Figure 4. cDNA analysis for c.3319A>C mutation of the ROBO3 gene. Reverse transcription (RT)-PCR showed leaky splicing with a weak wild-type band with an expected size of 576 bp and a stronger band of around 450 bp, yielded by the skipping of exon 22 of the ROBO3 gene, for patient 2 (lane 1). For the heterozygous father (lane 2), RT–PCR showed a stronger wild-type band and a weaker band for the exon 22 skipped product. Sequencing of the different products showed unaltered splicing for the larger product (upper electropherogram) and skipping of exon 22 of the ROBO3 gene (electropherogram below) for the smaller product leading to a frameshift and premature stop codon on the protein level.