Figure 3. Exon 16 splicing pattern associated with the c.1935G>A mutation. A: Analysis of endogenous RNA from lymphoid cells. We amplified a 346-bp fragment from exon 14 to exon 17 of MYO7A in a healthy control individual. An additional faint band appeared in the control; it corresponded to an mRNA species missing exon 16 (lane 2). In patient MB9, the splicing pattern showed a predominant exclusion of exon 16 (lane 1). Lane 3 refers to negative control and M indicates size marker. Direct sequencing of these two different transcripts revealed that a shorter band of 246 bp corresponds to an abnormal transcript without exon 16, whereas the other band of 374 bp corresponds to a normal transcript that contains exon 16. B: Ex vivo splicing assays were performed. Wild-type and mutant constructs were stably transfected in HeLa cells, and the exon 16 splicing pattern was analyzed by reverse transcription-polymerase chain reaction. Consistent with data presented in A, exon 16 is predominantly included from the wild-type construct (lane 1). The c.1935G>A mutation at the end of exon 16 resulted in massive skipping of the exon (lane 2). Lane 3 refers to negative control and M indicates size marker. UE and DE refer to upstream and downstream exons of the cassette, respectively.