c.3444+1G>A mutation affects the splicing and expression of CNGB1.

<p>(A) Schematic representation of the minigene construct used for the exon trapping experiment showing the position of the c.3444+1G>A mutation (marked by an arrowhead) and the deleted intronic <i>Xba</i>I-fragment. Vector backbone sequence is depicted in green. (B) Revese transcriptase PCR from HEK293T cells transfected with mutant and wild type minigene constructs. The electropherogram for the c.3444+1G>A mutant shows the skipping of exon 32. (C) Scheme showing the splice products. The length of the respective PCR products is indicated by double arrows. (D) Schematic comparison of the WT and mutant protein demonstrating the lack of the entire distal C-terminus and the last 10 aa of the αC helix in the context of the c.3444G>A mutation. Skipping of exon 32 causes a frameshift which results in addition of 68 unrelated amino acids after aa position 1075 of the CNGB1a protein (highlighted in grey). The numbers represent the length of the respective proteins (1245 aa for WT and 1143 for the mutant). (E) Western blot of membranes isolated from HEK293T cells transfected with CNGA1 and wild type or mutant CNGB1a probed with anti-B1 (<i>top panel</i>) or anti-ATPase (<i>bottom panel</i>). The weaker expression of the mutant protein was normalized in the presence of the proteasome inhibitors MG-132 and ALLN. CNBD: cyclic nucleotide-binding domain. Primers are shown as arrows. S1–S6: transmembrane segments; WT: wild type, Mut: c.3444+1G>A mutation.</p