PMC5279100_01
Alamut 2.0 documentation, splicing predictor: predictions window around the REEP1 c.595 + 1G>A variant. The top box represents the wild-type sequence, with a G at position c.595 + 1, and the bottom box represents the mutant sequences, with an A at c.595 + 1. Probabilities for the use of the 5'-splice sites are indicated for 5 different algorithms.
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F1
The ALAMUT v2.0 software (Interactive Biosoftware, Rouen, France) was used for in silico predicted scores of splicing and showed a marked decrease in donor splice site usage suggestive of exon 6 skipping (Fig. 1).
Amplification of the muted area from complementary DNA (A), and agarose gel electrophoresis showing truncated amplicon (B). bp = base pairs.
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F2
Agarose gel electrophoresis highlighted a truncated amplicon not found in a control sample (Fig. 2).
Complementary DNA sequencing with Sanger method of reverse transcription polymerase chain reaction products showing truncation of exon 6 in messenger RNA. (A) Wild-type allele, (B) mutant allele.
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F3
Direct sequencing of the PCR products confirmed complete deletion of the exon 6 at the mRNA level leading to a frame shift (Fig. 3), inferring modification of the C-terminal portion of the resulting protein (p.G140Afs*22).