RT-PCR analysis of mutations affecting splicing and transcript structure.

Each aberrant splicing product appearing on the agarose gel was isolated and sequenced to confirm the interpretation shown in the schematic. The genomic DNA mutations are depicted in green, the resulting cDNA aberration in red. Because no bone marrow was available from patients with the IVS4 + 3SD and IVS4-40SD mutations, the result shown is the predicted result. Note that patient 11 has a 5T insertion and patient 12 a 24-bp deletion, neither of which would be expected to alter splicing. Nevertheless, the 5T insertion produces at least 2 transcripts, one containing the 5T insertion and another unanticipatedly forcing utilization of the 2nd cryptic upstream splice donor site. For patient 12, whereas only 2 transcripts were identified (the normal and one containing the 24-bp deletion) following subcloning and sequencing, uncharacterized higher molecular weight products that presumably represent misprocessed primary transcripts are apparent.