A predicted missense mutation of RPGR causes aberrant RNA splicing with exon skipping

Abstract

Abstract: : Purpose: Mutations in the RPGR (retinitis pigmentosa GTPase regulator) isolated from RP3 region (Xp21.1) are responsible for up to 70% of X–linked (XL) retinitis pigmentosa (RP) families. RPGR exon ORF15 mutations also cause XL–atrophic macular degeneration and COD1 type XL–cone–rod dystrophy (CRD). During the RPGR mutation screening of DNA samples from our male patients with XL or isolated forms of RP or CRD, we identified a pathogenic nucleotide substitution (213G>A, last base of exon 2) that was predicted to cause a missense change (G52R) in the final protein. The purpose of this study was to determine whether this mutation could also alter the effectiveness of the adjacent splice junction. Methods: Total RNA was extracted from lymphocytes of the proband and his carrier mother. The cDNA was synthesized by reverse transcription and used for PCR amplification of the RPGR by use of a forward primer from exon 1 and a reverse primer from exon 3. After gel checking, the amplified PCR products were purified and directly sequenced in an ABI377 automated sequencer. Results: Gel analysis of the PCR products revealed that the proband had a single aberrant band smaller than expected size and his mother showed both the normal and aberrant bands. Sequence analysis of the aberrant transcript showed skipping of exon 2 (126 nt) which leads to an RPGR protein with an in–frame deletion of 42 amino acids, affecting part of the critical RCC1–like domain. Conclusions: The last base of exons is conserved as a G in 80% of 5 splice site consensus sequences, and yet when changed, it can cause complete disruption of normal splicing as shown in our case. Our data confirms that RNA analysis is the only way to determine the molecular pathology caused by some DNA sequence changes. Other groups have reported a different base change at the same nucleotide position (213G>T) and proposed that it would act as a nonsense mutation (G52X). It is likely that the real action of this variant is also at the splicing level. Other presumed missense mutations can also be splicing related if they are located in exonic splicing enhancers. The knowledge of the exact effects of different mutations is important for genotype–phenotype correlations and for clarifying the molecular mechanisms of genetic diseases.

Keywords: retinal degenerations: hereditary • genetics • mutations