Detection of<i>GBA</i>missense mutations and other variants using the Oxford Nanopore MinION

Leija‐Salazar, Melissa; Sedlazeck, Fritz J.; Mokretar, Katya; Mullin, Stephen; Toffoli, Marco; Athanasopoulou, Maria; Donald, Aimee; Sharma, Reena; Hughes, Derralyn; Schapira, Anthony H.V.; Proukakis, Christos

doi:10.1101/288068

Cited by 23 publications

(37 citation statements)

References 42 publications

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“…We successfully validated the protocol of Leija-Salazar [10] as an effective method for genotyping of GBA across the 8.9kb amplicon. Furthermore, we highlight the capability of this protocol to phase variants, due to the single molecule reads generated by nanopore sequencing, thereby enabling the establishment of direct molecular haplotypes.…”

Section: Discussionmentioning

confidence: 96%

“…For the first run ~60% of reads mapped to the GBA reference genome, and only a small fraction of the reads mapped to the pseudogene as indicated by Qualimap and visualization using Genome Ribbon ( Supplementary Figure 3). Coverage depth over GBA averaged >2000x across all samples as indicated by Qualimap, far in excess of the 100x coverage required for accurate GBA genotyping [10].…”

Section: Nanopore Sequencing Of Gba In Nzbri Cohortmentioning

confidence: 97%

“…DNA from peripheral blood samples was extracted using a modified salting-out method followed by a phenol-chloroform purification step [12]. For each sample, a PCR that generated an 8.9 kilobase (kb) amplicon encompassing the entire coding region of GBA was performed according to Leija-Salazar et al [10]. Agarose gel electrophoresis was used to verify the presence of the 8.9kb fragment for each sample.…”

Section: Dna Preparation and Pcrmentioning

confidence: 99%

“…To exclude false positives, we divided the Nanopolish quality score for the variant call by the total number of reads associated with the call. Adjusted quality scores >1.8 were considered true variants and <1.2 were considered false positives, the same threshold used by Leija-Salazar [10]. We further assessed the variant calls by Sanger sequencing validation (Supplementary Methods 1) and by manually checking the BAM files using IGV to ensure that the mutant reads were not a result of systematic error.…”

Section: Nanopore Sequence Data Analysismentioning

confidence: 99%

“…Although application of a long-PCR step targeted at GBA can effectively solve the pseudogene problem [9], 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, and indeed a recent report described a procedure for carrying out genotyping and haplotyping on the MinION sequencer (ONT) [10]. We have further refined this method and described its application for the discovery of GBA variants in a New Zealand longitudinal cohort of PD patients.…”

Section: Introductionmentioning

confidence: 99%

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Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson’s disease cohort

Graham

Pitcher

Liau

et al. 2019

Preprint

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Highlights:-Validated nanopore sequencing of the GBA gene for mutation detection.-9.2% of patients from a New Zealand Parkinson's disease cohort carry GBA mutations.-Significantly higher prevalence of dementia in patients carrying GBA mutations -Three novel mutations were detected including a putative splicing variant.-Single molecule reads of GBA amplicons revealed 74 different GBA haplotypes. Abstract Introduction:Mutations in the gene for glucocerebrosidase (GBA) cause Gaucher disease, an autosomal recessive lysosomal storage disorder. GBA mutation carriers have an elevated risk of Parkinson's disease (PD) compared with non-carriers and can experience a more aggressive disease. GBA analysis is technically challenging due to a related pseudogene and structural variations (SVs) that can occur at this locus. We have applied a recently developed nanopore sequencing method to analyze GBA variants in a clinically assessed New Zealand longitudinal cohort of PD. Method:We examined amplicons encompassing the coding region of GBA (8.9kb) from 229 PD cases using the GridION nanopore-sequencing platform. Mutations were Sanger validated. Results:We detected 23 mutations in 21 PD cases (9.2% of patients). We detected a strong PD risk allele p.N409S (rs76763715) in one case. Modest risk mutations included p.E365K (rs2230288) in 12 cases, and p.T408M (rs75548401) in seven cases, one of whom also had p.E365K. We additionally detected the possible risk alleles R78C (rs146774384) in one case, D179H (rs147138516) in one case which occurred on the same haplotype as an E365K allele, and one novel mutation p.L335=, that is predicted to impact splicing of GBA transcripts.Additionally, we found a higher prevalence of dementia among GBA mutation carriers. Conclusion:This work confirmed the utility of nanopore sequencing as a high-throughput method to identify known and novel GBA mutations, and to assign precise haplotypes. This work may contribute to understanding the effects of these mutations on disease pathogenesis, and to the development of more targeted treatments.

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Section: Discussionmentioning

confidence: 96%

Section: Nanopore Sequencing Of Gba In Nzbri Cohortmentioning

confidence: 97%

Section: Dna Preparation and Pcrmentioning

confidence: 99%

Section: Nanopore Sequence Data Analysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson’s disease cohort

Graham

Pitcher

Liau

et al. 2019

Preprint

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Intronic Haplotypes in the GBA Gene Do Not Predict Age at Diagnosis of Parkinson's Disease

et al. 2021

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Background: GBA mutations are a common risk factor for Parkinson's disease (PD). A recent study has suggested that GBA haplotypes, identified by intronic variants, can affect age at diagnosis of PD. Objectives: In this study, we assess this hypothesis using long reads across a large cohort and the publicly available Accelerating Medicines Partnership-Parkinson's Disease (AMP-PD) cohort. Methods: We recruited a PD cohort through the Remote Assessment of Parkinsonism Supporting Ongoing Development of Interventions in Gaucher Disease study (RAPSODI) and sequenced GBA using Oxford Nanopore technology. Genetic and clinical data on the full AMP-PD cohort were obtained from the online portal of the consortium. Results: A total of 1417 participants were analyzed. There was no significant difference in age at PD diagnosis between the two main haplotypes of the GBA gene. Conclusions: GBA haplotypes do not affect age at diagnosis of PD in the two independent cohorts studied.

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A novel mutation deep within intron 7 of the GBA gene causes Gaucher disease

Malekkou

Sevastou

Mavrikiou

et al. 2020

Molec Gen & Gen Med

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Background Mutations in the GBA gene that encodes the lysosomal enzyme acid β‐glucocerebrosidase cause Gaucher disease (GD), the most common lysosomal storage disorder. Most of the mutations are missense/nonsense, however, a few splicing mutations within or close to conserved consensus donor or acceptor splice sites have also been described. The aim of the study was to identify the mutation(s) in a Cypriot patient with type I GD. Methods The genomic DNA of the proband was screened for nine common mutations using Polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP) analysis. All exons and exon‐intron boundaries, and the 5’UTR and 3’UTR regions of the GBA gene, were investigated by Sanger sequencing. RNA analysis was performed using standard procedures, and the abnormal transcript was further cloned into pGEM‐T‐Easy plasmid vector and sequenced. The relevant intronic region was further sequenced by the Sanger method to identify the genetic variant. Results A novel point mutation, g.12599C > A (c.999 + 242C > A), was detected deep in intron 7 of the GBA gene. This type of mutation has been previously described for other diseases but this is the first time, as far as we know, that it is described for GD. This mutation creates a new donor splice site leading to aberrant splicing and resulting in the insertion of the first 239nt of intron 7 as a pseudoexon in the mRNA, creating a premature stop codon. Conclusion This study expands the mutation spectrum of GD and highlights the importance of RNA sequencing for the molecular diagnosis of patients bearing mutations in nonexonic regions.

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Detection ofGBAmissense mutations and other variants using the Oxford Nanopore MinION

Cited by 23 publications

References 42 publications

Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson’s disease cohort

Nanopore sequencing of the glucocerebrosidase (GBA) gene in a New Zealand Parkinson’s disease cohort

Intronic Haplotypes in the GBA Gene Do Not Predict Age at Diagnosis of Parkinson's Disease

A novel mutation deep within intron 7 of the GBA gene causes Gaucher disease

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