Fig. 3.

The ex vivo splicing assay. (a) Natural and putative splice sites in the region around TGFBR1 exon 5 with S&S splice site scores. The putative splicing donor site 9 bp upstream of the 5′ natural splice site and splice acceptor site 76 bp downstream from the 3′ natural splice site were predicted to contribute to the pathogenesis of LDS and MSSE, respectively. (b) The pTBNde(min) minigene system used to analyze the effect of TGFBR1 variants on ex vivo pre-mRNA splicing. (c) Splicing products amplified by RT–PCR using 2–3α and B2 primers shown in (b) were separated by 1.2% agarose gel electrophoresis. The identity of the spliced products was established by direct sequencing (d–f) and is schematically represented on the right. (d–f) Direct sequencing of cDNA from wild-type (top) and variant (bottom) alleles. In the small and large products of LDS variant alleles, the entire exon 5 (168 bp) (d) and last 9 bases of exon 5 (e) were missing, respectively. (f) For the MSSE variant allele, the first 76 bp bases of exon 5 was missing. FN-EDB, fibronectin-extra domain B; aa, amino acid position of TGFBR1