Figure 4.

RT-PCR analysis of the c.2607A>G, p.Ala869Ala mutation. Cultured cells from a heterozygous carrier were incubated with (−NMD) or without (+NMD) emetine to inhibit nonsense mediated decay. NMD was also inhibited in a normal cell line as a control. RNA was analysed by RT-PCR and sequenced in the forward (A) and reverse (B) directions. Text sequence represents the sequence seen in uninhibited cells (upper) and the additional sequence obtained from inhibited cells (lower). In (A) an additional sequence was located between exons 32 and 33 (in bold type), which corresponded to a pseudoexon in intron 32. In the reverse direction, the mutant sequence (arrowed) was present in cDNA from uninhibited (+NMD) cells. When nonsense mediated decay was inhibited an additional transcript was seen which corresponded to the use of the GTGCAC sequence (in bold) as a donor splice site.