Transcriptome analysis provides critical answers to the "variants of uncertain significance" conundrum

Abstract

While whole-genome and exome sequencing have transformed our collective understanding of genetics' role in disease pathogenesis, there are certain conditions and populations for whom DNA-level data fails to identify the underlying genetic etiology. Specifically, patients of non-White race and non-European ancestry are disproportionately affected by "variants of unknown/uncertain significance" (VUS), limiting the scope of precision medicine for minority patients and perpetuating health disparities. VUS often include deep intronic and splicing variants which are difficult to interpret from DNA data alone. RNA analysis can illuminate the consequences of VUS, thereby allowing for their reclassification as pathogenic versus benign. Here we review the critical role transcriptome analysis plays in clarifying VUS in both neoplastic and non-neoplastic diseases.

Keywords: deep intronic variants; genetic ancestry; splicing variants; variants of uncertain significance; variants of unknown significance.

Figure 1

Splice site motifs and variants. (a) A schematic of two exons separated by an intron in pre‐mRNA. Shown above the pre‐mRNA are the corresponding, relative‐weighted DNA nucleotide motifs at splicing donor and acceptor sites. Though the canonical/consensus boundary motifs are GT (or GU, in RNA, after the 3′ end of exon #1) and AG (before the 5′ end of exon #2), these are probabilistic. Other boundary motifs exist and positions up/downstream of the motifs are also important for splicing. RNA‐seq data can help isolate functionally relevant, potentially pathogenic splicing variants. Canonical boundary motifs are demarcated with dashed boxes. (b) Depicted are schematics of seven representative types of splicing mutations, their mechanisms in pre‐mRNA, and their potential consequences in mature mRNA. mRNA, messenger RNA. Figure created with BioRender.com

Figure 2

A hypothetical variant of unknown significance (VUS) leads to nonsense‐mediated decay (NMD), monoallelic allele‐specific expression (ASE), and loss of messenger RNA (mRNA) transcripts. Phasing of alleles (maternal vs. paternal) is indicated by pink (maternal) and blue (paternal) highlights. In the left‐hand panels we depict the hypothetical case of a loss‐of‐function intronic VUS (depicted as a yellow sunburst symbol) on the DNA‐level. Also shown schematically is a coding heterozygous single‐nucleotide polymorphism (SNP) upstream (shown as green and magenta color‐coded alleles). The VUS in this case causes intron fragment inclusion and when a premature stop codon is reached, premature truncation. The truncated mRNA is degraded via NMD, and complete loss of associated mRNA transcripts is observed. This is evidenced by the fact that in the bottom left sashimi plot, (1) we do not see expression of the magenta allele (a phasing proxy for the VUS), and (2) we do see monoallelic ASE of the green allele. ASE is the process whereby only one allele is expressed in RNA despite the fact that an individual is heterozygous at that position in their DNA. Key to recognizing whether an individual is displaying ASE is the identification of a coding heterozygous SNP up/downstream that can act as a phasing proxy. In contrast, in the right‐hand panels, we depict the scenario of a benign VUS whereby we do not see a loss of function in RNA; instead, in the sashimi plot, we see a roughly equal expression of both alleles (magenta and green) at the upstream heterozygous locus, as well as the correct “dosage” of expression. Figure created with BioRender.com.