Fig. 2: Molecular characterization of RAI1 intragenic deletion and expression analyses.

A MLPA profile’s probes for RAI1 gene reveal in the proband a heterozygous deletion of RAI1 involving exon 5. Each black dot displays the final probe ratio for each locus analyzed in the patient compared to controls. Standard deviations were set up according to the Coffalyzer DB software v131211. B Graphic representation of RAI1 deletion characterized in the patient at genomic and transcript levels. Top panel: electropherogram showing the deletion breakpoints at genomic level. The 3.4 kb RAI1 deletion is represented by a red bar involving part of IVS4, exon 5, IVS5 and part of exon 6 (chr17:17710076-17713445, hg19). Black bars above RAI1 exons refer to MLPA probes. Bottom panel: electropherogram of the aberrant RAI1 transcript, derived from the activation of a new splice-site in exon 6. C Wild-type and mutated RAI1 mRNAs and corresponding proteins with the insertion of 64 new amino acids highlighted in red. D Scatter plots obtained using TaqMan probe on exon junction 3-4, showing global RAI1 expression in patient (circles), brother (squares), mother (triangles pointing upward), father (triangles pointing downward) relatively to normal controls (rhombuses). The horizontal black bars indicate the range between mean ±2 standard deviation values. Data were normalized against TBP housekeeping gene. Similar results were obtained after normalization against GAPDH housekeeping gene (data not shown) and by using the Taqman probe on exon junction 2–3 (data not shown). E Scatter plots using the Syber green probe designed across exon junction 4–5, involved in the deletion showing specific expression of wt RAI1 transcript in:patient (circles), brother (squares) and mother (triangles pointing upward), respectively. Data were normalized against TBP. Similar results were obtained after normalization against GAPDH housekeeping gene (data not shown). F Family tree summarizing the RAI1 genotype and transcripts.