Systematic Approach Revealed SERPING1 Splicing-Affecting Variants to be Highly Represented in the Czech National HAE Cohort.

Hereditary angioedema due to C1 inhibitor deficiency (C1-INH-HAE) is a rare and life-threatening condition characterized by recurrent localized edema. We conducted a systematic screening of SERPING1 defects in a cohort of 207 Czech patients from 85 families with C1-INH-HAE. Our workflow involved a combined strategy of sequencing extended to UTR and deep intronic regions, advanced in silico prediction tools, and mRNA-based functional assays. This approach allowed us to detect a causal variant in all families except one and to identify a total of 56 different variants, including 5 novel variants that are likely to be causal. We further investigated the functional impact of two splicing variants, namely c.550 + 3A > C and c.686-7C > G using minigene assays and RT-PCR mRNA analysis. Notably, our cohort showed a considerably higher proportion of detected splicing variants compared to other central European populations and the LOVD database. Moreover, our findings revealed a significant association between HAE type 1 missense variants and a delayed HAE onset when compared to null variants. We also observed a significant correlation between the presence of the SERPING1 variant c.-21 T > C in the trans position to causal variants and the frequency of attacks per year, disease onset, as well as Clinical severity score. Overall, our study provides new insights into the genetic landscape of C1-INH-HAE in the Czech population, including the identification of novel variants and a better understanding of genotype-phenotype correlations. Our findings also highlight the importance of comprehensive screening strategies and functional analyses in improving the C1-INH-HAE diagnosis and management.

Myeloid lineage cells evince distinct steady-state level of certain gene groups in dependence on hereditary angioedema severity.

Hereditary angioedema (HAE) is a rare genetic disorder with variable expressivity even in carriers of the same underlying genetic defect, suggesting other genetic and epigenetic factors participate in modifying HAE severity. Recent knowledge indicates the role of immune cells in several aspects of HAE pathogenesis, which makes monocytes and macrophages candidates to mediate these effects. Here we combined a search for HAE phenotype modifying gene variants with the characterization of selected genes' mRNA levels in monocyte and macrophages in a symptom-free period. While no such gene variant was found to be associated with a more severe or milder disease, patients revealed a higher number of dysregulated genes and their expression profile was significantly altered, which was typically manifested by changes in individual gene expression or by strengthened or weakened relations in mutually co-expressed gene groups, depending on HAE severity. SERPING1 showed decreased expression in HAE-C1INH patients, but this effect was significant only in patients carrying mutations supposedly activating nonsense-mediated decay. Pro-inflammatory CXC chemokine superfamily members CXCL8, 10 and 11 were downregulated, while other genes such as FCGR1A, or long non-coding RNA NEAT1 were upregulated in patients. Co-expression within some gene groups (such as an NF-kappaB function related group) was strengthened in patients with a severe and/or mild course compared to controls. All these findings show that transcript levels in myeloid cells achieve different activation or depression levels in HAE-C1INH patients than in healthy controls and/or based on disease severity and could participate in determining the HAE phenotype.

PLAUR splicing pattern in hereditary angioedema patients' monocytes and macrophages.

BACKGROUND: The PLAUR gene encodes the urokinase-like plasminogen activator receptor (uPAR) and may undergo alternative splicing. Excluding cassette exons 3, 5 and 6 from the transcript results in truncated protein variants whose precise functions have not been elucidated yet. The PLAUR gene is one of several expressed in myeloid cells, where uPAR participates in different cellular processes, including the contact activation system and kallikrein-kinin system, which play an important role in hereditary angioedema (HAE) pathogenesis. A hypothesis about the PLAUR splicing pattern impact on HAE severity was tested. METHODS AND RESULTS: The RT-PCR quantified by capillary electrophoresis was used. Although no significant difference in alternative transcript frequency was observed between healthy volunteers and HAE patients, a significant increase in all cassette exon inclusion variants was revealed during monocyte-to-macrophage differentiation. CONCLUSIONS: PLAUR alternative splicing in monocytes and macrophages neither was different between HAE patients and healthy controls, nor reflected disease severity. However, the results showed an PLAUR splicing pattern was changing during monocyte-to-macrophage differentiation, but the significance of these changes is unknown and awaits future clarification.

Histamine intolerance.

Histamine intolerance (HIT) is a non-immunological disorder associated with an impaired ability to metabolize ingested histamine. Manifestation of HIT includes gastrointestinal and non-gastrointestinal symptoms. Clinical symptoms of HIT are non-specific and can imitate different diseases such as allergies, food intolerance, mastocytosis and other. The diagnosis of HIT is difficult. There are several candidate tests to detect DAO insufficiency, but their informative value is questionable. Currently, a positive clinical effect of a low-histamine diet is the most important for establishing the diagnosis. Equally in the treatment, a low-histamine diet is the most crucial approach. Other therapeutic options such as DAO supplementation treatment with antihistamines or probiotics are considered as complementary treatments. Our article provides a review on histamine intolerance, focusing on etiology and the diagnostic and treatment possibilities.

An investigational oral plasma kallikrein inhibitor for on-demand treatment of hereditary angioedema: a two-part, randomised, double-blind, placebo-controlled, crossover phase 2 trial.

BACKGROUND: Guidelines recommend effective on-demand therapy for all individuals with hereditary angioedema. We aimed to assess the novel oral plasma kallikrein inhibitor, sebetralstat, which is in development, for on-demand treatment of hereditary angioedema attacks. METHODS: In this two-part phase 2 trial, individuals with type 1 or 2 hereditary angioedema aged 18 years or older were recruited from 25 sites, consisting of specialty outpatient centres, across nine countries in Europe and the USA. Individuals were eligible if they had experienced at least three hereditary angioedema attacks in the past 93 days, were not on prophylactic therapy, and had access to and the ability to self-administer conventional attack treatment. In part 1 of the trial, participants were given a single 600 mg open-label oral dose of sebetralstat to assess safety, pharmacokinetics, and pharmacodynamics of the dose. Part 2 was a randomised, double-blind, placebo-controlled, two-sequence, two-period (2 × 2) crossover trial; participants were randomly assigned (1:1) to either sequence 1, in which they were given a single dose of 600 mg of sebetralstat to treat the first eligible attack and a second dose of placebo to treat the second eligible attack, or sequence 2, in which they were given placebo to treat the first eligible attack and then 600 mg of sebetralstat to treat the second eligible attack. Participants and investigators were masked to treatment assignment. The primary endpoint was time to use of conventional attack treatment within 12 h of study drug administration, which was assessed in all participants who were randomly assigned to treatment and who received study drug for two attacks during part 2 of the study. Safety was assessed in all participants who received at least one dose of study drug, starting in part 1. This study is registered with ClinicalTrials.gov, NCT04208412, and is completed. FINDINGS: Between July 2, 2019, and Dec 8, 2020, 84 individuals were screened and 68 were enrolled in part 1 and received sebetralstat (mean age 38·3 years [SD 13·2], 37 [54%] were female, 31 [46%] were male, 68 [100%] were White). 42 (62%) of 68 participants completed pharmacokinetic assessments. Sebetralstat was rapidly absorbed, with a geometric mean plasma concentration of 501 ng/mL at 15 min. In a subset of participants (n=6), plasma samples obtained from 15 min to 4 h after study drug administration had near-complete protection from ex vivo stimulated generation of plasma kallikrein and cleavage of high-molecular-weight kininogen. In part 2, all 68 participants were randomly assigned to sequence 1 (n=34) or sequence 2 (n=34). 53 (78%) of 68 participants treated two attacks (25 [74%] in the sequence 1 group and 28 [82%] in the sequence 2 group). Time to use of conventional treatment within 12 h of study drug administration was significantly longer with sebetralstat versus placebo (at quartile 1: >12 h [95% CI 9·6 to >12] vs 8·0 h [3·8 to >12]; p=0·0010). There were no serious adverse events or adverse event-related discontinuations. INTERPRETATION: Oral administration of sebetralstat was well tolerated and led to rapid suppression of plasma kallikrein activity, resulting in increased time to use of conventional attack treatment and faster symptom relief versus placebo. Based on these results, a phase 3 trial to evaluate the efficacy and safety of two dose levels of sebetralstat in adolescent and adult participants with hereditary angioedema has been initiated (NCT05259917). FUNDING: KalVista Pharmaceuticals.

Risk Factors for Severe COVID-19 and Hospital Admission in Patients With Inborn Errors of Immunity - Results From a Multicenter Nationwide Study.

Despite the progress in the understanding how COVID-19 infection may impact immunocompromised patients, the data on inborn errors of immunity (IEI) remain limited and ambiguous. Therefore, we examined the risk of severe infection course and hospital admission in a large cohort of patients with IEI. In this multicenter nationwide retrospective survey-based trial, the demographic, clinical, and laboratory data were collected by investigating physicians from 8 national referral centers for the diagnosis and treatment of IEI using a COVID-19-IEI clinical questionnaire. In total, 81 patients with IEI (including 16 with hereditary angioedema, HAE) and confirmed SARS-CoV-2 infection were enrolled, and were found to have a 2.3-times increased (95%CI: 1.44-3.53) risk ratio for hospital admission and a higher mortality ratio (2.4% vs. 1.7% in the general population). COVID-19 severity was associated with the presence of clinically relevant comorbidities, lymphopenia, and hypogammaglobulinemia, but not with age or BMI. No individuals with HAE developed severe disease, despite a hypothesized increased risk due to perturbed bradykinin metabolism. We also demonstrated a high seroconversion rate in antibody-deficient patients and the safety of anti-spike SARS CoV-2 monoclonal antibodies and convalescent plasma. Thus, IEI except for HAE, represent significant risk factors for a severe COVID-19. Therefore, apart from general risk factors, immune system dysregulation may also be involved in the poor outcomes of COVID-19. Despite the study limitations, our results support the findings from previously published trials.

Recombinant human C1 esterase inhibitor for hereditary angioedema attacks: A European registry.

BACKGROUND: Hereditary angioedema (HAE) due to C1 esterase inhibitor deficiency (C1-INH-HAE) is characterized by recurrent swelling attacks. A European treatment registry was established to review the adverse event profile and efficacy of recombinant human C1 esterase inhibitor (rhC1-INH) for HAE attacks. METHODS: Individuals with C1-INH-HAE were enrolled following a decision to treat with rhC1-INH and provision of written informed consent. Medical history and baseline HAE information were collected at screening. Healthcare providers entered data on HAE attacks, response to treatment, and adverse events using a web-based questionnaire. RESULTS: From July 1, 2011, through December 1, 2019, 71 patients with C1-INH-HAE (30 male/41 female; mean age, 47.3 years; age range, 19-78 years) in 9 countries reported 2356 attacks and were treated with rhC1-INH. Before registry entry, patients, including 20 (28.2%) who were on maintenance therapy/prophylaxis at registry enrollment, experienced a mean of 25 HAE attacks per year (median, 16 [range, 0-185]). Most treated HAE attacks were abdominal (46.1%), followed by peripheral (38.3%), oro-facial-pharyngeal (14.8%), urogenital (3.2%), and laryngeal (2.6%). The mean rhC1-INH dose was 3307 U (43.3 U/kg). Patients reported symptom improvement within 4 h for 97.8% of attacks (2305/2356) with rhC1-INH; most attacks (99.8%; 2351/2356) required only 1 dose. Five attacks were treated with a second dose (total rhC1-INH dose administered for attack, 4200 U). No hypersensitivity, thrombotic/thromboembolic events, or drug-related serious adverse events were reported. CONCLUSION: The rhC1-INH treatment registry provided real-world data on the treatment of 2356 HAE attacks that were consistent with clinical trial data of rhC1-INH in patients with C1-INH-HAE.

Acquired Angioedema with C1 Inhibitor Deficiency: Occurrence, Clinical Features, and Management: A Nationwide Retrospective Study in the Czech Republic Patients.

INTRODUCTION: Acquired angioedema with C1 inhibitor deficiency (AAE-C1-INH) is rare but a potentially life-threatening disease. There are no official prevalence data, nor approved therapies for this condition. OBJECTIVE: In this study, we aimed to collect and analyze clinical data on patients with AAE-C1-INH in the Czech Republic. METHODS: We have conducted a retrospective analysis of AAE-C1-INH patients from Czech referral centers for the treatment of hereditary angioedema with C1 inhibitor deficiency. The inclusion criteria involved recurrent episodes of angioedema with the first manifestation at or after the age of 40, negative family history of angioedema, and C1 inhibitor function 50% or less. RESULTS: A total of 14 patients (7 males and 7 females) met the inclusion criteria for AAE-C1-INH. The median age of the symptom onset was 59.5 years, and the median diagnosis delay was 1 year. The most common clinical manifestation was facial edema (100%) and upper airway swelling (85.7%). All patients responded to the acute attack treatment with icatibant and plasma-derived or recombinant C1 inhibitor concentrate. Lymphoid malignancy was identified in 9 patients (64%), monoclonal gammopathy of uncertain significance in 3 (21%), and in 1 patient autoimmune disease (ulcerative colitis) was considered causative (7%). We were not able to identify any underlying disease only in 1 patient (7%). In 6 of 7 patients (86%) treated for lymphoma, either a reduction in the frequency of angioedema attacks or both angioedema symptoms' disappearance and complement parameter normalization was observed. CONCLUSIONS: The prevalence of AAE-C1-INH in the Czech Republic is about 1:760,000. This rare condition occurs in approximately 8% of the patients with angioedema with C1 inhibitor deficiency. AAE-C1-INH is strongly associated with lymphoproliferative disorders, and treating these conditions may improve the control of angioedema symptoms.

Oral once-daily berotralstat for the prevention of hereditary angioedema attacks: A randomized, double-blind, placebo-controlled phase 3 trial.

BACKGROUND: Berotralstat (BCX7353) is an oral, once-daily inhibitor of plasma kallikrein in development for the prophylaxis of hereditary angioedema (HAE) attacks. OBJECTIVE: Our aim was to determine the efficacy, safety, and tolerability of berotralstat in patients with HAE over a 24-week treatment period (the phase 3 APeX-2 trial). METHODS: APeX-2 was a double-blind, parallel-group study that randomized patients at 40 sites in 11 countries 1:1:1 to receive once-daily berotralstat in a dose of 110 mg or 150 mg or placebo (Clinicaltrials.gov identifier NCT03485911). Patients aged 12 years or older with HAE due to C1 inhibitor deficiency and at least 2 investigator-confirmed HAE attacks in the first 56 days of a prospective run-in period were eligible. The primary efficacy end point was the rate of investigator-confirmed HAE attacks during the 24-week treatment period. RESULTS: A total of 121 patients were randomized; 120 of them received at least 1 dose of the study drug (n = 41, 40, and 39 in the 110-mg dose of berotralstat, 150-mg of dose berotralstat, and placebo groups, respectively). Berotralstat demonstrated a significant reduction in attack rate at both 110 mg (1.65 attacks per month; P = .024) and 150 mg (1.31 attacks per month; P < .001) relative to placebo (2.35 attacks per month). The most frequent treatment-emergent adverse events that occurred more with berotralstat than with placebo were abdominal pain, vomiting, diarrhea, and back pain. No drug-related serious treatment-emergent adverse events occurred. CONCLUSION: Both the 110-mg and 150-mg doses of berotralstat reduced HAE attack rates compared with placebo and were safe and generally well tolerated. The most favorable benefit-to-risk profile was observed at a dose of 150 mg per day.

Deep Intronic Mutation in SERPING1 Caused Hereditary Angioedema Through Pseudoexon Activation.

PURPOSE: Hereditary angioedema (HAE) is a rare autosomal dominant life-threatening disease characterized by low levels of C1 inhibitor (type I HAE) or normal levels of ineffective C1 inhibitor (type II HAE), typically occurring as a consequence of a SERPING1 mutation. In some cases, a causal mutation remains undetected after using a standard molecular genetic analysis. RESULTS: Here we show a long methodological way to the final discovery of c.1029 + 384A > G, a novel deep intronic mutation in intron 6 which is responsible for HAE type I in a large family and has not been identified by a conventional diagnostic approach. This mutation results in de novo donor splice site creation and subsequent pseudoexon inclusion, the mechanism firstly described to occur in SERPING1 in this study. We additionally discovered that the proximal part of intron 6 is a region potentially prone to pseudoexon-activating mutations, since natural alternative exons and additional cryptic sites occur therein. Indeed, we confirmed the existence of at least two different alternative exons in this region not described previously. CONCLUSIONS: In conclusion, our results suggest that detecting aberrant transcripts, which are often low abundant because of nonsense-mediated decay, requires a modified methodological approach. We suggest SERPING1 intron 6 sequencing and/or tailored mRNA analysis to be routinely used in HAE patients with no mutation identified in the coding sequence.

Neutrophils Are Dysregulated in Patients with Hereditary Angioedema Types I and II in a Symptom-Free Period.

Neutrophils impact on processes preceding the formation of bradykinin, a major swelling mediator in hereditary angioedema (HAE), yet their potential role in HAE pathogenesis has not been sufficiently studied. We assessed the relative mRNA expression of 10 genes related to neutrophil activation using RNA extracted from the peripheral blood neutrophils of 23 HAE patients in a symptom-free period and 39 healthy donors. Increased relative mRNA expression levels of CD274, IL1B, IL1RN, IL8, MMP9, and TLR4, together with a lack in their mutual correlations detected in HAE patients compared to healthy controls, suggested a preactivated state and dysregulation of patients' neutrophils. Patients' neutrophil-alerted state was further supported by increased CD11b, decreased CD16 plasma membrane deposition, and increased relative CD274+ and CD87+ neutrophil counts, but not by increased neutrophil elastase or myeloperoxidase plasma levels. As CD274 mediates inhibitory signals to different immune cells, neutrophils were cocultured with T-cells/PBMC. The decrease in CD25+ and IFN-γ + T-cell/PBMC ratio in patients indicated the patients' neutrophil suppressive functions. In summary, the results showed neutrophils' alerted state and dysregulation at the transcript level in patients with HAE types I and II even in a symptom-free period, which might make them more susceptible to edema formation. Neutrophils' T-cell suppressive capacity in HAE patients needs to be further investigated.

Treatment of Hereditary Angioedema Attacks with Icatibant and Recombinant C1 Inhibitor During Pregnancy.

PURPOSE: Hereditary angioedema (HAE) is a rare disease caused by a C1 inhibitor (C1-INH) deficit. Clinically, HAE is manifested by repeated episodes of localized subcutaneous or submucosal oedema attacks. Managing HAE patients in pregnancy is challenging, since there are only limited data on the safety and efficacy of various therapeutic approaches. METHODS: We present our clinical experience treating acute HAE attacks during pregnancy in six consecutive patients. RESULTS: During the pregnancies, 79 HAE attacks occurred. The most frequent were abdominal 53 (67.1%) followed by peripheral 21 (26.6%), facial 10 (12.7%), and laryngeal 10 (12.7%) oedemas; 13 (16.5%) attacks were combined. Fifty (63.3%) attacks were treated with recombinant human C1-INH (rhC1-INH); 17 (21.5%) with plasma-derived, pasteurized, nanofiltered C1-INH (pnfC1-INH); 13 (16.5%) with icatibant; and 1 (1.3%) with plasma-derived, nanofiltered C1-INH (nfC1-INH). Treatment had to be repeated in 5 attacks (6.3%). All six deliveries (one caesarean section and five spontaneous vaginal deliveries) were complication free. All pregnancies went to the full term and the patients delivered healthy babies with a birth weight ranging from 2850 to 3690 g. No congenital abnormalities were detected in the neonates. No abortions occurred. CONCLUSIONS: Our results show good C1-INH or icatibant treatment efficacy for HAE attacks in pregnancy. The treatment by the first drug used was effective in 93.7% of all attacks. In 6.3% of attacks, a second treatment had to be used. No adverse effects were observed.

Recombinant human C1 esterase inhibitor for prophylaxis of hereditary angio-oedema: a phase 2, multicentre, randomised, double-blind, placebo-controlled crossover trial.

BACKGROUND: Hereditary angio-oedema is a recurrent, oedematous disorder caused by deficiency of functional C1 inhibitor. Infusions of plasma-derived C1 esterase inhibitor deter attacks of hereditary angio-oedema, but the prophylactic effect of recombinant human C1 esterase inhibitor has not been rigorously studied. We aimed to assess the efficacy of recombinant human C1 esterase inhibitor for prophylaxis of hereditary angio-oedema. METHODS: We conducted this phase 2, multicentre, randomised, double-blind, placebo-controlled crossover trial at ten centres in Canada, the Czech Republic, Israel, Italy, Macedonia, Romania, Serbia, and the USA. We enrolled patients aged 13 years or older with functional C1-inhibitor concentrations of less than 50% of normal and a history of four or more attacks of hereditary angio-oedema per month for at least 3 months before study initiation. Patients were randomly assigned centrally (1:1:1:1:1:1), via an interactive response technology system with fixed allocation, to receive one of six treatment sequences. During each sequence, patients received intravenous recombinant human C1 esterase inhibitor (50 IU/kg; maximum 4200 IU) twice weekly, recombinant human C1 esterase inhibitor once weekly and placebo once weekly, and placebo twice weekly, each for 4 weeks with a 1 week washout period between crossover. All patients, investigators, and study personnel who participated in patient care were masked to group allocation during the study. The primary efficacy endpoint was the number of attacks of hereditary angio-oedema observed in each 4 week treatment period. Attack symptoms were recorded daily. The primary efficacy analysis was done in the intention-to-treat population. Safety was assessed in all patients who received at least one injection of study medication. This study is registered with ClinicalTrials.gov, number NCT02247739. FINDINGS: Between Dec 29, 2014, and May 3, 2016, we enrolled 35 patients, of whom 32 (91%) underwent randomisation (intention-to-treat population) and 26 (81%) completed the study. The mean number of attacks of hereditary angio-oedema over 4 weeks was significantly reduced with recombinant human C1 esterase inhibitor twice weekly (2·7 attacks [SD 2·4]) and once weekly (4·4 attacks [3·2]) versus placebo (7·2 attacks [3·6]), with mean differences of -4·4 attacks (p<0·0001) and -2·8 attacks (p=0·0004), respectively. We recorded adverse events in ten (34%) of 29 patients given twice-weekly recombinant human C1 esterase inhibitor, 13 (45%) of 29 patients given the once-weekly regimen, and eight (29%) of 28 patients given placebo. Headache (twice-weekly treatment) and nasopharyngitis (once-weekly treatment) were the most common adverse events. Two (7%) adverse events (fatigue and headache) were deemed possibly related to treatment with recombinant human C1 esterase inhibitor, but both resolved without additional treatment. No thrombotic or thromboembolic events, systemic allergic reactions (including anaphylaxis), or neutralising antibodies were reported. INTERPRETATION: Prophylaxis with recombinant human C1 esterase inhibitor provided clinically relevant reductions in frequency of hereditary angio-oedema attacks and was well tolerated. In view of the pharmacokinetic profile of recombinant human C1 esterase inhibitor, our results suggest that efficacy of C1-inhibitor replacement therapy might not be a direct function of plasma trough concentrations of C1 inhibitor. FUNDING: Pharming Technologies.

[Current treatment options for hereditary angioedema]. / Soucasné moznosti lécby hereditárního angioedému.

Hereditární angioedém je vzácné dominantne dedicné onemocnení zpusobené deficitem inhibitoru C1-esterázy (C1-INH). Onemocnení se klinicky projevuje recidivami lokalizovaných otoku podkozí a sliznic. Nemoc je hendikepující a muze být i smrtelná. Charakteristická je extrémní variabilita v cetnosti a závaznosti symptomu. Clánek se zabývá organizací péce o pacienty a zahrnuje aktuální lécebné moznosti onemocnení. Strategie lécby zahrnuje krátkodobou a dlouhodobou profylaxi a lécbu atak. V soucasné dobe lécebné moznosti zahrnují atenuované androgeny, antifibrinolytika, rekombinantní (rhC1-INH) a plazma derivované (pdC1-INH) C1-INH, antagonistu bradykininového receptoru a inhibitor kalikreinu. V Ceské republice je péce od roku 2011 soustredena do 4 diagnosticko-terapeutických center.Klícová slova: bradykininový receptor - C1 inhibitor - hereditární angioedém - komplementový systém.