Figure 3

Analysis by RT- PCR of the endogenous splicing pattern of control and patients SFCP, SFC3, SFC6 and SFC13 derived fibroblasts after transfection with different U1 isoforms. (A), (C) and (E) The constitutive splicing of exons 2, 6 and 15 of the HGSNAT gene was not altered in control fibroblasts after overexpression of U1-WT or any of the modified U1 constructs. (B) In the patients SFCP and SFC3, bearing the homozygous mutation c.234 + 1G > A, only the fully adapted U1 (2.5 μg of U1-sup4) resulted in partial correction of exon 2 skipping. The same result was obtained with 1 or 3.5 μg of the U1-sup4 construct (data not shown). (D) and (F) For patients SFC6 and SFC13, bearing genotypes c.633 + 1G >A/c.1334T > C and c.1542 + 4dupA/c.1150C > T, respectively, the transfection of 2.5 or 3.5 μg of U1-WT or any generated U1 suppressor vector did not produced any change in the endogenous aberrant splicing pattern. Sequencing results for all RT-PCR products are illustrated by schematic drawings. M: molecular weight marker; NT: non-treated cells; C-: negative control; RFP: red fluorescent protein.