Detection of single nucleotide and copy number variants in the Fabry disease-associated GLA gene using nanopore sequencing

Albina Nowak,Omer Murik,Gheona Altarescu,Tzvia Mann,David A Zeevi

doi:10.1038/s41598-021-01749-7

Albina Nowak, Omer Murik + Show 3 more

Open Access

https://doi.org/10.1038/s41598-021-01749-7

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Abstract

More than 900 variants have been described in the GLA gene. Some intronic variants and copy number variants in GLA can cause Fabry disease but will not be detected by classical Sanger sequence. We aimed to design and validate a method for sequencing the GLA gene using long-read Oxford Nanopore sequencing technology. Twelve Fabry patients were blindly analyzed, both by conventional Sanger sequence and by long-read sequencing of a 13 kb PCR amplicon. We used minimap2 to align the long-read data and Nanopolish and Sniffles to call variants. All the variants detected by Sanger (including a deep intronic variant) were also detected by long-read sequencing. One patient had a deletion that was not detected by Sanger sequencing but was detected by the new technology. Our long-read sequencing-based method was able to detect missense variants and an exonic deletion, with the added advantage of intronic analysis. It can be used as an efficient and cost-effective tool for screening and diagnosing Fabry disease.

Highlights

Fabry disease (FD; OMIM: 301500) is an X-linked lysosomal storage disorder caused by deficiency of the alpha galactosidase A enzyme, resulting in accumulation of glycosphingolipids, globotriaosylceramide (GL-3, Gb3, CTH) and globotriaosylsphingosine (Lyso-GL-3, lyso-Gb3)[1]
FD can be caused by several types of molecular variants in this gene: missense (57%), nonsense (11%), partial deletions (6%), insertions (6%), and defects in the processing of RNA, which lead to aberrant splicing (6%)[6]
All patients were previously diagnosed by measuring the alpha galactosidase A enzyme activity and Sanger sequencing

Summary

Introduction

Fabry disease (FD; OMIM: 301500) is an X-linked lysosomal storage disorder caused by deficiency of the alpha galactosidase A enzyme, resulting in accumulation of glycosphingolipids, globotriaosylceramide (GL-3, Gb3, CTH) and globotriaosylsphingosine (Lyso-GL-3, lyso-Gb3)[1]. E.g., frame-shift variants such as deletions and duplications, non-sense and some missense and splicing variants lead to a zero or very low α-GAL activity and, the classic phenotype. Such patients are at high risk of developing a small-fiber neuropathy, progressive proteinuric kidney disease, chronic diarrhea and abdominal pains, fibrotic cardiac disease resulting in rhythm and conduction disturbances, progressive hypertrophic cardiomyopathy, and cerebrovascular stroke[11]. Other missense and splicing variants lead to a significant residual α-GAL activity and, the lateonset phenotype Such patients suffer from cardiac or, more rarely, renal disease

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