Figure 4.

Impact on transcript processing of splicing mutations. Exons are indicated by boxes and introns are represented by horizontal bars (not to scale). Location of specific primers used in RT-PCR is indicated by arrows. Dotted lines represent the anomalous splicing outcome and sequences of the abnormal products are shown (electropherogram). Genomic mutations (in red) are harboured in a heterozygous state and the consensus transcript was also detected in patients’ samples further confirmed by specific band sequencing. (A–C) Reverse transcription-polymerase chain reaction (RT-PCR) products obtained from RNA of control and patients’ blood samples (agarose electrophoretic results) and schematic representation of the aberrant splicing events. (A) CASK c.2589+2T>G (MD-208) induces the complete skipping of exon 26, corresponding to the lower 402 bp band. (B) PDGFB c.602-1G>C (MD-173) causes the loss of the last exon of the gene plus 28 bp of the contiguous 3′ untranslated region (3′UTR) region presenting a 367 bp aberrant product absent in the healthy control. (C) CPLANE1 c.7588+3G>A (MD-216) resulted in single exon 37 skipping leading to an alternative transcript 55 bp shorter than the expected. (D) PMPCA exon 6 minigene assay. Agarose gel shows the band pattern of transcripts obtained after overexpression of pSPL3-derived constructs: wild-type (WT) allele, mutant allele c.633+1G>A and empty vector ø. SD6 and SA2 are the resident exons of the pSPL3 reporting vector. In the absence of an inserted fragment, a product of 263 bp is obtained. The analysed variant causes the skipping of exon 6. bp, base pair; Ct, control; FW, forward primer; RV, reverse primer.