Figure 3.

Molecular genetic characterization of the splice effect of the c.718+23G>A variant in TULP1. (a) Schematic representation of the TULP1 gene and enlargement of the wild‐type and mutant DNA sequences at the exon–intron boundary of exon 7 of TULP1. The SpliceSiteFinder‐like (SSFL, range 0–100), MaxEntSCan (MES, range 0–12), GeneSplicer (GS, range 0–24) and Human Splicing Finder (HSF, range 0–100) scores for the splice donor site are indicated above the gene. The red “A” highlights the variant c.718+23G>A identified in both siblings. The red numbers represent altered scores compared to the wild‐type. The green circle implies a newly recognized SC35 motif. Dotted circles indicate exonic splice silencers no longer present by prediction tools (b) Schematic representation of the mutant pCI‐NEO‐RHO vector, containing exon 4–11 of the TULP1 gene used to transfect HEK293T cells with a wild‐type or mutant midigene. (c) RT‐PCR products of the wild‐type and mutant midigene showing the expected 832‐bp wild‐type fragment and a 852‐bp fragment of the mutant midigene corresponding to a 20‐nucleotide elongation of the mRNA encoded by exon 7. The wild‐type fragment was absent in the cells transfected with the mutant midigene. RT‐PCR analysis of RHO exon 5 was performed as a control for efficient transfection. (d) Sanger sequence analysis of the RT‐PCR fragments confirmed the wild‐type and the 20‐bp elongation of exon 7 in the mutant