation for Parkinson's Research; Katan Gaucher disease, GBA, long‐read sequencing, mutation detection, mutation phasing, Oxford Nanopore Trust MinION, Parkinson’s disease This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. © 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc. Mol Genet Genomic Med. 2019;e564. wileyonlinelibrary.com/journal/mgg3 | 1 of 12 https://doi.org/10.1002/mgg3.564 2 of 12 | LEIJA‐SALAZAR ET AL. 1 | INTRODUCTION used for applications ranging from pathogen sequencing in the field (Quick et al., 2016) to sequencing a whole human The GBA gene (OMIM #606463) encodes the lysosomal genome (Jain et al., 2018). It is still not routinely used in enzyme Glucocerebrosidase, deficiency of which leads human disease diagnostics, but has been successfully used to accumulation of glucosylceramide. Biallelic (homozy- for SNV detection in CYP2D6, HLA‐A, and HLA‐B (Sović et gous or compound heterozygous) mutations in GBA cause al., 2016); TP53 in cancer (Crescenzio Francesco Minervini Gaucher disease (GD), the most common lysosomal storage et al., 2016); and BCR‐ABL1 in leukemia (Crescenzio F. disorder (Schapira, Chiasserini, Beccari, & Parnetti, 2016). Minervini et al., 2017). SNPs were successfully typed in Heterozygous GBA mutations are a significant risk factor for chromosome 20 in a recent whole genome sequencing study Parkinson's disease (PD; Mullin & Schapira, 2015; Sidransky of the NA12878 genome (Jain et al., 2018). et al., 2009), with evidence of longitudinal changes in many In the present study, we present and validate an efficient carriers suggestive of prodromal PD (Beavan et al., 2015). laboratory and bioinformatic protocol for GBA analysis using GBA mutations are also associated with dementia with the MinION. In addition to disease‐causing variants, it can Lewy bodies (Geiger et al., 2016) and multiple system at- detect intronic ones and provide phasing information. The rophy (MSA; Mitsui et al., 2015), related conditions which MinION protocol can thus provide further insights into GBA also demonstrate aggregation of the alpha‐synuclein pro- than other sequencing technologies and is ready to be consid- tein. At present, more than 300 mutations have been linked ered for diagnostic use. to Gaucher disease (Hruska, LaMarca, Scott, & Sidransky, 2008), and the number of studies analyzing the prevalence and phenotype of GBA mutations in PD is rapidly increasing 2 | MATERIALS AND METHODS (Adler et al., 2017; Alcalay et al., 2015; Berge‐Seidl et al., 2017; Liu et al., 2016). 2.1 | Overview, DNA extraction, and PCR GBA comprises eleven exons and ten introns over ~8 kb Samples successfully used in this study were derived from on chromosome 1q21. A nearby pseudogene GBAP has 102 individuals. All samples shown to carry mutations are 96% exonic sequence homology to the GBA coding region. shown in Supporting Information Table S1. We used samples The region also contains the Metaxin gene (MTX1) and its from saliva of 93 living individuals, and from brain from nine pseudogene. The existence of these two pseudogenes confers (seven PD and one MSA patients, and one control). Brain an increased risk for recombination between homologous samples were provided by Queen Square and Parkinson's UK regions, which can generate complex alleles. The homology brain banks. SNV analyses were performed blinded to disease between GBA and GBAP is highest between exons 8 and 11, status and any previous sequencing results from the patient where most of the pathogenic mutations have been reported, or relatives. All individuals had given written informed con- usually resulting from recombination events (Hruska et al., sent. Ethics approval was provided by the National Research 2008). Ethics Service London—Hampstead Ethics Committee, with The complex regional genomic structure complicates additional permission for study of brains from the research PCR and DNA sequencing, and some exons are also prob- tissue banks provided by the UK National Research Ethics lematic in exome sequencing (Mandelker et al., 2016) and Service (07/MRE09/72). DNA was isolated from brain using whole genome sequencing (Bodian et al., 2016). Established phenol–chloroform (Nacheva et al., 2017) and from saliva analysis protocols usually involve PCR of up to three frag- using Oragene DNA Kit. ments, carefully designed to not amplify GBAP (Neumann We enriched for GBA by amplifying an 8.9‐kb sequence, et al., 2009), followed by Sanger sequencing of coding which covered all coding exons, the introns between them, and exons. Illumina targeted sequencing protocols have also re- part of the 3’ UTR region (chr1: 155,202,296–155,211,206; cently been developed (Liu et al., 2016; Zampieri, Cattarossi, Supporting Information Figure S1). We customized previously Bembi, & Dardis, 2017). In recent years, long reads produced reported primers (Jeong et al., 2011) to carry Oxford Nanopore by sequencing DNA molecules in real time have become adapters and barcodes for multiplexing. Primer sequences commercially available and have several advantages over were npGBA‐F: 5’‐TTTCTGTTGGTGCTGATATTGC short reads (Goodwin, McPherson, & McCombie, 2016). TCCTAAAGTTGTCACCCATACATG‐3’ and npcMTX1: Oxford Nanopore sequencing technology analyses a single 5’‐ ACTTGCCTGTCGCTCTATCTTCCCAACCTTTCTTC DNA molecule while it passes through a pore, producing CTTCTTCTCAA‐3’. characteristic changes in current depending on the sequence Two DNA polymerases with appropriate optimized (Ip et al., 2015). The Oxford Nanopore MinION is currently PCR conditions were used to amplify the GBA target re- the most portable long‐read sequencer. It can be plugged gion (Supporting Information Table S2): Expand Long into a computer through a USB connection and provides se- Template PCR (Roche) and Kapa Hi‐Fi Polymerase (Kapa quencing data and runs metrics data in real time. It has been Biosystems). Amplicons were purified by Qiaquick PCR LEIJA‐SALAZAR ET AL. | 3 of 12 Purification Kit (Qiagen), and DNA concentration was mea- a hidden Markov
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