Posted By: HGG Advances
Each month, the editors of Human Genetics and Genomics Advances interview an early-career researcher who has published work in the journal. This month we check in with Rachel Oh to discuss her paper “A systematic assessment of the impact of rare canonical splice site variants on splicing using functional and in silico methods“.
HGGA: What motivated you to start working on this project?
RO: Accurate variant interpretation is essential to both genome diagnostics and screening in medical genetics. Canonical splice site variants (CSSVs) are considered “null variants” (in the same category as nonsense, frameshift, initiation codon, single or multi-exon deletion variants) in a gene where loss-of-function (LoF) is a known mechanism of disease. Thus, CSSVs are assigned very strong evidence for pathogenicity (PVS1). In recent years, there have been numerous computational tools developed to predict the impact of DNA variants on splicing; however, none are designed specifically for CSSVs. Our group was interested in assessing the precise consequences of rare CSSVs in blood-expressed genes on splicing via cDNA sequencing and compare the results with splicing predictions from in silico tools such as SpliceAI (currently considered the “gold standard” tool). As a medical genetics resident, I saw this project as an excellent opportunity to learn more about the fascinating nature of splicing and gain skillsets in analyzing genome and RNA sequencing data.
HGGA: What about this paper/project most excites you?
RO: There are many things that I think are exciting. First, we found that nearly one in four CSSVs might not cause LoF and that in silico predictions using established tools such as SpliceAI were often discordant with RNA sequencing data. Interestingly, we found that while the in silico predictions were sometimes able to predict the overall outcome (frameshift vs. non-frameshift), the altered splicing events (cryptic splice site usage, intron inclusion, or exon skipping) were often incorrectly assumed, highlighting the need for RNA studies to accurately understand the impact of CSSVs. Finally, in the absence of any evidence-based guidance, we assumed for all in silico predictions that exon skipping would be the typical impact of an acceptor site loss and that intron retention/inclusion would be the typical impact of a donor site loss. Our results further supported, however, that this reasoning is overly simplistic and often false, underscoring our gaps in knowledge of complex splicing processes in human cells.
HGGA: What do you hope is the impact of this work for the human genetics community?
RO: I would encourage geneticists to inquire when genome sequencing identifies novel rare CSSVs in genes associated with rare, poorly characterized conditions with non-specific phenotypes, such as autism or developmental delay, and consider appropriate clinical evaluation and RNA/functional studies to further evaluate the pathogenicity of the CSSV in question. This may be especially important in the context of newborn genomic screening and sequencing programs where phenotypes may be absent/variable. Furthermore, understanding the impact of the CSSVs on splicing and gene expression accurately will shed light on potential targeted therapies where knowing the exact mechanisms of disease is critical.
HGGA: What are some of the biggest challenges you’ve faced as a young scientist?
RO: I think knowing what I don’t know is definitely easier than not knowing what I don’t know. This is where I have personally benefitted from having a mentor who can help me focus on the areas of knowledge that are lacking and provide insights on whether the questions I am asking are valuable/relevant questions. I think as humans, we also like to believe that we know what we know. However, this project has opened my eyes to the potential downfalls of certain heuristics and to see that our attempts to generalize/reduce complex information can result in missing important details or exceptions to the rule. It was an important reminder for me to keep an open mind to there being multiple possible explanations/mechanisms behind complex biological processes. Lastly, the rapidly evolving landscape of genetics and genomic technologies (as we identified a number of newly developed in silico tools/algorithms even during the manuscript writing process) is always a challenge to stay on top of. I hope to strive to keep abreast of relevant findings in genetics and genomics research to be able to recognize which of the scientific advances may apply to a particular patient/family in my genetics clinic.
HGGA: And for fun, what is one of the most fascinating things in genetics you’ve learned about in the past year or so?
RO: I recently learned about repeat associated non-AUG (RAN) translation in the context of neurogenetic disorders. This novel mechanism underlying some of the most debilitating neurological disorders (now collectively to be thought of as “RANopathies”) has identified potential new therapeutic targets (including repurposing of metformin in clinical trials for C9orf72-related amyotrophic lateral sclerosis (ALS)), highlighting the importance of delineating the exact pathogenetic mechanisms to improve treatment options and clinical outcomes.
Rachel Youjin Oh, MD, is a fourth year resident in the Medical Genetics and Genomics program at the University of Toronto and the Hospital for Sick Children.