The impact of the substitutions in exon 3’s last nucleotide (position c.550) on splicing was previously functionally evaluated using RNA analysis and was established as pathogenic [75]. In the same splice site, we detected a novel variant, c.550 + 3A > C, in a patient P02201 and as the variant had not been previously reported, we tried to functionally evaluate it. Unfortunately, none of the patient’s family members were available for testing. The variant potentially affecting the donor splice site (5'ss) of exon 3 was analyzed in silico by MaxEnt Score [76], and the ratio between mutated and wild-type 5'ss sequence was 0.41—a number suggesting a substantial effect on splicing. According to Le Guédard-Méreuze et al. [77], substitution + 3A > C is prone to cause a splicing defect even if the donor splice site does not contain any further nucleotide changes. We performed minigene analysis (detailed procedure described in Supplement Methods) to confirm the deleterious effect on splicing and it showed aberrant splicing in nearly 100% of mutated minigene construct transcripts (Fig. 3).Fig. 3Minigene splicing analysis of a novel variant c.550 + 3A > C. The wild-type and mutant genomic fragments of SERPING1 comprising exon 3 and flanking upstream (229 bp) and downstream (255 bp) intron sequences, were cloned into pET01 vector and HepG2 cells were transfected with these minigenes. Capillary electrophoresis of RT-PCR products showed aberrant splicing in nearly 100% of mutated minigene construct transcripts. Several different aberrant transcripts were detected. The most abundant was intron 3 retention followed by exon 3 skipping. Transcripts using cryptic donor splice sites − 10 and + 27 were found in the mutant minigene analysis, whereas they were not detected in the wild type at all. a Minigene analysis results: column A shows transcript proportions resulting from control minigene construct representing c.550 + 3A; column C shows transcript proportions resulting from minigene construct representing c.550 + 3C. b Scheme of the transcripts detected in the minigene analysis. c Scheme of the pET minigene construct. Cryptic splice sites found in the analysis and their exact sequences are displayed. MaxEnt Score values (MES) are specified beneath each splice siteFull size imageVariant c.686-7C > G was detected in a mother and her daughter (P03001-P03002), both affected with HAE symptoms. This variant had been described before [7], but its impact had not been yet fully evaluated. Therefore, we extracted RNA from both patients’ blood and analyzed samples by RT-PCR and fragment analysis (Supplementary Methods), which showed complete impairment caused by the variant (Fig. 4).Fig. 4mRNA analysis of patients carrying variant c.686-7C > G. Capillary electrophoresis was performed on PCR products amplified by primers annealing to exons 4 and 7. a Healthy control shows only one wild type peak. b Both patient sample results show 2 peaks—reference transcript peak and a peak corresponding to a 6 bp longer transcript using a de novo created acceptor splice site. Aberrant and normal transcript proportion was roughly equal, which corresponds to complete impairment caused by the variant. The variant preserves a reading frame and leads to incorporation of additional two amino acids (proline-alanine) into the polypeptide chain

The impact of the substitutions in exon 3’s last nucleotide (position c.550) on splicing was previously functionally evaluated using RNA analysis and was established as pathogenic [75]. In the same splice site, we detected a novel variant, c.550 + 3A > C, in a patient P02201 and as the variant had not been previously reported, we tried to functionally evaluate it. Unfortunately, none of the patient’s family members were available for testing. The variant potentially affecting the donor splice site (5'ss) of exon 3 was analyzed in silico by MaxEnt Score [76], and the ratio between mutated and wild-type 5'ss sequence was 0.41—a number suggesting a substantial effect on splicing. According to Le Guédard-Méreuze et al. [77], substitution + 3A > C is prone to cause a splicing defect even if the donor splice site does not contain any further nucleotide changes. We performed minigene analysis (detailed procedure described in Supplement Methods) to confirm the deleterious effect on splicing and it showed aberrant splicing in nearly 100% of mutated minigene construct transcripts (Fig. 3).Fig. 3Minigene splicing analysis of a novel variant c.550 + 3A > C. The wild-type and mutant genomic fragments of SERPING1 comprising exon 3 and flanking upstream (229 bp) and downstream (255 bp) intron sequences, were cloned into pET01 vector and HepG2 cells were transfected with these minigenes. Capillary electrophoresis of RT-PCR products showed aberrant splicing in nearly 100% of mutated minigene construct transcripts. Several different aberrant transcripts were detected. The most abundant was intron 3 retention followed by exon 3 skipping. Transcripts using cryptic donor splice sites − 10 and + 27 were found in the mutant minigene analysis, whereas they were not detected in the wild type at all. a Minigene analysis results: column A shows transcript proportions resulting from control minigene construct representing c.550 + 3A; column C shows transcript proportions resulting from minigene construct representing c.550 + 3C. b Scheme of the transcripts detected in the minigene analysis. c Scheme of the pET minigene construct. Cryptic splice sites found in the analysis and their exact sequences are displayed. MaxEnt Score values (MES) are specified beneath each splice siteFull size imageVariant c.686-7C > G was detected in a mother and her daughter (P03001-P03002), both affected with HAE symptoms. This variant had been described before [7], but its impact had not been yet fully evaluated. Therefore, we extracted RNA from both patients’ blood and analyzed samples by RT-PCR and fragment analysis (Supplementary Methods), which showed complete impairment caused by the variant (Fig. 4).Fig. 4mRNA analysis of patients carrying variant c.686-7C > G. Capillary electrophoresis was performed on PCR products amplified by primers annealing to exons 4 and 7. a Healthy control shows only one wild type peak. b Both patient sample results show 2 peaks—reference transcript peak and a peak corresponding to a 6 bp longer transcript using a de novo created acceptor splice site. Aberrant and normal transcript proportion was roughly equal, which corresponds to complete impairment caused by the variant. The variant preserves a reading frame and leads to incorporation of additional two amino acids (proline-alanine) into the polypeptide chain