Splicing in the Diagnosis of Rare Disease: Advances and Challenges

Abstract

Mutations which affect splicing are significant contributors to rare disease, but are frequently overlooked by diagnostic sequencing pipelines. Greater ascertainment of pathogenic splicing variants will increase diagnostic yields, ending the diagnostic odyssey for patients and families affected by rare disorders, and improving treatment and care strategies. Advances in sequencing technologies, predictive modeling, and understanding of the mechanisms of splicing in recent years pave the way for improved detection and interpretation of splice affecting variants, yet several limitations still prohibit their routine ascertainment in diagnostic testing. This review explores some of these advances in the context of clinical application and discusses challenges to be overcome before these variants are comprehensively and routinely recognized in diagnostics.

Short amplicon reverse transcription‐polymerase chain reaction detects aberrant splicing in genes with low expression in blood missed by ribonucleic acid sequencing analysis for clinical diagnosis

“…Although aberrant splicing is associated with disease phenotype, the phenotypic severity may depend on the type of aberrant splicing. For example, in‐frame aberrant splicing may have milder phenotypic effects than a frameshift or termination codon induced by aberrant splicing, depending on the relevant gene's pathogenetic mechanisms (Lord & Baralle, 2021). However, further studies are needed on a gene‐by‐gene basis to determine what usage level of alternative splicing is pathogenically significant.…”

Targeted RNAseq Improves Clinical Diagnosis of Very Early-Onset Pediatric Immune Dysregulation

“…One of the largest limitations of RNAseq for rare disease diagnostics is that the ability to capture a variant is dependent on that gene’s expression level in the sequenced tissue type. This generally leads to arguments that the disease-relevant tissue is a necessity for RNAseq and/or that sequencing depth should be at least 50–100 M reads per sample [ 5 , 38 , 39 ]. The recent minimum read sequencing depth (MRSD) study identified whole blood (over LCL, cultured fibroblasts, and skeletal muscle) as the worst option for most gene panels [ 63 ].…”

“…In some cases, targeting an affected but not causal gene results in worse patient outcomes [ 23 ]. Since diagnostic yield from exome sequencing remains well under 50%, the accessibility of immune cells for genomic profiling of peripheral blood samples obtained by standard phlebotomy raises prospects of RNA-based analyses, specifically RNAseq, that might identify aberrant molecular events such as altered splicing or gene expression [ 5 , 35 , 36 , 37 , 38 , 39 ]. The relevant mutations might not be observable in exome sequences, or may be of uncertain significance.…”

A Deep Exon Cryptic Splice Site Promotes Aberrant Intron Retention in a Von Willebrand Disease Patient

“…Aberrant splicing is a common cause of human genetic disease with various studies estimating that 15-60% of disease-causing mutations disrupt pre-mRNA splicing ( [1,2]; reviewed in [3]). Moreover, 5-20% of cancer-predisposing mutations adversely affect splicing [4].…”