AG-exclusion zones revisited: lessons to learn from 91 intronic NF1 3' splice site mutations outside the canonical AG-dinucleotides.

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.