A Novel Heterozygous Intronic Mutation in the FBN1 Gene Contributes to FBN1 RNA Missplicing Events in the Marfan Syndrome

Figures

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  Figure 1. Schematic representation of in silico analysis of the c.2678-15C>A variant identified in intron 22 of the FBN1 gene by various computational tools. (a) Representation of the position of the canonical (c.2678, black triangle) and cryptic (c.2678-13, grey triangle) splice site identified in the reference (RS) and mutated sequence (MS), respectively. Arrow: the C to A point mutation (c.2678-15C>A). The intron 22 (i22) sequence is shown in lower case and exon 23 (E23) sequence in upper case. (b) The predicted splice score of the canonical site decreases by 29.8% and 49.7% in the mutated sequence (grey parallelogram) versus the reference sequence (black parallelogram) as revealed by the MES and GSP algorithm, respectively. (c) The predicted splice scores of a new cryptic splice site due to the c.2678-15C>A mutation.

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  Figure 2. RT-PCR analysis of minigene-derived transcripts. (a) Schematic representation of FLAG (F) and Myc- (M-) tagged reference (FBN1-Ref) and mutant (FBN1-Mut) minigene plasmids (PL). Exons (E) are denoted with white boxes and introns (i) with solid black horizontal lines. The approximate location of the primers for downstream RT-PCR analysis is shown (for primer sequences see Table 1). (b) Expression of FBN1-Ref and FBN1-Mut minigenes in HeLa and COS-7 cells as revealed by RT-PCR, using primers (521 and 530) targeting E22 and Myc. A representative of two independent experiments for each transfection is shown. RPL19 amplification was carried out as an input RNA control for the RT-PCR. L: size reference ladder. MC: mock cells. (c) Schematic representation of PCR products corresponding either to correct splicing (374 bp band) or to partial inclusion (dark grey box) of intron 22 (387 bp band) as revealed by sequencing (see Supp. Figure S3). (d) Partial inclusion of intron 22 in transcripts was assayed by using different combinations of primers targeting FLAG (primer 528 shown in (a)), exon 22/exon 23 junction (primer 533), or exon 23/retained intron 22 inside (primer 535). (e) A diagram…

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  Figure 3. Western blot analysis of minigene-derived proteins. (a) FLAG/Myc-tagged reference (FBN1-Ref) and mutant (FBN1-Mut) minigene plasmids (PL) were transfected into HeLa cells as indicated at the top of each lane, and cell lysates were analyzed by Western blotting with a mouse monoclonal anti-FLAG (upper panel) or anti-Myc (lower panel) antibody. The results from experiments performed on four batches of cells for each transfection are shown. Lane 13: cells transfected with empty vector. The Western blot detection of the FLAG-tagged bacterial alkaline phosphatase (BAP) was used as a marker of equivalent transfection efficiency and equal loading. MW values (kDa) of the bands detected are shown in brackets. (b) COS-7 cells were transfected as indicated at the top of each lane and analyzed by Western blotting with a mouse monoclonal anti-FLAG antibody (upper panel). Protein load (lower panel): membrane stained with Amido Black 10B after immunostaining. Other indications as in (a).

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  Figure 4. Treatment of transfected cells with MG132 leads to detection of truncated proteins derived from mutant FBN1 constructs. (a) FLAG/Myc-tagged reference (FBN1-Ref; PL 1283) and mutant (FBN1-Mut; PL 1288) minigene plasmids (PL) were transfected into HeLa cells as indicated at the top of each lane. Lane 9: mock cells. Either the proteasome inhibitor MG132 or the DMSO vehicle was applied to transfected cells as indicated in Materials and Methods. Whole-cell extracts of transfected cells were analyzed by Western blotting with a mouse monoclonal anti-FLAG antibody. Black arrows: MW values (kDa) of the bands detected. (b) A diagram of the protein products as predicted by sequence analysis of the corresponding cDNAs. Deduced MW values (kDa) are also indicated. Dark grey box: partial inclusion of intron 22; F: FLAG epitope; M: Myc epitope; PTC: premature termination codon.