Dominant-negative SERPING1 variants cause intracellular retention of C1 inhibitor in hereditary angioedema
Didde Haslund, … , Lene N. Nejsum, Jacob Giehm Mikkelsen
Didde Haslund, … , Lene N. Nejsum, Jacob Giehm Mikkelsen
Published November 6, 2018
Citation Information: J Clin Invest. 2019;129(1):388-405. https://doi.org/10.1172/JCI98869.
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Dominant-negative SERPING1 variants cause intracellular retention of C1 inhibitor in hereditary angioedema

Abstract

Hereditary angioedema (HAE) is an autosomal dominant disease characterized by recurrent edema attacks associated with morbidity and mortality. HAE results from variations in the SERPING1 gene that encodes the C1 inhibitor (C1INH), a serine protease inhibitor (serpin). Reduced plasma levels of C1INH lead to enhanced activation of the contact system, triggering high levels of bradykinin and increased vascular permeability, but the cellular mechanisms leading to low C1INH levels (20%–30% of normal) in heterozygous HAE type I patients remain obscure. Here, we showed that C1INH encoded by a subset of HAE-causing SERPING1 alleles affected secretion of normal C1INH protein in a dominant-negative fashion by triggering formation of protein-protein interactions between normal and mutant C1INH, leading to the creation of larger intracellular C1INH aggregates that were trapped in the endoplasmic reticulum (ER). Notably, intracellular aggregation of C1INH and ER abnormality were observed in fibroblasts from a heterozygous carrier of a dominant-negative SERPING1 gene variant, but the condition was ameliorated by viral delivery of the SERPING1 gene. Collectively, our data link abnormal accumulation of serpins, a hallmark of serpinopathies, with dominant-negative disease mechanisms affecting C1INH plasma levels in HAE type I patients, and may pave the way for new treatments of HAE.

Authors

Didde Haslund, Laura Barrett Ryø, Sara Seidelin Majidi, Iben Rose, Kristian Alsbjerg Skipper, Tue Fryland, Anja Bille Bohn, Claus Koch, Martin K. Thomsen, Yaseelan Palarasah, Thomas J. Corydon, Anette Bygum, Lene N. Nejsum, Jacob Giehm Mikkelsen

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Figure 7

Correlation of reduced C1INH secretion in skin-derived fibroblasts derived from a patient carrying the c.551_685del mutation with C1INH accumulation in ER.

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Correlation of reduced C1INH secretion in skin-derived fibroblasts deriv...
(A) C1INH in medium from control (NHDF-03, NHDF-05, NHDF-15) and patient-derived fibroblasts. (B) Evaluation of SERPING1 mRNA expression levels in control and patient-derived fibroblasts by qPCR. Equal expression of normal and mutant SERPING1 alleles in fibroblasts derived from the patient carrying the c.551_685del mutation. Inset: Agarose gel showing PCR products from PCR amplification (using primers spanning the deletion) on cDNA synthesized from RNA purified from either control NHDF-03 fibroblasts or patient-derived fibroblasts. (C) Restricted C1INH secretion induced by lentiviral delivery of SERPING1[c.551_685del]. Control NHDF-03 fibroblasts were transduced with lentiviral vectors encoding C1INHGly162_Pro206del (or eGFP as control) at an estimated MOI of 1. (D) Structure of the ER in control fibroblasts and fibroblasts derived from the c.551_685del patient. The fibroblasts were transfected with 900 ng plasmid encoding the Tomato fluorescence gene fused to an ER-targeting sequence to visualize ER (red). Cells were fixated 48 hours after transfections, and nuclei visualized with Hoechst (blue). Scale bar: 10 μm. (E) Accumulation of endogenous C1INH within the ER of patient-derived fibroblasts. The fibroblasts were transfected as in D, fixated, and incubated with Hoechst to visualize the nuclei (blue) and C1INH antibody to visualize both normal and mutated C1INH (green). Scale bars: 10 μm. (A–C) The experiment was carried out in triplicate (n = 3), and data are mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, patient-derived fibroblasts compared with NHDF-05 (A) or NHDF-15 (B) or transduced compared with untransduced NHDF-03 (C). Statistical analyses were performed by 1-way ANOVA with Dunnett’s multiple comparison test. (D–E) Data are representative of findings from more than 3 biological replicates. (A–E) Similar results were seen in at least 2 independent experiments.