Fig. 2: Effect of the variant c.531 + 1482 A > G on transcript level. | European Journal of Human Genetics

Fig. 2: Effect of the variant c.531 + 1482 A > G on transcript level.

From: Genome sequencing-based discovery of a novel deep intronic APC pathogenic variant causing exonization

Fig. 2

A RT-PCR product of the variant carrier yielded a larger extra band on 1.5% agarose gel electrophoresis. The same result is seen on Agilent DNA1000 chip. Black arrow points out the band for the normal transcript, red arrow indicates the larger band of the aberrantly spliced transcript and blue arrows show the heteroduplexes. L: ladder, C1, C2, C3: controls. B Sanger sequencing of the RT-PCR amplicon revealed a 56-bp exonization of intron 5 of APC. Forward sequence and reverse sequence superpositions show the 5’ and the 3’ ends of the exonized stretch, respectively. C Graphical representation of the exonization of a 56-bp stretch of intron 5 of APC as a result of the germline variant c.531 + 1482 A > G. Red dot shows the germline intronic base change, dark green boxes are canonical exons and light green box is the exonized intronic region. Orange AG and GT dinucleotides are the cryptic donor and acceptor sites activated as a result of the variant. The exonized region sequence is highlighted below, visualized by a screenshot of the UCSC Genome Browser (https://genome-euro.ucsc.edu). Exon and intron sizes are not to scale. D Fluorescent capillary electrophoresis performed by selective amplification of the normal and aberrantly spliced products. The 195 bp peak is consistent with the aberrant fragment and is present exclusively in the variant carrier. E Allelic imbalance measured at the positions of coding heterozygote variants c.1458 T > C and c.1635G > A of the proband by comparing electrophoretic peak height ratios of variant and reference signs in cDNA and gDNA. Calculated ratios are indicated below the figures (see Methods for calculation formula). The variant positions are depicted from their reverse sequences. F Tagging SNP test carried out for the detection of the degree of the aberrant splice event. The RT-PCR products are amplified selectively from the wild-type transcript. The arrows point out the positions of the exonic germline heterozygote variants and indicate, that only the reference nucleotide is present at these positions at the RNA-level, therefore the normal transcript comes from only one allele.

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