The intronic mutation c.1889-6G>A affects GGCX splicing. (A) Chromatograms of the sequencing results of the patient’s genomic DNA near the junction of intron 13 and exon 14. A heterozygous mutation of c.1889-6G>A was identified; it is indicated by an asterisk and an arrow. The splicing-acceptor site (AG) of the wild-type and the mutant sequences is underlined. (B) Schematic diagram of the minigene splicing assay. Polymerase chain reaction (PCR) fragments of exon 14 flanked by intronic sequences were cloned into a pSPL3 minigene splicing assay vector. These constructs were transfected into HEK293 cells, and the splicing products were amplified by reverse transcription polymerase chain reaction (RT-PCR) for sequencing analysis. (C) DNA electrophoresis of the splicing products from the empty vector, the wild-type GGCX, and the c.1889-6G>A mutant. (D) Chromatograms of the sequencing results of the splicing products of the wild-type GGCX and the c.1889-6G>A mutant. Intronic insertion sequences are indicated in red. (E) Amino acid sequences of the C-terminus of the 2 identified GGCX truncation mutants. Additional sequences added to the C-terminus of GGCX due to reading frame shift are highlighted in red. bp, base pair; LPAS, late poly(A) signal; SA, splice acceptor; SD, splice donor; SV40, simian virus 40.