SERPING1P1003Ac.120_121delAGp.Gly41Argfs*16Increased exon 3 skipping (band B) → p.Asp18Glyfs*26 (NMD?), decreased down IRDecreased expression of full-length mutant allele compared to wt allele
Patient: P100
Variant: c.120_121delAG p.Gly41Argfs*16
CAID: CA2580612030
Functional effect: Decreased expression of full-length mutant allele compared to wt allele. Observed in blood.
SERPING1ID2704Ac.551-2A > Gnewdis AcExon 4 skipping (band B), use of cr: c.604 (band A)p.Gly184_Pro228delp.Ala185 Leufs*54 (NMD?)Normal exon inclusion (band A) *, exon 4 skipping (band B), exon 4 + 5 skipping (band C)p.Gly184_Pro228delp.Gly184_Ser296del
Patient: ID270
Variant: c.551-2A > G
CAID: CA380696603
Functional effect: Normal exon inclusion (band A) *, exon 4 skipping (band B), exon 4 + 5 skipping (band C). Observed in blood.
SERPING1H024Ac.685 + 2_685 + 13delnewdis DExon 4 skipping (band C), use of cr: c.649 (band B), use of cr: c.685 + 132 (band A)p.Gly184_Pro228delp.Gly217_Pro228delp.Asp229Glyfs*11 (NMD?)Normal exon inclusion (band A) *, exon 4 skipping (band B), exon 4 + 6 skippingp.Gly184_Pro228delp.[Gly184_Pro228del;Ala297Glyfs*25] (NMD?)
Patient: H02
Variant: c.685 + 2_685 + 13del
CAID: CA2580612034
Functional effect: Normal exon inclusion (band A) *, exon 4 skipping (band B), exon 4 + 6 skipping. Observed in blood.
SERPING1P133Ac.550G > Tnew (p.Gly184Trp)dis DExon 3 skipping (cca 95%) (band B), down IR (band A)p.Asp18Glyfs*26 (NMD?)p.Gly184Cysfs*72 (NMD?)N.A.–
Patient: P13
Variant: c.550G > T (p.Gly184Trp)
CAID: CA380695715
Functional effect: N/A
SERPING1ID3963Ac.305_317delCCACCCAACCAACnewp.Pro102Tyrfs*41Decreased exon 3 skipping (band B) → p.Asp18Glyfs*26 (NMD?), increased down IRMonoallelic expression of full-length allele (wt only)
Patient: ID396
Variant: c.305_317delCCACCCAACCAACnew p.Pro102Tyrfs*41
ClinVarID/CAID: 1460243
Functional effect: Monoallelic expression of full-length allele (wt only)
SERPING1ID267Ac.1225_1249 + 19delnew (p.Met409Asnfs*14)dis DExon 7 skipping (band D), use of cr: c.1206 (band C), use of cr: c.1249 + 20 (HeLa and HepG2 only), use of cr: c.1249 + 64, use of cr: c.1249 + 67 (HeLa and HepG2 only), use of cr: c.1249 + 137 (band B), down IR (band A)p.Val344Asnfs*14p.Val403Asnfs*14 p.Met409_Leu416delp.Met409Glyfs*14p.Met409Glyfs*14p.Met409Glyfs*14p.Met409Glyfs*14Normal exon inclusion (band B) *,exon 7 skipping (band C),use of cr: c.1249 + 64use of cr: c.1249 + 67use of cr: c.1249 + 137 (band A)p.Val344Asnfs*14p.Met409Glyfs*14p.Met409Glyfs*14p.Met409Glyfs*14
Patient: ID26
Variant: c.1225_1249 + 19del (p.Met409Asnfs*14)
CAID: CA2580612035
Functional effect: Normal exon inclusion (band B) *, exon 7 skipping (band C), use of cryptic splice site: c.1249 + 64; use of cryptic splice site: c.1249 + 67; use of cryptic splice site: c.1249 + 137 (band A). Observed in blood.
SERPING1ID2664Ac.685delGnewdis D + dn D (shifted)Exon 4 skipping (band D), use of cr: c.649 (band C), use of dn: c.684 (band B), use of cr: c.685 + 132 (band A)p.Gly184_Pro228delp.Gly217_Pro228delp.Asp229Thrfs*4 (NMD?)p.Asp229Valfs*48 (NMD?)Normal exon inclusion (band A) *, exon 4 skipping (band B), exon 4 + 5 skippingp.Gly184_Pro228delp.Gly184_Ser296del
Patient: ID266
Variant: c.685delG
CAID: CA2580612031
Functional effect: Normal exon inclusion (band A) *, exon 4 skipping (band B), exon 4 + 5 skipping. Observed in blood.
SERPING1ID10213Ac.550G > A (p.Gly184Arg)dis DExon 3 skipping (cca 95%) (band B), down IR (band A)p.Asp18Glyfs*26 (NMD?)p.Gly184Serfs*72 (NMD?)Monoallelic expression (wt only)–
Patient: ID1021
Variant: c.550G > A (p.Gly184Arg)
CAID: CA219267
Functional effect: Monoallelic expression (wt only). Observed in blood.
SERPING1ID4085Ac.686-12A > Gnewdn Ac + dis AcUse of dn: c.686–11 (100%)p.Asp229Alafs*8 (NMD?)Normal exon inclusion *, use of dn: c.686-11p.Asp229Alafs*8 (NMD?)
Patient: ID408
Variant: c.686-12A > G
CAID: CA2580612032
Functional effect: Normal exon inclusion *, use of de novo splice site: c.686-11. Observed in blood.
marked exon 2 skipping in these assays, but also yielded a large fraction of normal transcripts.
Cell transfection assays showed marked exon 2 skipping in these assays, but also yielded a large fraction of normal transcripts.
c.51+3A>G mutation cosegregates with low C1 inhibitor protein levels in one family. alternative splicing of exon 3, found in peripheral blood cell mRNA but not in the liver or in hepatoma cell lines and we show that, in cultured monocytes of a patient carrying the c.51+3A>G mutation, this alternative splicing is shifted from exon 3 exclusion to skipping of both exons 2 and 3
Variants: c.51+3A>G; c.51+5G>A
CAIDs: CA2579981804; error in CAR-unable to register
Twelve human C1 inhibitor P1 variants were constructed by site-directed mutagenesis of the codon for arginine 444 and were expressed in COS-1 cells to analyze the functional properties.
Transiently transfected COS-1 cells were assayed for the presence of C1 inhibitor antigen with the mAb RII RIA (21). Expression levels of all P1 variants were comparable to wild type. . Lysates of transfected COS cells did contain recombinant C1 inhibitor P1 stop antigen, but in substantially lower amounts than the other P1 variants. This indicates that the premature stop codon results in an aberrantly folded protein, which is degraded intracellularly.
P1 His retained the ability to complex with target proteases, but considerably higher amounts of this variant was required compared to recombinant wild-type C1 inhibitor.
P1 Cys was shown to be nonfunctional in kinetic analyses.
Conclusion: P1-Cys variant is nonfunctional toward Cls, beta-factor XIIa, kallikrein, and plasmin. The low activity of P1-His toward Cls is probably not physiologically relevant.
Variants: c.1396C>T; c.1397G>A
CAIDs: CA116521; CA116519
RT-PCR analysis of mRNA samples from five unrelated patients with mutations affecting the 3′ end of exon 3 (c.550G>C, c.550G>A, and c. 550+5 G>A) performed by our group, showed an additional transcript lacking exon 3. Second, the semi-quantitative comparison of the expression of both variants in controls and patients on agarose gels, revealed an apparently higher expression of the exon 3 skipped variant in these patients than in controls
Patient: 5 unrelated patients
Variants: c.550G>C; c.550G>A; c.550+5G>A
CAIDs: CA380695714; CA219267; CA599664126
FunctionalEffect: RT-PCR of mRNA samples from patients blood showed an additional transcript lacking exon 3. Expression in agarose gels revealed a higher expression of the exon 3 skipped variant in these pts vs controls.
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
550+3A>C: Minigene analysis showed aberrant splicing in nearly 100% of mutated minigene construct transcripts, confirmed exon 3 splicing disruption
686-7C>G: RT-PCR and fragment analysis. Both patient sample results show 2 peaks (WT shows only 1)—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
Variants: c.550+3A>C; c.686-7C>G
CAIDs: CA2573332696;