Assessment of the effects of NF1 variants on NF1 pre-mRNA splicing
We investigated the effects on NF1 pre-mRNA splicing of 34 variants that had been identified in individuals suspected of NF1 (Figure 1). In 13 cases, the results of the in vitro exon trap experiments were confirmed by analysis of RNA from a blood sample from the corresponding individual; in 4 cases subject RNA was analyzed directly, without performing exon trapping (Figure 1B). For the remaining subjects, RNA was not available.
We detected one or more abnormal splice products, either in vitro , in subject RNA, or in both for 25 variants (74 %). Exon skipping (type I defect) [Anna and Monika, 2018] was observed for 18 variants; 2 variants resulted in incorporation of a pseudo-exon causing premature truncation of the NF1 ORF (type II defect); utilization of a non-canonical splice site, resulting in exon truncation (type III defect) was observed for 4 variants; and 3 variants caused intron retention (type IV defect). In 5 cases, abnormal splicing resulted in an in-frame deletion (Supplementary Information, Table S1; Figure 1C). Of these, the c.288+3 A>T, r.205_288del, p.(Arg69_Gly96del) and c.2710A>T, r.2707_2850del, p.(Cys904_Val951del) deletions were selected to determine whether the deletion affected NF activity (see below). In 7 cases, a missense change completely prevented canonical NF1 pre-mRNA splicing, making assessment of NF function redundant. In 9 cases, we did not observe an effect of the variant onNF1 pre-mRNA splicing, either in vitro or in subject RNA (Supplementary Information, Table S1). These cases included 5 missense variants, of which 2 could be subjected to functional assessment (see below). In total, analysis of NF1 pre-mRNA splicing assisted in the classification of 25 variants as likely pathogenic (Supplementary Information, Table S1).