Effect of GGCX mutations on its pre-mRNA splicing. (A) Minigene splicing assay of GGCX mutations. GGCX mutations and the wild-type sequences of the affected exons were transiently transcribed in HEK293 cells. The splicing products were amplified by reverse transcriptase-PCR and visualized by 2.5% agarose gel electrophoresis. Previously reported missense mutations in GGCX’s coding regions were labeled in red. M, DNA ladder marker; Control, splicing product from the empty vector. Exon 2, exon 3, exon 11+12, and exon 15 are the splicing products from the wild-type GGCX sequence of the corresponding exons. (B) Sequencing results of the splicing products of wild-type GGCX and of the c.1609 G>T mutant (bottom band). The mis-splicing site in exon 11 is indicated with a red arrowhead. (C) Sequencing results of the splicing products of wild-type GGCX and the c.1609 G>T mutant (top band). The 12-bp intronic insertion sequences are indicated in red. The c.1609 G>T mutation in the mis-splicing sequence is indicated with an asterisk. (D) Amino acid sequences of the 2 mis-splicing products of the c.1609 G>T mutation. The G537-a sequence corresponds to the mis-splicing of the 25-bp deletion, as shown in panel B. The G537-b sequence corresponds to the mis-splicing of the 12-bp insertion, as shown in panel C. Additional sequences introduced to GGCX are highlighted in red. The asterisk in the G537-a sequence indicates an introduced stop codon because of the frame-shift translation.