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Abstract Background Hereditary angioedema (HAE) with C1‐inhibitor deficiency (C1‐INH‐HAE) is a rare disease caused by low level (type I) or dysfunction (type II) of the C1‐inhibitor protein with subsequent reduction of certain complement protein levels. Methods To develop and test the reliability of a two‐tier method based on C1‐INH and C4 quantitation followed by genetic analysis from dried blood spot (DBS) for establishing the diagnosis of C1‐INH‐HAE. C1‐INH and C4 proteins have been quantified in human plasma using a classical immuno‐assay and in DBS using a newly developed proteolytic liquid chromatography–mass spectrometry method. Genetic analysis was carried out as reported previously (PMID: 35386643) and by a targeted next‐generation sequencing panel, multiplex ligation‐dependent probe amplification and in some cases whole genome sequencing. Results DBS quantification of C1‐INH and C4 showed the same pattern as plasma, offering the possibility of screening patients with AE symptoms either locally or remotely. Genetic analysis from DBS verified each of the previously identified SERPING1 mutations of the tested C1‐INH‐HAE patients and revealed the presence of other rare variations in genes that may be involved in the pathogenesis of AE episodes. Conclusions C1‐INH/C4 quantification in DBS can be used for screening of hereditary AE and DNA extracted from dried blood spots is suitable for identifying various types of mutations of the SERPING1 gene.

Rationale & Objective: Pregnancy, delivery, and neonatal outcomes in women with complement-mediated thrombotic microangiopathy (cTMA) have not been well described. A better understanding of these outcomes is necessary to provide women with competent pregnancy counseling. Study Design: Cohort study. Setting and Participants: Women with a history of cTMA and pregnancies enrolled into the Vienna thrombotic microangiopathy cohort. Exposure: New onset or relapses of cTMA. Outcomes: Pregnancy, delivery, and neonatal outcomes of pregnancies in women (a) before cTMA manifestation, (b) complicated by pregnancy-associated cTMA (P-cTMA), and (c) after first manifestation of cTMA or P-cTMA. Analytical Approach: Mixed models were used to adjust the comparison of pregnancy, delivery, and neonatal outcomes between conditions (before, with, and after cTMA) for repeated pregnancies using the mother’s ID as random factor. In addition, the fixed factors, mother’s age and neonate’s sex, were used for adjustment. For (sex-adjusted and age-adjusted) centile outcomes, only the mother’s age was used. Adjusted odds ratios were derived from a generalized linear mixed model with live birth as the outcome. Least squares means and pairwise differences between them were derived from the linear mixed models for the remaining outcomes. Results: 28 women reported 74 pregnancies. Despite higher rates of fetal loss before the diagnosis of P-cTMA and preterm births with P-cTMA, most of the women were able to conceive successfully. Neonatal development in all 3 conditions of pregnancies was excellent. Pregnancy and neonatal outcomes were better in women with a pregnancy after the diagnosis of cTMA. Limitations: Although our data set comprises a considerable number of 74 pregnancies, the effective sample size is lower because only 28 mothers with multiple pregnancies were observed. The statistical power for detecting clinically relevant effects was probably low. A recall bias for miscarriages cannot be ruled out. Conclusions: Prepregnancy counseling of women with a history of cTMA can be supportive of their desire to become pregnant.

IntroductionWhile complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood.MethodsWe therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome.ResultsWe show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p

IntroductionAs the global pandemic continues, new complications of COVID-19 in pediatric population have turned up, one of them being hemolytic uremic syndrome (HUS), a complement-mediated thrombotic microangiopathy (CM-TMA) characterized by triad of thrombocytopenia, microangiopathic hemolytic anemia and acute kidney injury (AKI). With both multisystem inflammatory syndrome in children (MIS-C) and HUS sharing complement dysregulation as one of the key factors, the aim of this case report is to highlight differences between these two conditions and also emphasize the importance of complement blockade as a treatment modality.Case reportWe describe a 21-month-old toddler who initially presented with fever and confirmed COVID-19. His condition quickly deteriorated and he developed oliguria, accompanied with diarrhea, vomiting and oral intake intolerance. HUS was suspected, supported with compelling laboratory findings, including decreased platelets count and C3 levels, elevated LDH, urea, serum creatinine and sC5b-9 and presence of schistocytes in peripheral blood, negative fecal Shiga toxin and normal ADAMTS13 metalloprotease activity. The patient was given C5 complement blocker Ravulizumab and started to display rapid improvement.ConclusionAlthough reports of HUS in the setting of COVID-19 continue to pour in, the questions of exact mechanism and similarities to MIS-C remain. Our case for the first time accentuates the use of complement blockade as a valuable treatment option in this scenario. We sincerely believe that reporting on HUS as a complication of COVID-19 in children will give rise to improved diagnosis and treatment, as well as better understanding of both of these intricating diseases.

Atypical hemolytic uremic syndrome (aHUS), also called complement-mediated hemolytic uremic syndrome (CM-HUS), is a rare disease caused by dysregulation in the alternative complement activation pathway. It is a life-threatening condition causing ischemia of a number of organs, and it typically causes acute kidney injury. This disorder may be triggered by various factors including viral or bacterial infections, pregnancy, surgery, and injuries. In about 60% of cases, the genetic origin of the disease can be identified—commonly mutations affecting complementary factor H and MCP protein. Eculizumab, a monoclonal antibody to the C5 component of the complement, represents the current effective treatment.We describe a case of a young woman with a previous history of polyvalent allergies, who developed atypical hemolytic uremic syndrome after vaccination with mRNA vaccine against SARS-CoV-2. The disease manifested by scleral bleeding, acute renal insufficiency, anemia, and thrombocytopenia. The patient was treated with plasma exchanges without sufficient effect; remission occurred only after starting treatment with eculizumab. Genetic examination showed that the patient is a carrier of multiple inherited risk factors (a rare pathogenic variant in CFH, MCPggaac haplotype of the CD46 gene, and the risk haplotype CFH H3). The patient is currently in hematological remission with persistent mild renal insufficiency, continuing treatment with eculizumab/ravulizumab. By this case report, we meant to point out the need for careful monitoring of people after vaccination, as it may trigger immune-mediated diseases, especially in those with predisposing factors.

De novo thrombotic microangiopathy (TMA) is associated with poor kidney graft survival, and as we previously described, it is a recipient driven process with suspected genetic background. Direct Sanger sequencing was performed in 90 KTR with de novo TMA and 90 corresponding donors on selected regions in CFH, CD46, C3, and CFB genes that involve variations with a functional effect or confer a risk for aHUS. Additionally, 37 recipients of paired kidneys who did not develop TMA were analyzed for the MCPggaac haplotype. Three-years death-censored graft survival was assessed using Kaplan-Meier and Cox regression models. The distribution of haplotypes in all groups was in the Hardy-Weinberg equilibrium and there was no clustering of haplotypes in any group. In the TMA group, we found that MCPggaac haplotype carriers were at a significantly higher risk of graft loss compared to individuals with the wild-type genotype. Worse 3-year death-censored graft survival was associated with longer cold ischemia time (HR 1.20, 95% CI 1.06, 1.36) and recipients’ MCPggaac haplotype (HR 3.83, 95% CI 1.42, 10.4) in the multivariable Cox regression model. There was no association between donor haplotypes and kidney graft survival. Similarly, there was no effect of the MCPggaac haplotype on 3-year graft survival in recipients of paired kidneys without de novo TMA. Kidney transplant recipients carrying the MCPggaac haplotype with de novo TMA are at an increased risk of premature graft loss. These patients might benefit from therapeutic strategies based on complement inhibition.

BackgroundHereditary Angioedema with C1-inhibitor deficiency (C1-INH-HAE) is a rare disease characterized by recurrent subcutaneous and/or submucosal edematous (HAE) episodes, which may occur at any age. The mean age of the symptom onset is 10–12 years. Diagnostic protocols differ by age group and family history.MethodsWe retrospectively analyzed clinical and laboratory data (C4-, C1-INH concentration and function) from 49 pediatric patients diagnosed with C1-INH deficiency at our Angioedema Center between 2001 and 2020. Moreover, we analyzed the connection between complement parameters and symptom onset.ResultsFrom the 49 pediatric patients [boy/girl: 23/26, the average age of diagnosis: 6.7 years (min: 0-max: 18.84)], the majority (36/49, 73%) was diagnosed as the result of family screening. Of all the enrolled patients, 34% (17/49) experienced symptoms before the diagnosis. During the observational period, 33% (16/49) of the patients remained asymptomatic, while 33% (16/49) became symptomatic. The average age at symptom onset was 7.8 years (min: 0.5–max: 18). Only 27% (13/49) of pediatric patients were diagnosed after referrals to our center because of typical symptoms. From those patients diagnosed with family screening, 4/36 experienced symptoms at or before the time of the diagnosis. In the case of five newborns from the family screening group, umbilical cord blood samples were used for complement testing. In the case of 3/36 patients, the first complement parameters did not clearly support the disease, but the presence of the mutation identified in the family verified the diagnosis. Complement results were available from 11 patients who became symptomatic during the observational period. Complement parameters 1 year prior to and after the onset of symptoms were compared, and significantly lower concentrations of C1-INH (p = 0.0078) were detected after the onset of symptoms compared to the preceding (symptom-free) period.DiscussionThe majority of pediatric patients were diagnosed as a result of family screening before the onset of symptoms. Early diagnosis allows supplying the patients with special acute treatment for HAE attacks, which may occur at any time. Our results highlight the importance of DNA analysis in pediatric patients in case of a known mutation in the family, and an ambiguous result of complement testing.

BackgroundHereditary angioedema (HAE) due to C1-inhibitor (C1-INH) deficiency (C1-INH-HAE) is a rare autosomal dominant disorder, characterized by recurrent, unpredictable edematous symptoms involving subcutaneous, and/or submucosal tissue. C1-INH-HAE may be caused by more than 700 different mutations in the gene encoding C1-INH (SERPING1) that may lead to decreased protein synthesis or to functional deficiency.MethodsConcentrations of C1-INH, C4, C1q, and anti-C1-INH antibodies, as well as functional C1-INH activity were determined in subjects suffering from edematous symptoms and admitted to the Hungarian Angioedema Center of Reference and Excellence. In those patients, who were diagnosed with C1-INH-HAE based on the complement measurements, SERPING1 was screened by bidirectional sequencing following PCR amplification and multiplex ligation-dependent probe amplification. For detecting large deletions, long-range PCRs covering the entire SERPING1 gene by targeting 2–7 kb long regions were applied.ResultsAltogether 197 individuals with C1-INH deficiency belonging to 68 families were identified. By applying Sanger sequencing or copy number determination of SERPING1 exons, 48 different mutations were detected in 66/68 families: 5 large and 15 small insertions/deletions/delins, 16 missense, 6 nonsense, and 6 intronic splice site mutations. Two novel variations (p.Tyr199Ser [c.596A>C] and the duplication of exon 7) were shown to cosegregate with deficient C1-inhibitor level and activity, while two other variations were detected in single patients (c.797_800delinsCTTGGAGCTCAAGAACTTGGAGCT and c.812dup). A series of long PCRs was applied in the remaining 2 families without an identified mutation and a new, 2606 bp long deletion including the last 91 bp of exon 6 (c.939_1029+2515del) was identified in all affected members of one pedigree. In the remaining one family, a deep intronic SERPING1 variation (c.1029+384A>G) was detected by a targeted next-generation sequencing panel as reported previously.ConclusionsSequencing and copy number determination of SERPING1 exons uncover most pathogenic variants in C1-INH-HAE patients, and further methods are worth to be applied in cases with unrevealed genetic background. Since knowledge of the genetic background may support the establishment of the correct and early diagnosis of C1-INH-HAE, identification of causative mutations and reporting data supporting the interpretation on the pathogenicity of these variants is of utmost importance.

BackgroundFactor H-related protein 5 (FHR-5) is a member of the complement Factor H protein family. Due to the homology to Factor H, the main complement regulator of the alternative pathway, it may also be implicated in the pathomechanism of kidney diseases where Factor H and alternative pathway dysregulation play a role. Here, we report the first observational study on CFHR5 variations along with serum FHR-5 levels in immune complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) and C3 glomerulopathy (C3G) patients together with the clinical, genetic, complement, and follow-up data.MethodsA total of 120 patients with a histologically proven diagnosis of IC-MPGN/C3G were enrolled in the study. FHR-5 serum levels were measured in ELISA, the CFHR5 gene was analyzed by Sanger sequencing, and selected variants were studied as recombinant proteins in ELISA and surface plasmon resonance (SPR).ResultsEight exonic CFHR5 variations in 14 patients (12.6%) were observed. Serum FHR-5 levels were lower in patients compared to controls. Low serum FHR-5 concentration at presentation associated with better renal survival during the follow-up period; furthermore, it showed clear association with signs of complement overactivation and clinically meaningful clusters.ConclusionsOur observations raise the possibility that the FHR-5 protein plays a fine-tuning role in the pathogenesis of IC-MPGN/C3G.

Abstract Background Acquired or genetic abnormalities of the complement alternative pathway are the primary cause of C3glomerulopathy(C3G) but may occur in immune-complex-mediated membranoproliferative glomerulonephritis (IC-MPGN) as well. Less is known about the presence and role of C4nephritic factor(C4NeF) which may stabilize the classical pathway C3-convertase. Our aim was to examine the presence of C4NeF and its connection with clinical features and with other pathogenic factors. Results One hunfe IC-MPGN/C3G patients were enrolled in the study. C4NeF activity was determined by hemolytic assay utilizing sensitized sheep erythrocytes. Seventeen patients were positive for C4NeF with lower prevalence of renal impairment and lower C4d level, and higher C3 nephritic factor (C3NeF) prevalence at time of diagnosis compared to C4NeF negative patients. Patients positive for both C3NeF and C4NeF had the lowest C3 levels and highest terminal pathway activation. End-stage renal disease did not develop in any of the C4NeF positive patients during follow-up period. Positivity to other complement autoantibodies (anti-C1q, anti-C3) was also linked to the presence of nephritic factors. Unsupervised, data-driven cluster analysis identified a group of patients with high prevalence of multiple complement autoantibodies, including C4NeF. Conclusions In conclusion, C4NeF may be a possible cause of complement dysregulation in approximately 10–15% of IC-MPGN/C3G patients.

Haemolytic Uraemic Syndrome associated with Streptococcus pneumoniae infections (SP-HUS) is a clinically well-known entity that generally affects infants, and could have a worse prognosis than HUS associated to E. coli infections. It has been assumed that complement genetic variants associated with primary atypical HUS cases (aHUS) do not contribute to SP-HUS, which is solely attributed to the action of the pneumococcal neuraminidase on the host cellular surfaces. We previously identified complement pathogenic variants and risk polymorphisms in a few Hungarian SP-HUS patients, and have now extended these studies to a cohort of 13 Spanish SP-HUS patients. Five patients presented rare complement variants of unknown significance, but the frequency of the risk haplotypes in the CFH-CFHR3-CFHR1 region was similar to the observed in aHUS. Moreover, we observed desialylation of Factor H (FH) and the FH-Related proteins in plasma samples from 2 Spanish and 4 Hungarian SP-HUS patients. To analyze the functional relevance of this finding, we compared the ability of native and “in vitro” desialylated FH in: (a) binding to C3b-coated microtiter plates; (b) proteolysis of fluid-phase and surface-bound C3b by Factor I; (c) dissociation of surface bound-C3bBb convertase; (d) haemolytic assays on sheep erythrocytes. We found that desialylated FH had reduced capacity to control complement activation on sheep erythrocytes, suggesting a role for FH sialic acids on binding to cellular surfaces. We conclude that aHUS-risk variants in the CFH-CFHR3-CFHR1 region could also contribute to disease-predisposition to SP-HUS, and that transient desialylation of complement FH by the pneumococcal neuraminidase may have a role in disease pathogenesis.

BackgroundThe acquired form of idiopathic thrombotic thrombocytopenic purpura (TTP) is an autoimmune disease, in which the underlying deficiency of the ADAMTS13 protease is caused by autoantibodies, predominantly of the IgG isotype. Certain HLA-DR-DQ haplotypes were associated with the risk of developing TTP.ObjectivesTo investigate the development of the ADAMTS13-specific antibody response during the course of the disease, we analyzed the concentration, subclass distribution, and inhibitory potential of anti-ADAMTS13 IgG autoantibodies in samples of TTP patients drawn during the first acute phase, in remission, and during relapse. Additionally, we compared the anti-ADAMTS13 IgG levels between patients carrying and not carrying risk and protective HLA-DR-DQ haplotypes.Patients and MethodsWe determined the anti-ADAMTS13 IgG concentration and subclass distribution in 101 antibody-positive samples of 81 acquired TTP patients by ELISA methods. The presence and semi-quantitative amount of anti-ADAMTS13 inhibitors were determined in 97 of 100 deficient samples, and the specific inhibitory potential of anti-ADAMTS13 autoantibodies was determined in 49 selected samples, by mixing ADAMTS13-activity assays. HLA-DR-DQ typing and haplotype prediction were performed in 70 of the above patients.ResultsWe found that IgG1 and IgG4 were the predominant subclasses, present in almost all samples. While IgG1 was the dominant subclass in almost half of the samples taken during the first acute episode, IgG4 was dominant in all samples taken during or following a relapse. The inhibitory potential of the samples correlated with levels of the IgG4 subclass. Anti-ADAMTS13 antibodies of IgG4-dominant samples had higher specific inhibitory potentials than IgG1-dominant samples, independently of disease stage. Interestingly, we found that patients carrying the protective DR7-DQ2 and DR13-DQ6 haplotypes had higher anti-ADAMTS13 IgG levels.ConclusionOur results indicate that IgG4 becomes the dominant subtype at some point of the disease course, apparently before the first relapse, parallel to the increase in inhibitory potential of the anti-ADAMTS13 autoantibodies. Furthermore, we found an association between the genetic background and the antibody response in TTP.

Systematic evaluation of the potential relationship between the common genetic variants of CYP21A2 and hormone levels.The relationships of CYP21A2 intron 2 polymorphisms and haplotypes with diverse baseline and stimulated blood hormone levels were studied in 106 subjects with non-functioning adrenal incidentaloma (NFAI). The rationale for using NFAI subjects is dual: i) their baseline hormone profiles do not differ from those of healthy subjects and ii) hormone levels after stimulation tests are available.The carriers (N = 27) of a well-defined CYP21A2 haplotype cluster (c5) had significantly elevated levels of cortisol (p = 0.0110), and 17-hydroxyprogesterone (p = 0.0001) after ACTH stimulation, and 11-deoxycortisol after metyrapone administration (p = 0.0017), but the hormone values were in normal ranges. In addition, the carriers (N = 33) of the C allele of the rs6462 polymorphism had a higher baseline aldosterone level (p = 0.0006). The prevalence of these genetic variants of CYP21A2 did not differ between NFAI and healthy subjects.The common CYP21A2 variants presumably exert the same effect on hormone levels in the healthy and disease-affected populations. Therefore, they may contribute to complex diseases such as some cardiovascular diseases, and may influence the genotype-phenotype correlation in patients with congenital adrenal hyperplasia (CAH) including the individual need for hormone substitution.

The human steroid 21-hydroxylase gene (CYP21A2) participates in cortisol and aldosterone biosynthesis, and resides together with its paralogous (duplicated) pseudogene in a multiallelic copy number variation (CNV), called RCCX CNV. Concerted evolution caused by non-allelic gene conversion has been described in great ape CYP21 genes, and the same conversion activity is responsible for a serious genetic disorder of CYP21A2, congenital adrenal hyperplasia (CAH). In the current study, 33 CYP21A2 haplotype variants encoding 6 protein variants were determined from a European population. CYP21A2 was shown to be one of the most diverse human genes (HHe=0.949), but the diversity of intron 2 was greater still. Contrary to previous findings, the evolution of intron 2 did not follow concerted evolution, although the remaining part of the gene did. Fixed sites (different fixed alleles of sites in human CYP21 paralogues) significantly accumulated in intron 2, indicating that the excess of fixed sites was connected to the lack of effective non-allelic conversion and concerted evolution. Furthermore, positive selection was presumably focused on intron 2, and possibly associated with the previous genetic features. However, the positive selection detected by several neutrality tests was discerned along the whole gene. In addition, the clear signature of negative selection was observed in the coding sequence. The maintenance of the CYP21 enzyme function is critical, and could lead to negative selection, whereas the presumed gene regulation altering steroid hormone levels via intron 2 might help fast adaptation, which broadly characterizes the genes of human CNVs responding to the environment.

Existing evidence indicates that modifier genes could change the phenotypic outcome of the causal SERPING1 variant and thus explain the expression variability of hereditary angioedema due to C1-inhibitor deficiency (C1-INH-HAE). To further examine this hypothesis, we investigated the presence or absence of 18 functional variants of genes encoding proteins involved in the metabolism and function of bradykinin, the main mediator of C1-INH-HAE attacks, in relation to three distinct phenotypic traits of patients with C1-INH-HAE, i.e., the age at disease onset, the need for long-term prophylaxis (LTP), and the severity of the disease. Genetic analyses were performed by a validated next-generation sequencing platform. In total, 233 patients with C1-INH-HAE from 144 unrelated families from five European countries were enrolled in the study. Already described correlations between five common functional variants [F12-rs1801020, KLKB1-rs3733402, CPN1-rs61751507, and two in SERPING1 (rs4926 and rs28362944)] and C1-INH-HAE severity were confirmed. Furthermore, significant correlations were found between either the age at disease onset, the LTP, or the severity score of the disease and a series of other functional variants (F13B-rs6003, PLAU-rs2227564, SERPINA1-rs28929474, SERPINA1-rs17580, KLK1-rs5515, SERPINE1-rs6092, and F2-rs1799963). Interestingly, correlations uncovered in the entire cohort of patients were different from those discovered in the cohort of patients carrying missense causal SERPING1 variants. Our findings indicate that variants other than the SERPING1 causal variants act as independent modifiers of C1-INH-HAE severity and could be tested as possible prognostic biomarkers.

BACKGROUND: Heroin dependence is a debilitating psychiatric disorder with complex inheritance. Since the dopaminergic system has a key role in rewarding mechanism of the brain, which is directly or indirectly targeted by most drugs of abuse, we focus on the effects and interactions among dopaminergic gene variants. OBJECTIVE: To study the potential association between allelic variants of dopamine D2 receptor (DRD2), ANKK1 (ankyrin repeat and kinase domain containing 1), dopamine D4 receptor (DRD4), catechol-O-methyl transferase (COMT) and dopamine transporter (SLC6A3) genes and heroin dependence in Hungarian patients. METHODS: 303 heroin dependent subjects and 555 healthy controls were genotyped for 7 single nucleotide polymorphisms (SNPs) rs4680 of the COMT gene; rs1079597 and rs1800498 of the DRD2 gene; rs1800497 of the ANKK1 gene; rs1800955, rs936462 and rs747302 of the DRD4 gene. Four variable number of tandem repeats (VNTRs) were also genotyped: 120 bp duplication and 48 bp VNTR in exon 3 of DRD4 and 40 bp VNTR and intron 8 VNTR of SLC6A3. We also perform a multivariate analysis of associations using Bayesian networks in Bayesian multilevel analysis (BN-BMLA). FINDINGS AND CONCLUSIONS: In single marker analysis the TaqIA (rs1800497) and TaqIB (rs1079597) variants were associated with heroin dependence. Moreover, -521 C/T SNP (rs1800955) of the DRD4 gene showed nominal association with a possible protective effect of the C allele. After applying the Bonferroni correction TaqIB was still significant suggesting that the minor (A) allele of the TaqIB SNP is a risk component in the genetic background of heroin dependence. The findings of the additional multiple marker analysis are consistent with the results of the single marker analysis, but this method was able to reveal an indirect effect of a promoter polymorphism (rs936462) of the DRD4 gene and this effect is mediated through the -521 C/T (rs1800955) polymorphism in the promoter.