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).