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Presence of C1-inhibitor polymers in a subset of patients suffering from hereditary angioedema.

Madsen DE, Hansen S, Gram J, Bygum A, Drouet C, Sidelmann JJ - PLoS ONE (2014)

Bottom Line: Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels.Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers.In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients.

View Article: PubMed Central - PubMed

Affiliation: University of Southern Denmark, Institute of Public Health, Unit for Thrombosis Research, Esbjerg, Denmark.

ABSTRACT
Hereditary angioedema (HAE) is a potentially life-threatening disease caused by mutations in the gene encoding the serine protease inhibitor (serpin) C1 inhibitor (C1-inh). The mutations cause decreased functional plasma levels of C1-inh, which triggers unpredictable recurrent edema attacks. Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels. However, a few reports have demonstrated that some mutations cause C1-inh polymerization in vitro, and it is speculated that C1-inh polymers may exist in patient plasma, challenging the current classification of HAE patients. To investigate the presence of C1-inh polymers in patient plasma samples, we developed an immunological method, where monoclonal antibodies produced against polymerized C1-inh were applied in native PAGE western blotting. Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers. Identical C1-inh polymerization phenotypes were observed in four affected family members from one of these families. Genotyping of the families revealed that the polymerogenic mutations of two families were located in proximity to the reactive center loop insertion site in C1-inh (p.Ile271Thr and p.Ser258_Pro260del),and one mutation affected helix C (p.Thr167Asn). In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients.

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Related in: MedlinePlus

Native PAGE WB analysis of EDTA plasma samples from 31 HAE patients.“ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.
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pone-0112051-g003: Native PAGE WB analysis of EDTA plasma samples from 31 HAE patients.“ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.

Mentions: Thirty-one patient EDTA plasma samples representing each HAE family were analyzed for the presence of C1-inh polymers using native PAGE WB (Fig. 3). Polymerized C1-inh was identified in plasma samples from patients with ID. no. 5, 6, 28 and 33 (Mut. no. 6), ID. no. 20 (Mut. no. 13) and ID. no. 23 (Mut. no. 21). The polymers ranged in size from dimers to tetramers, with only Mut. no. 21 giving rise to a tetrameric band. The dominant oligomeric species among the polymer positive patients was the trimeric band. The four patients carrying Mut. no. 6 were analyzed in parallel (Fig. 4), and displayed identical polymerization patterns, different from those observed for Mut. no. 13 and 21. In ID. nos. 28, 33, 20 and 23 a low molecular weight form of C1-inh was also observed. This band was not observed in ID. nos. 5 and 6, although these patients carry the same mutation as ID. no. 28 and 33. ID. no. 23 presented with a band between the mono- and dimeric species. This band might represent C1-inh in association with a protease.


Presence of C1-inhibitor polymers in a subset of patients suffering from hereditary angioedema.

Madsen DE, Hansen S, Gram J, Bygum A, Drouet C, Sidelmann JJ - PLoS ONE (2014)

Native PAGE WB analysis of EDTA plasma samples from 31 HAE patients.“ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4219832&req=5

pone-0112051-g003: Native PAGE WB analysis of EDTA plasma samples from 31 HAE patients.“ID. no.” depicts in house patient number, “Mut. no.” depicts individual mutation numbers, “P” depicts C1-inh polymers formed at 65°C for 35 min, “M” depicts monomeric C1-inh, “LP” denotes low molecular weight C1-inh polymers formed using gel filtration and ion exchange chromatography, “C” depicts an EDTA plasma pool, “G” depicts that the genotype of the patient is unknown., “*” indicates that CPDA plasma was analyzed instead of EDTA plasma.
Mentions: Thirty-one patient EDTA plasma samples representing each HAE family were analyzed for the presence of C1-inh polymers using native PAGE WB (Fig. 3). Polymerized C1-inh was identified in plasma samples from patients with ID. no. 5, 6, 28 and 33 (Mut. no. 6), ID. no. 20 (Mut. no. 13) and ID. no. 23 (Mut. no. 21). The polymers ranged in size from dimers to tetramers, with only Mut. no. 21 giving rise to a tetrameric band. The dominant oligomeric species among the polymer positive patients was the trimeric band. The four patients carrying Mut. no. 6 were analyzed in parallel (Fig. 4), and displayed identical polymerization patterns, different from those observed for Mut. no. 13 and 21. In ID. nos. 28, 33, 20 and 23 a low molecular weight form of C1-inh was also observed. This band was not observed in ID. nos. 5 and 6, although these patients carry the same mutation as ID. no. 28 and 33. ID. no. 23 presented with a band between the mono- and dimeric species. This band might represent C1-inh in association with a protease.

Bottom Line: Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels.Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers.In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients.

View Article: PubMed Central - PubMed

Affiliation: University of Southern Denmark, Institute of Public Health, Unit for Thrombosis Research, Esbjerg, Denmark.

ABSTRACT
Hereditary angioedema (HAE) is a potentially life-threatening disease caused by mutations in the gene encoding the serine protease inhibitor (serpin) C1 inhibitor (C1-inh). The mutations cause decreased functional plasma levels of C1-inh, which triggers unpredictable recurrent edema attacks. Subjects suffering from HAE have been classified in type I patients with decreased functional and antigenic levels of C1-inh, and type II patients with decreased functional but normal antigenic C1-inh levels. However, a few reports have demonstrated that some mutations cause C1-inh polymerization in vitro, and it is speculated that C1-inh polymers may exist in patient plasma, challenging the current classification of HAE patients. To investigate the presence of C1-inh polymers in patient plasma samples, we developed an immunological method, where monoclonal antibodies produced against polymerized C1-inh were applied in native PAGE western blotting. Using this approach we analyzed genuine plasma samples from 31 Danish HAE families, and found that plasma samples from three genotypically distinct HAE type I families (classified upon C1-inh plasma concentrations) contained C1-inh polymers. Identical C1-inh polymerization phenotypes were observed in four affected family members from one of these families. Genotyping of the families revealed that the polymerogenic mutations of two families were located in proximity to the reactive center loop insertion site in C1-inh (p.Ile271Thr and p.Ser258_Pro260del),and one mutation affected helix C (p.Thr167Asn). In conclusion, we demonstrate that C1-inh polymers are present in the plasma of a subgroup of HAE type I patients.

Show MeSH
Related in: MedlinePlus