The Retinitis Pigmentosa Mutation c.3444+1G>A in CNGB1 Results in Skipping of Exon 32

Retinitis pigmentosa (RP) is a severe hereditary eye disorder characterized by progressive degeneration of photoreceptors and subsequent loss of vision. Two of the RP associated mutations were found in the CNGB1 gene that encodes the B subunit of the rod cyclic nucleotide-gated channel (CNGB1a). One of them (c.3444+1G>A) is located at the donor site of exon 32 and has been proposed to result in a frameshift and truncation of the last 28 aa of the corresponding protein. However, this ambiguous conclusion was not verified by experimental data. Recently, another study reported that the last 28 aa of CNGB1a harbor a motif required for the proper targeting of this subunit to rod photoreceptor outer segments. This suggests that defective targeting is the major cause for the RP phenotype in affected patients. Here, we investigated the splicing of c.3444+1G>A by exon trapping experiments and could demonstrate that instead of the proposed truncation of the last 28 aa this mutation leads to replacement of the last 170 aa of CNGB1a by 68 unrelated amino acids. The 170 aa deletion covers the complete distal C-terminus including the last 10 aa of an important alpha (αC) helix within the ligand-binding domain of CNGB1a. When expressed in a heterologous expression system the corresponding mutant full-length CNGB1a subunit was more susceptible to proteosomal degradation compared to the wild-type counterpart. In conclusion, our experimental data do not support the hypothesis proposed by the original study on the c.3444+1G>A mutation. Based on this, we suggest that apart from the defective targeting other mechanisms may be responsible for the RP phenotype in affected individuals

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