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C1-inhibitor deficiency and angioedema: molecular mechanisms and clinical progress

C1 inhibitor (C1-INH, also known as SERPING1) can be deficient in plasma as a result of genetic or acquired conditions, and this causes an episodic, local increase in vascular permeability in the subcutaneous and submucosal layers, identified as angioedema (hereditary or acquired). Bradykinin, the mediator of the increase in vascular permeability, is released on inappropriate activation of the contact system, which is controlled by C1 inhibitor. Therapy aims to reverse or prevent angioedema. Advances in understanding the complex effects of C1-INH deficiency at the molecular level have led to new molecular-targeted approaches. Three new treatments, an inhibitor of kallikrein to prevent bradykinin release, an antagonist of the bradykinin receptor to prevent its action and a recombinant human C1-INH produced in transgenic animals, are under clinical evaluation currently. Here, we review the molecular mechanisms underlying angioedema due to C1-inhibitor deficiency and clinical progress using molecular-targeted interventions.

The use of plasma-derived C1 inhibitor in the treatment of hereditary angioedema

C1-inhibitor (C1-INH) deficiency is the genetic defect underlying hereditary angioedema (HAE). Subjects with HAE suffer from recurrent angioedema that may result in death when it affects the larynx, severe abdominal pain when it affects the gastrointestinal mucosa and disfiguration when it affects the skin. The use of plasma-derived C1-INH concentrates to revert angioedema in HAE patients started in the 1970s. Since that time, three different preparations arrived onto the market, two of them are still present. Controlled studies and a large clinical experience indicate that C1-INH concentrate should be considered the treatment of choice for disabling angioedema attacks at any site. Efficacy has also been shown in preventing angioedema induced by invasive medical manoeuvres. Limited experience with repeated weekly infusions indicates that C1-INH can be used for long-term prophylaxis in selected patients. The safety profile is excellent and there are no reports of transmission of viral infections with the preparations available at present. C1-INH is licensed only in a limited number of countries. Clinical trials are ongoing at present to expand registration.

Human neuroserpin: structure and time-dependent inhibition

Human neuroserpin (hNS) is a protein serine protease inhibitor expressed mainly in the nervous system, where it plays key roles in neural development and plasticity by primarily targeting tissue plasminogen activator (tPA). Four hNS mutations are associated to a form of autosomal dominant dementia, known as familial encephalopathy with neuroserpin inclusion bodies. The medical interest in and the lack of structural information on hNS prompted us to study the crystal structure of native and cleaved hNS, reported here at 3.15 and 1.85 A resolution, respectively. In the light of the three-dimensional structures, we focus on the hNS reactive centre loop in its intact and cleaved conformations relative to the current serpin polymerization models and discuss the protein sites hosting neurodegenerative mutations. On the basis of homologous serpin structures, we suggest the location of a protein surface site that may stabilize the hNS native (metastable) form. In parallel, we present the results of kinetic studies on hNS inhibition of tPA. Our data analysis stresses the instability of the hNS-tPA complex with a dissociation half-life of minutes compared to a half-life of weeks observed for other serpin-cognate protease complexes.

Interaction of C1 inhibitor with thrombin on the endothelial surface.

Thrombin, the central bioregulatory enzyme of haemostasis, also has a potent vasopermeability effect on the surface of endothelial cells, and has therefore been considered a major link between the activation of the coagulation pathway and inflammation. C1 inhibitor inhibits thrombin with a low second-order rate constant that can be increased by heparin. The aim of this study was to investigate whether the C1 inhibitor-induced inhibition of thrombin is potentiated on the endothelial surface. The interaction of C1 inhibitor and thrombin was evaluated in an in-vitro system of human umbilical vein endothelial cells (HUVECs) to which purified C1 inhibitor and thrombin have been added. The role of heparins and selectins has been tested by adding heparinase and Mab to selectins. Kinetic analysis under pseudo-first-order conditions showed that the inhibitory effect of C1 inhibitor on thrombin is greater on the surface of endothelial cells. After incubating nanomolar concentrations of thrombin and micromolar concentrations of C1 inhibitor in a purified system, thrombin activity remained significant, but was almost totally suppressed in the presence of HUVECs. The abolition of such suppression by heparinase and Mab to selectins supports the involvement of heparin and selectins in C1 inhibitor–thrombin interaction. Furthermore, the second-order rate constant was 25 ± 3 /s per mol/l in our purified system, but increased to 100 ± 9 /s per mol/l in the presence of HUVECs. Our results indicate that C1 inhibitor can inhibit thrombin activity on vascular endothelium via binding to selectins and potentiation by heparins. This may contribute to the modulation of thrombin activity on vasopermeability and on coagulation especially when the major natural anticoagulant pathways are impaired.

First independent replication of the involvement of LARS2 in Perrault syndrome by whole-exome sequencing of an Italian family

Perrault syndrome (MIM #233400) is a rare autosomal recessive disorder characterized by ovarian dysgenesis and primary ovarian insufficiency in females, and progressive hearing loss in both genders. Recently, mutations in five genes (HSD17B4, HARS2, CLPP, LARS2 and C10ORF2) were found to be responsible for Perrault syndrome, although they do not account for all cases of this genetically heterogeneous condition. We used whole-exome sequencing to identify pathogenic variants responsible for Perrault syndrome in an Italian pedigree with two affected siblings. Both patients were compound heterozygous for two novel missense variants within the mitochondrial leucyl-tRNA synthetase (LARS2): NM_015340.3:c.899C>T(p.Thr300Met) and c.1912G>A(p.Glu638Lys). Both variants cosegregated with the phenotype in the family. p.Thr300 and p.Glu638 are evolutionarily conserved residues, and are located, respectively, within the editing domain and immediately before the catalytically important KMSKS motif. Homology modeling using as template the E. coli leucyl-tRNA synthetase provided further insights on the possible pathogenic effects of the identified variants. This represents the first independent replication of the involvement of LARS2 mutations in Perrault syndrome, contributing valuable information for the further understanding of this disease.

The spectrum of factor XI deficiency in Italy

Factor XI (FXI) deficiency is a rare inherited bleeding disorder invariably caused by mutations in the FXI gene. The disorder is rather frequent in Ashkenazi Jews, in whom around 98% of the abnormal alleles is represented by Glu117X and Phe283Leu mutations. A wide heterogeneity of causative mutations has been previously reported in a few FXI deficient patients from Italy. In this article, we enlarge the knowledge on the genetic background of FXI deficiency in Italy. Over 4 years, 22 index cases, eight with severe deficiency and 14 with partial deficiency, have been evaluated. A total of 21 different mutations in 30 disease‐associated alleles were identified, 10 of which were novel. Among them, a novel Asp556Gly dysfunctional mutation was also identified. Glu117X was also detected, as previously reported from other patients in Italy, while again Phe283Leu was not identified. A total of 34 heterozygous relatives were also identified. Bleeding tendency was present in very few cases, being inconsistently related to the severity of FXI deficiency in plasma. In conclusion, at variance with other populations, no single major founder effect is present in Italian patients with FXI deficiency.

The expanding spectrum of PRPS1-associated phenotypes: three novel mutations segregating with X-linked hearing loss and mild peripheral neuropathy

Next-generation sequencing is currently the technology of choice for gene/mutation discovery in genetically-heterogeneous disorders, such as inherited sensorineural hearing loss (HL). Whole-exome sequencing of a single Italian proband affected by non-syndromic HL identified a novel missense variant within the PRPS1 gene (NM_002764.3:c.337G>T (p.A113S)) segregating with post-lingual, bilateral, progressive deafness in the proband’s family. Defects in this gene, encoding the phosphoribosyl pyrophosphate synthetase 1 (PRS-I) enzyme, determine either X-linked syndromic conditions associated with hearing impairment (eg, Arts syndrome and Charcot-Marie-Tooth neuropathy type X-5) or non-syndromic HL (DFNX1). A subsequent screening of the entire PRPS1 gene in 16 unrelated probands from X-linked deaf families led to the discovery of two additional missense variants (c.343A>G (p.M115V) and c.925G>T (p.V309F)) segregating with hearing impairment, and associated with mildly-symptomatic peripheral neuropathy. All three variants result in a marked reduction (>60%) of the PRS-I activity in the patients’ erythrocytes, with c.343A>G (p.M115V) and c.925G>T (p.V309F) affecting more severely the enzyme function. Our data significantly expand the current spectrum of pathogenic variants in PRPS1, confirming that they are associated with a continuum disease spectrum, thus stressing the importance of functional studies and detailed clinical investigations for genotype–phenotype correlation.

Molecular bases of neuroserpin function and pathology

Abstract Serpins build a large and evolutionary widespread protein superfamily, hosting members that are mainly Ser-protease inhibitors. Typically, serpins display a conserved core domain composed of three main β-sheets and 9–10 α-helices, for a total of approximately 350 amino acids. Neuroserpin (NS) is mostly expressed in neurons and in the central and peripheral nervous systems, where it targets tissue-type plasminogen activator. NS activity is relevant for axogenesis, synaptogenesis and synaptic plasticity. Five (single amino acid) NS mutations are associated with severe neurodegenerative disease in man, leading to early onset dementia, epilepsy and neuronal death. The functional aspects of NS protease inhibition are linked to the presence of a long exposed loop (reactive center loop, RCL) that acts as bait for the incoming partner protease. Large NS conformational changes, associated with the cleavage of the RCL, trap the protease in an acyl-enzyme complex. Contrary to other serpins, this complex has a half-life of approximately 10 min. Conformational flexibility is held to be at the bases of NS polymerization leading to Collins bodies intracellular deposition and neuronal damage in the pathological NS variants. Two main general mechanisms of serpin polymerization are currently discussed. Both models require the swapping of the RCL among neighboring serpin molecules. Specific differences in the size of swapped regions, as well as differences in the folding stage at which polymerization can occur, distinguish the two models. The results provided by recent crystallographic and biophysical studies allow rationalization of the functional and pathological roles played by NS based on the analysis of four three-dimensional structures.

Embelin binds to human neuroserpin and impairs its polymerisation

Neuroserpin (NS) is a serpin inhibitor of tissue plasminogen activator (tPA) in the brain. The polymerisation of NS pathologic mutants is responsible for a genetic dementia known as familial encephalopathy with neuroserpin inclusion bodies (FENIB). So far, a pharmacological treatment of FENIB, i.e. an inhibitor of NS polymerisation, remains an unmet challenge. Here, we present a biophysical characterisation of the effects caused by embelin (EMB a small natural compound) on NS conformers and NS polymerisation. EMB destabilises all known NS conformers, specifically binding to NS molecules with a 1:1 NS:EMB molar ratio without unfolding the NS fold. In particular, NS polymers disaggregate in the presence of EMB, and their formation is prevented. The NS/EMB complex does not inhibit tPA proteolytic activity. Both effects are pharmacologically relevant: firstly by inhibiting the NS polymerisation associated to FENIB, and secondly by potentially antagonizing metastatic processes facilitated by NS activity in the brain.

Hereditary angioedema in a Jordanian family with a novel missense mutation in the C1-inhibitor N-terminal domain.

Hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE) is an autosomal dominant disease caused by mutations in the SERPING1 gene. A Jordanian family, including 14 individuals with C1-INH-HAE clinical symptoms, was studied. In the propositus and his parents, SERPING1 had four mutations leading to amino acid substitutions. Two are known polymorphic variants (c.167T>C; p.Val34Ala and c.1438G>A; p.Val458Met), the others are newly described. One (c.203C>T; p.Thr46Ile) is located in the N-terminal domain of the C1-inhibitor protein and segregates with angioedema symptoms in the family. The other (c.800C>T; p.Ala245Val) belongs to the serpin domain, and derives from the unaffected father. DNA from additional 24 family members were screened for c.203C>T mutation in the target gene. All individuals heterozygous for the c.203C>T mutation had antigenic and functional plasma levels of C1-inhibitor below 50% of normal, confirming the diagnosis of type I C1-INH-HAE. Angioedema symptoms were present in 14 of 16 subjects carrier for the c.203T allele. Among these subjects, those carrying the c.800T variation had more severe and frequent symptoms than subjects without this mutation. This family-based study provides the first evidence that multiple amino acid substitutions in SERPING1 could influence C1-INH-HAE phenotype.