New Approach for Detection of Normal Alternative Splicing Events and Aberrant Spliceogenic Transcripts with Long-Range PCR and Deep RNA Sequencing.

Vita Šetrajčič Dragoš,Gašper Klančar,Ana Blatnik,Srdjan Novaković,Vida Stegel,Mateja Krajc

doi:10.3390/biology10080706

Vita Šetrajčič Dragoš, Gašper Klančar + Show 4 more

Open Access

https://doi.org/10.3390/biology10080706

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Journal: Biology	Publication Date: Jul 23, 2021
Citations: 2	License type: CC BY 4.0

Affiliation: Institute of Oncology Ljubljana, University of Ljubljana

Abstract

Simple SummaryRNA splicing defects, caused by genetic variants, are a common molecular mechanism of disease. To detect variants that cause splicing impairment, mRNA-based studies must be performed. Classical mRNA assays are time-consuming, which is why we have validated a new reliable straightforward approach to detect normal alternative splicing events and also splicing aberrations. Using our approach, we were able to reclassify three variants of uncertain significance in NBN and STK11 genes, which is of great importance for a proper clinical management of the patients.RNA sequencing is a promising technique for detecting normal and aberrant RNA isoforms. Here, we present a new single-gene, straightforward 1-day hands-on protocol for detection of splicing alterations with deep RNA sequencing from blood. We have validated our method’s accuracy by detecting previously published normal splicing isoforms of STK11 gene. Additionally, the same technique was used to provide the first comprehensive catalogue of naturally occurring alternative splicing events of the NBN gene in blood. Furthermore, we demonstrate that our approach can be used for detection of splicing impairment caused by genetic variants. Therefore, we were able to reclassify three variants of uncertain significance: NBN:c.584G>A, STK11:c.863-5_863-3delCTC and STK11:c.615G>A. Due to the simplicity of our approach, it can be incorporated into any molecular diagnostics laboratory for determination of variant’s impact on splicing.

Highlights

Alternative splicing is a process in which a single gene’s pre-mRNA undergoes processing into multiple mature mRNA isoforms
All that a laboratory requires is a set of primers that align to the 5 and 3 UTR region of the gene of interest, long-range PCR amplification, Nextera XT library preparation kit and access to an Next generation sequencing (NGS) instrument
RNA sequencing (RNAseq) data can be analyzed on any desktop computer, without the need for high computational power

Summary

Introduction

Alternative splicing is a process in which a single gene’s pre-mRNA undergoes processing into multiple mature mRNA isoforms. Understanding the naturally occurring alternative splicing isoforms of clinically relevant genes is of great importance for correct interpretation of splicing assays. RNA splicing defects are a common molecular mechanism of disease, and there are studies demonstrating that RNA sequencing (RNAseq) considerably improves diagnostics yield [2,3,4]. In the study by Yamada et al, the authors showed that the detection rate of deleterious variants increased by 19% if combination of exome and transcriptome analysis was performed, compared with exome sequencing alone [2]. Identification of splicing defects is of high importance to improve patient’s management. An additional drawback of the above-mentioned conventional RNA assays is that the maximal fragment’s length suitable for analysis is limited to approximately 1000bp, which makes it impossible to investigate variants detected in long exons such as exon in BRCA1 and exon in BRCA2 gene. For small diagnostic laboratories, whole transcriptome sequencing can be financially demanding, and it requires high computational power and storage capacity for RNAseq data analysis [6,7]

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