Ann Lab Med.  2024 Mar;44(2):188-191. 10.3343/alm.2023.0163.

Reverse Transcription-PCR-based Sanger Sequencingconfirmed Exon-skipping Effect of a Novel GEN1 Intronic Variant (c.1408+4A>G)

• Kim J
• Yu J
• Lee S
• Kim S
• Lee IS
• Lee W
• Chun S

• KMID: 2553392
• DOI: http://doi.org/10.3343/alm.2023.0163

Figure

• 

  Fig. 1 Genomic alterations in GEN1 detected using a targeted NGS panel, and agarose gel electrophoresis of RT-PCR products. (A) Integrative Genomics Viewer display of the c.1408+4A>G variant in GEN1 (B) Schematic diagram of the adenine (A) to guanine (G) substitution in the consensus sequence at a splice donor site in intron 13 (R denotes a purine [A or G]). (C) Agarose gel image showing only the normal splice product (a, 500 bp) in the control, and both aberrant (b, 356 bp) and normal splice products in the patient. A 500-bp amplicon was designed to partially cover exons 11 and 14 and fully cover exons 12 and 13 in the control using a target-specific primer set. Abbreviations: NGS, next-generation sequencing; RT-PCR, reverse transcription-PCR; VAF, variant allele frequency.

• 

  Fig. 2 Schematic diagram of transcript analysis of the control and patient. (A) The c.1408+4A>G variant causes aberrant RNA splicing, resulting in the skipping of exon 13 (r.1265_1408del). (B) The r.1265_1408del mutant translates into a protein with a 49-amino-acid deletion and an insertion of a glycine in the deleted region (p.Glu422_Arg470delinsGly). Residues 422–464 within the deleted region are part of the chromodomain (residues 390–464). (C) Sanger sequencing chromatograms demonstrating heterozygosity for the variant that causes exon 13 skipping (forward sequence).

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