Figure 3.

Sequence analysis of in vivo splicing in patient 1 and his parents. (A) RT-PCR amplification products using primers in exons 4 and 6 of CYP11A1. Lanes 1 and 2, patient 1 and his mother, showing two bands corresponding to transcripts containing exon 5 (upper band) and skipping exon 5 (lower band); this was confirmed by Sanger sequencing. In contrast, in lane 3, the father showed only the upper, exon 5 containing, transcript. (B) Partial chromatograms from Sanger sequencing of the upper bands in the patient, mother, and father. For the c.990G>A change (Right), the patient and father’s sequence revealed only the WT c.990G (white arrows), suggesting that the c.990A variant results in exon skipping and destruction of the truncated mRNA by NMD. For c.940G>A (Left), the patient’s sequence only has the mutant c.940A allele A inherited from his mother (black arrow), corroborating the NMD destruction of the c.990A allele inherited from his father; if it were present, it would give a heterozygous base at this position. In contrast, the mother’s sequence shows both WT G and a small peak of the mutant A (black arrow), consistent with skipping of mutant exon 5 in most mutant transcripts but revealing the presence of some transcripts containing exon 5.