FIG. 2.

Characterization of the paternal c.14646+2563C>T mutation. (a) RT-PCR performed on CF and MF cells differentiated during 4 days. RT-PCR amplification was performed on the region c.14438–15030 of RYR1 including exons 101 and 102. Size analysis of the amplified fragments showed the presence of a normal-sized fragment of 593 bp and of an abnormal 692 bp fragment in MF cells (lane 2), while only the normal-sized fragment is present in CF cells (lane 1). (b) Scheme of the minigene constructs used for the analysis of the c.14646+2563C>T mutation. RYR1 intronic regions containing sequence of the pseudo-exon, with (gray) or without (white) the mutation, were inserted into a pCIneo vector between exons −1 and +1. An RT-PCR amplification using a forward primer in exon −1 and a reverse primer in exon +1 (arrows) was performed on HEK 293 cells transfected with each minigene construct. Size analysis of the amplified fragments showed the presence of a unique fragment of 620 bp in HEK293 cells transfected with the C construct (lane 1), corresponding to the normal splicing, and of two fragments of 620 and 719 bp in HEK293 cells transfected with M construct (lane 2), corresponding to the inclusion of the pseudo-exon. The identity of these fragments was confirmed by sequencing. CF, control fetal; MF, mutant fetal; RT-PCR, reverse transcriptase–polymerase chain reaction.