Abstract
Uncovering frequent motives of action by which variants impair 3′ splice site (3′ss) recognition and selection is essential to improve our understanding of this complex process. Through several mini‐gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722‐11T>G, although expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG‐exclusion zone (AGEZ) between the authentic 3′ss AG and the branch point. Evaluation of 90 additional noncanonical intronic NF1 3′ss mutations confirmed that 63% of all mutations and 89% (49/55) of the single‐nucleotide variants upstream of positions ‐3 interrupt the AGEZ. Of these AGEZ‐interrupting mutations, 24/49 lead to exon skipping suggesting that absence of AG in this region is necessary for accurate 3′ss selection already in the initial steps of splicing. The analysis of 91 noncanonical NF1 3′ss mutations also shows that 90% either introduce a novel AG in the AGEZ, cause a Y>R transversion at position ‐3 or remove ≥2 Ys in the AGEZ. We confirm in a validation cohort that these three motives distinguish spliceogenic from splice‐neutral variants with 85% accuracy and, therefore, are generally applicable to select among variants of unknown significance those likely to affect splicing.
Highlights
Through several mini‐gene experiments, we demonstrate that the pyrimidine (Y) to purine (R) transversion NM_000267.3(NF1):c.1722‐11T>G, expected to weaken the polypyrimidine tract, causes exon skipping primarily by introducing a novel AG in the AG‐exclusion zone (AGEZ) between the authentic 3′ss AG and the branch point
Differences in the composition of trans‐acting splicing factors in the two cell lines may explain why the use of this cryptic 3′ss is more pronounced when the minigene construct C4 is transfected into the SH‐SY5Y cells and exon skipping is more prevalent in the HEK293 cells. These results suggest that c.1722‐11T>G exerts its splice effect mainly by creating an AG‐dinucleotide within the AGEZ, which seems to have a stronger impact on the recognition and/or use of the authentic 3′ss than the loss of a pyrimidine of the polypyrimidine tract (PPT)
Through the in‐depth analysis of the neurofibromatosis type 1 (NF1) variant c.1722‐11T>G and the subsequent evaluation of 91 disease‐causing NF1 variants located at the 3′end of an intron but outside the canonical AG‐dinucleotide we show here that creation of a novel AG within the AGEZ, that is the intronic sequence between the branch point (BP) and the AG of the authentic 3′ss, is a frequent action motive of intronic sequence variants with a splice effect
Summary
Even “leaky” NF1 splice mutations, that is splice mutations leading to a splice effect only in a proportion of, but not in all, transcripts from this allele, may result in an attenuated but still clinically recognizable NF1 phenotype This has been observed in a number of patients with typical cutaneous NF1 features such as multiple café au lait maculae (CALM) and cutaneous neurofibromas (see e.g., Fernandez‐Rodriguez et al, 2011), including a case that will be discussed in this report. Direct complementary DNA (cDNA) sequencing of the entire coding sequence starting with RNA extracted from short‐ term lymphocyte cultures treated with puromycin to prevent nonsense‐ mediated RNA decay, proved to be highly sensitive for the detection of noncanonical splice mutations (Messiaen & Wimmer, 2008) Having applied this mutation analysis strategy for roughly two decades, we have identified a large number of noncanonical NF1 splice mutations fully characterized at the transcript level. These three motives will be useful to select for further transcript analysis that intronic VUS found at a gDNA level that are most likely to have a splice effect
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