RNA-Seq Perspectives to Improve Clinical Diagnosis.

Sergio Lois,Javier Benítez,Juan Carlos Trivino,Guillermo Marco-Puche

doi:10.3389/fgene.2019.01152

Abstract

In recent years, high-throughput next-generation sequencing technology has allowed a rapid increase in diagnostic capacity and precision through different bioinformatics processing algorithms, tools, and pipelines. The identification, annotation, and classification of sequence variants within different target regions are now considered a gold standard in clinical genetic diagnosis. However, this procedure lacks the ability to link regulatory events such as differential splicing to diseases. RNA-seq is necessary in clinical routine in order to interpret and detect among others splicing events and splicing variants, as it would increase the diagnostic rate by up to 10–35%. The transcriptome has a very dynamic nature, varying according to tissue type, cellular conditions, and environmental factors that may affect regulatory events such as splicing and the expression of genes or their isoforms. RNA-seq offers a robust technical analysis of this complexity, but it requires a profound knowledge of computational/statistical tools that may need to be adjusted depending on the disease under study. In this article we will cover RNA-seq analyses best practices applied to clinical routine, bioinformatics procedures, and present challenges of this approach.

Highlights

In recent years, the use of next-generation sequencing (NGS) for the diagnosis of Mendelian or rare genetic disorders has entered routine clinical practice
The increasing ability to sequence entire genomes in a cost-effective manner has allowed the identification of approximately 260 novel rare genetic diseases per year (Boycott et al, 2017)
Different studies reported on how the application of RNA sequencing (RNA-seq) can help to shed light on the possible pathogenicity of variants of unknown significance (VUS) identified through DNA sequencing studies such as whole-exome sequencing (WES) and whole-genome sequencing (WGS), as it provides direct insight into the transcriptional alterations caused by VUS and improves diagnostic rates (Cummings et al, 2017; Kremer et al, 2017)

Summary

INTRODUCTION

The use of next-generation sequencing (NGS) for the diagnosis of Mendelian or rare genetic disorders has entered routine clinical practice. In terms of evolutionary conservation, about 50% of the synonymous positions in codons of conserved alternatively spliced mRNAs are under selection pressure, suggesting that conserved alternative exons and their flanking introns are strongly enriched in splicing regulatory elements (Blencowe, 2006) In this regard, it has been estimated that up to 25% of synonymous substitutions can disrupt normal splicing in the same way as non-synonymous variants or premature termination codons (Pagani et al, 2005), suggesting that those regions should be routinely examined. Some authors demonstrate the utility of RNA-seq to diagnose 10% of patients with mitochondrial diseases and identify candidate genes for the remaining 90% (Kremer et al, 2017)

SECTION 1: TOWARDS CLINICAL APPLICATION OF RNA SEQUENCING

Findings

SECTION 3: ISSUES TO BE ADDRESSED IN THE TRANSCRIPTOMIC APPROACH