Efficacy of GalNAc C3 siRNAs in factor H-deficient mice with C3 glomerulopathy.

Complement alternative pathway (AP) dysregulation drives C3 glomerulopathy (C3G), a rare renal disorder characterized by glomerular C3 deposition and glomerular damage, for which no effective treatments are available. Blockade of complement C3 is emerging as a viable therapeutic option. In an earlier study we showed that SLN500, a small interfering RNA targeting liver C3 synthesis, was able to limit AP dysregulation and glomerular C3d deposits in mice with partial factor H (FH) deficiency (Cfh+/- mice). Here, we assessed the pharmacological effects of SLN501 - an optimized SLN500 version - in mice with complete FH deficiency (Cfh-/- mice) that exhibit a more severe C3G phenotype. SLN501 effectively prevented liver C3 synthesis, thus limiting AP dysregulation, glomerular C3d deposits and the development of ultrastructural alterations. These data provide firm evidence of the use of siRNA-mediated liver C3 gene silencing as a potential therapy for treating C3G patients with either partial or complete FH loss of function.

CNS Inflammation as the First Sign of Complement Factor I Deficiency: A Severe Myelitis Treated With Intense Immunotherapy and Eculizumab

OBJECTIVES: Complement factor I (CFI) deficiency is a rare autosomal recessive inborn error of immunity. In this report, we highlight that complete CFI deficiency may present with isolated and severe CNS inflammation without associated systemic features nor prior non-CNS episodes. This inflammation may respond to complement blockade therapy. METHODS: This is a case description of a young girl with severe longitudinal transverse myelitis treated with aggressive immunotherapy that included eculizumab. Published cases of CFI-associated CNS inflammation were reviewed and discussed. RESULTS: A primary immunodeficiency panel revealed 2 germline pathogenic variants in the CFI gene. Further complement testing of the index case and her family confirmed complete CFI deficiency. DISCUSSION: We describe a unique case of severe spinal inflammation secondary to complete CFI deficiency. Although rare, isolated CNS inflammation may be the primary manifestation of complete CFI deficiency. To halt the uncontrolled complement-mediated inflammation associated with CFI deficiency, prompt targeted blockade of the complement pathway using eculizumab may be life changing in the acute phase. Long-lasting blockade of the complement pathway is also essential to prevent relapse in this subgroup of patients.

Involvement of the complement system in immune thrombocytopenia: review of the literature.

Immune thrombocytopenia (ITP) is a thrombocytopenic condition induced by autoimmune mechanisms and includes secondary ITP with underlying diseases such as connective tissue diseases (CTD). In recent years, it has been elucidated that the subsets of the ITP are associated with complement abnormalities but much remains unclear. To perform a literature review and identify the characteristics of complement abnormalities in ITP. PUBMED was used to collect the literature published up to June 2022 related to ITP and complement abnormalities. Primary and secondary ITP (CTD-related) were examined. Out of the collected articles, 17 were extracted. Eight articles were related to primary ITP (pITP) and 9 to CTD-related ITP. Analysis of the literature revealed that the ITP severity was inversely correlated with serum C3, C4 levels in both ITP subgroups. In pITP, a wide range of complement abnormalities was reported, including abnormalities of initial proteins, complement regulatory proteins, or the end products. In CTD-related ITP, reported complement abnormalities were limited to the initial proteins. Activation of the early complement system, mainly through activation of C3 and its precursor protein C4, was reported for both ITPs. On the other hand, more extensive complement activation has been reported in pITP.

Pearls & Oy-sters: Homozygous Complement Factor I Deficiency Presenting as Fulminant Relapsing Complement-Mediated CNS Vasculitis.

A 36-year-old man presented multiple times with fever, headache, alteration of mental status, and focal neurologic deficits. MRI revealed extensive white matter lesions that were partially reversed between episodes. Workup revealed persistently low complement factor C3, low factor B, and absent alternative complement pathway activity. Biopsy revealed neutrophilic vasculitis. Genetic testing revealed a homozygous variant in complement factor I (CFI), which was thought to be pathogenic. CFI regulates complement-mediated inflammation, and deficiency in this factor leads to unchecked alternative pathway activity and decrease in C3 and factor B through consumption. The patient has remained stable since starting IL-1ß inhibition. Complement factor I is a rare disorder that should be considered in patients with atypical relapsing neurologic disease associated with neutrophilic pleocytosis.

Initial manifestations in Patients with Inborn Errors of Immunity Based on Onset Age: a Study from a Nationwide Survey in Japan.

PURPOSE: Patients with inborn errors of immunity (IEI) manifest various initial symptoms; however, those that are critical for the early diagnosis of IEI have not been identified. Also, the significance of the ten warning signs of primary immunodeficiency (PID) among infants has not been established. This study aimed to conduct a nationwide survey of IEI in Japan and investigated the initial manifestations based on onset age. METHODS: Among 1298 patients, data regarding the initial manifestation were available from 505 patients. Patients with autoinflammatory diseases, complement deficiency, and phenocopies of IEI were excluded. RESULTS: The ten warning signs were positive in 67.3% of the cases. The positivity rate was low (20.5%) in patients with immune dysregulation. Although the positivity rate was low (36.6%) in patients aged less than 3 months, they were highly positive for family history of IEI (26.8%). Infectious symptoms were the most commonly observed in all age groups and in all disease categories. Symptoms of "immune dysregulation" were present in approximately 15% of the patients. Regarding the anatomical category, almost all initial symptoms were "systemic" infections in patients with X-linked severe combined immunodeficiency. Moreover, "respiratory" symptoms were the most common in patients with IEI aged ≥ 1 year and accounted for more than 50% in all age groups in patients with common variable immunodeficiency. CONCLUSION: These results highlight the significance of the 10 warning signs and may serve as clinical indicators for early diagnosis, considering the initial presentation of IEI.

The complement system and human autoimmune diseases.

Genetic deficiencies of early components of the classical complement activation pathway (especially C1q, r, s, and C4) are the strongest monogenic causal factors for the prototypic autoimmune disease systemic lupus erythematosus (SLE), but their prevalence is extremely rare. In contrast, isotype genetic deficiency of C4A and acquired deficiency of C1q by autoantibodies are frequent among patients with SLE. Here we review the genetic basis of complement deficiencies in autoimmune disease, discuss the complex genetic diversity seen in complement C4 and its association with autoimmune disease, provide guidance as to when clinicians should suspect and test for complement deficiencies, and outline the current understanding of the mechanisms relating complement deficiencies to autoimmunity. We focus primarily on SLE, as the role of complement in SLE is well-established, but will also discuss other informative diseases such as inflammatory arthritis and myositis.

Genetic Diagnosis of Inborn Errors of Immunity in an Emerging Country: a Retrospective Study of 216 Moroccan Patients.

PURPOSE: Genetic testing provides great support to validate the clinical diagnosis of inborn errors of immunity (IEI). However, the high cost and advanced technology make these tests inaccessible to a large proportion of patients in low-income countries. In the present study, we aim to evaluate the Moroccan experience in genetic testing and to report the main molecular features and difficulties encountered in genetic diagnosis. METHODS: We performed a multi-center retrospective analysis of all patients with a molecular diagnosis and registered in the national registry between 2010 and 2022. To estimate the impact of the newly identified mutations, we calculated the Combined Annotation Dependent Depletion (CADD) score and the mutation significance cutoff (MSC) for each variant. RESULTS: A total of 216 (29%) patients received a genetic diagnosis out of 742 patients with IEI included in the registry. All genetic tests were performed in the context of thesis projects (40%) or international collaborations (60%). A set of 55 genetic defects were identified, including 7 newly reported: SNORA31, TBX21, SPPL2A, TYK2, RLTPR, ZNF341, and STAT2 GOF. Genetic diagnoses were more frequent in the defects of innate and intrinsic immunity with a percentage of 78%, while antibody deficiencies had a lower frequency with a percentage of 17.5%. Only one genetic diagnosis has been made in the complement deficiency group. The most commonly used molecular techniques were Sanger sequencing (37%) followed by targeted gene sequencing (31%). CONCLUSION: The thesis projects and collaborations were beneficial as they allowed us to provide a definitive genetic diagnosis to 29% of the patients and to contribute to the identification of new genetic defects and mutations. These results offer insight into the progress made in genetic diagnoses of IEI in Morocco, which would provide a baseline for improving the clinical management of patients with IEI.

Complement C1s deficiency in a male Caucasian patient with systemic lupus erythematosus: a case report.

Deficiencies of the early complement components of the classical pathway (CP) are well-documented in association with systemic lupus erythematosus (SLE) or SLE-like syndromes and severe pyogenic infections. Among these, complete C1s deficiency has been reported in nine cases so far. Here, we describe a 34-year-old male patient who presented with severe, recurrent infections since childhood, including meningitides with pneumococci and meningococci, erysipelas, subcutaneous abscess, and recurrent infections of the upper airways. The patient also exhibited adult-onset SLE, meeting 7/11 of the ACR criteria and 34 of the 2019 EULAR/ACR classification criteria, along with class IV-G (A) proliferative lupus nephritis (LN). A screening of the complement cascade showed immeasurably low CH50, while the alternative pathway (AP) function was normal. Subsequent determination of complement components revealed undetectable C1s with low levels of C1r and C1q, normal C3, and slightly elevated C4 and C2 concentrations. The patient had no anti-C1q antibodies. Renal biopsy showed class IV-G (A) LN with complement C1q positivity along the glomerular basement membranes (GBMs) and weak deposition of IgG, IgM, and complement C3 and C4 in the mesangium and GBM. In an ELISA-based functional assay determining C4d deposition, the patient's absent complement activity was fully restored by adding C1s. The genome of the patient was analyzed by whole genome sequencing showing two truncating variants in the C1S gene. One mutation was located at nucleotide 514 in exon 5, caused by a nucleotide substitution from G to T, resulting in a nonsense mutation from Gly172 (p.Gly172*). The other mutation was located at nucleotide 750 in exon 7, where C was replaced by a G, resulting in a nonsense mutation from Tyr250 (p.Tyr250*). Both mutations create a premature stop codon and have not previously been reported in the literature. These genetic findings, combined with the absence of C1s in the circulation, strongly suggest a compound heterozygote C1s deficiency in our patient, without additional defect within the complement cascade. As in a previous C1s deficiency case, the patient responded well to rituximab. The present case highlights unanswered questions regarding the CP's role in SLE etiopathogenesis.

Strong Association of Combined Genetic Deficiencies in the Classical Complement Pathway With Risk of Systemic Lupus Erythematosus and Primary Sjögren's Syndrome.

OBJECTIVE: Complete genetic deficiency of the complement component C2 is a strong risk factor for monogenic systemic lupus erythematosus (SLE), but whether heterozygous C2 deficiency adds to the risk of SLE or primary Sjögren's syndrome (SS) has not been studied systematically. This study was undertaken to investigate potential associations of heterozygous C2 deficiency and C4 copy number variation with clinical manifestations in patients with SLE and patients with primary SS. METHODS: The presence of the common 28-bp C2 deletion rs9332736 and C4 copy number variation was examined in Scandinavian patients who had received a diagnosis of SLE (n = 958) or primary SS (n = 911) and in 2,262 healthy controls through the use of DNA sequencing. The concentration of complement proteins in plasma and classical complement function were analyzed in a subgroup of SLE patients. RESULTS: Heterozygous C2 deficiency-when present in combination with a low C4A copy number-substantially increased the risk of SLE (odds ratio [OR] 10.2 [95% confidence interval (95% CI) 3.5-37.0]) and the risk of primary SS (OR 13.0 [95% CI 4.5-48.4]) when compared to individuals with 2 C4A copies and normal C2. For patients heterozygous for rs9332736 with 1 C4A copy, the median age at diagnosis was 7 years earlier in patients with SLE and 12 years earlier in patients with primary SS when compared to patients with normal C2. Reduced C2 levels in plasma (P = 2 × 10-9 ) and impaired function of the classical complement pathway (P = 0.03) were detected in SLE patients with heterozygous C2 deficiency. Finally, in a primary SS patient homozygous for C2 deficiency, we observed low levels of anti-Scl-70, which suggests a risk of developing systemic sclerosis or potential overlap between primary SS and other systemic autoimmune diseases. CONCLUSION: We demonstrate that a genetic pattern involving partial deficiencies of C2 and C4A in the classical complement pathway is a strong risk factor for SLE and for primary SS. Our results emphasize the central role of the complement system in the pathogenesis of both SLE and primary SS.

Moyamoya Disease Associated with a Deficiency of Complement Component 6.

OBJECTIVES: Complement component 6 (C6) deficiency is a very rare genetic defect that leads to significantly diminished synthesis, secretion, or function of C6. In the current report, we demonstrate a previously undescribed, homozygous missense mutation in exon 17 of the C6 gene (c.2545A>G p.Arg849Gly) in a 35-year-old Japanese woman with moyamoya disease and extremely low levels of CH50 (<7.0 U/mL). MATERIALS AND METHODS: The complement gene analysis using hybridization capture-based next generation sequencing was performed. CH50 was determined in patient's plasma mixed with plasma from a healthy donor or purified human C6 protein. Western blot was performed on patient's plasma using polyclonal antibodies against C6, with healthy donor's plasma and purified human C6 protein as positive controls while C6-depleted human serum as a negative control. The carriage of ring finger protein 213 variant (c.14576G>A p.Arg4859Lys), a susceptibility gene for moyamoya disease, was examined by direct sequencing. RESULTS: CH50 mixing test clearly showed a deficiency pattern, being rescued by addition of only 1% healthy donor's plasma or 1 µg/mL purified human C6 protein (1/50-1/100 of physiological concentration). Western blot revealed the absence of C6 protein in the patient's plasma, confirming a quantitative deficiency of C6. The ring finger protein 213 variant was not detected. CONCLUSIONS: Our data implies that unrecognized complement deficiencies would be harbored in cerebrovascular diseases with unknown etiologies.

Secondary Complement Deficiency Impairs Anti-Microbial Immunity to Klebsiella pneumoniae and Staphylococcus aureus During Severe Acute COVID-19.

A high incidence of secondary Klebsiella pneumoniae and Staphylococcus aureus infection were observed in patients with severe COVID-19. The cause of this predisposition to infection is unclear. Our data demonstrate consumption of complement in acute COVID-19 patients reflected by low levels of C3, C4, and loss of haemolytic activity. Given that the elimination of Gram-negative bacteria depends in part on complement-mediated lysis, we hypothesised that secondary hypocomplementaemia is rendering the antibody-dependent classical pathway activation inactive and compromises serum bactericidal activity (SBA). 217 patients with severe COVID-19 were studied. 142 patients suffered secondary bacterial infections. Klebsiella species were the most common Gram-negative organism, found in 58 patients, while S. aureus was the dominant Gram-positive organism found in 22 patients. Hypocomplementaemia was observed in patients with acute severe COVID-19 but not in convalescent survivors three months after discharge. Sera from patients with acute COVID-19 were unable to opsonise either K. pneumoniae or S. aureus and had impaired complement-mediated killing of Klebsiella. We conclude that hyperactivation of complement during acute COVID-19 leads to secondary hypocomplementaemia and predisposes to opportunistic infections.

Complement Genetics for the Practicing Allergist Immunologist: Focus on Complement Deficiencies.

Complement deficiencies have been considered to be rare for many decades, but this assumption is changing year by year. Recognition of these conditions significantly increases thanks to the availability of different testing approaches and due to clinical awareness. Furthermore, sequencing technologies (including Sanger sequencing, targeted gene panels, and whole exome/genome sequencing) may facilitate the identification of the underlying disease-causing genetic background. On the other hand, functional characterization of the identified possibly pathogenic variations and performing family studies, as illustrated by some of our cases, remain similarly important to establish a precise clinical diagnosis facilitating the most appropriate management. Here, we present 4 illustrative cases with complement deficiencies of diverse etiologies and also provide an educative, step-by-step description on how to identify the underlying cause of complement deficiency based on the results of complement laboratory testing.

Genetic workup as a complementary tool for the diagnosis of primary complement component deficiencies: a multicenter experience.

Diagnosis of primary complement deficiencies requires a high index of suspicion. Thus, susceptible patients are often underdiagnosed and untreated. Here, we present a multicenter experience with two novel inborn errors of the classical complement system. This is a retrospective multicenter analysis of computerized medical records of children (<18 years) admitted in the period between 2012 and 2018 at Shaare Zedek Medical Center in Jerusalem and Edmond and Lily Safra Children's Hospital, Tel-Hashomer Medical Center, in Ramat Gan, Israel. Patients were genetically diagnosed by a complementary immune workup. We identified 5 patients (3 males) from four different families harboring two novel mutations in the complement components C6-C8. Genetic mutations were identified by whole-exome sequencing or by sequencing of the coding exons of a single gene based on the findings in the immune workup. Clinical manifestations consisted of meningitis with or without meningococcemia. The immune workup demonstrated nearly absent levels of CH50, compatible with a complement pathway defect. Diagnosis delay ranged between 0 and 30 years. CONCLUSION: Awareness of risk factors for primary complement deficiencies, even at the first infectious episode, should facilitate prompt immune and genetic workup, commencing diagnosis and proper treatment for the patient and family. WHAT IS KNOWN: • Deficiencies in the classical terminal complement components increase susceptibility to invasive meningococcal infections. • Recurrent meningococcal infections mandate a diagnostic workup of the complement system. WHAT IS NEW: • Genetic workup can be utilized for prompt diagnosis of complement deficiencies. • High rates of consanguinity, even in the presence of a single meningococcal infection, should promote immune and genetic workups.

Clinical Outcome and Underlying Genetic Cause of Functional Terminal Complement Pathway Deficiencies in a Multicenter UK Cohort.

BACKGROUND: Terminal complement pathway deficiencies often present with severe and recurrent infections. There is a lack of good-quality data on these rare conditions. This study investigated the clinical outcome and genetic variation in a large UK multi-center cohort with primary and secondary terminal complement deficiencies. METHODS: Clinicians from seven UK centers provided anonymised demographic, clinical, and laboratory data on patients with terminal complement deficiencies, which were collated and analysed. RESULTS: Forty patients, median age 19 (range 3-62) years, were identified with terminal complement deficiencies. Ten (62%) of 16 patients with low serum C5 concentrations had underlying pathogenic CFH or CFI gene variants. Two-thirds were from consanguineous Asian families, and 80% had an affected family member. The median age of the first infection was 9 years. Forty-three percent suffered meningococcal serotype B and 43% serotype Y infections. Nine (22%) were treated in intensive care for meningococcal septicaemia. Two patients had died, one from intercurrent COVID-19. Twenty-one (52%) were asymptomatic and diagnosed based on family history. All but one patient had received booster meningococcal vaccines and 70% were taking prophylactic antibiotics. DISCUSSION: The genetic etiology and clinical course of patients with primary and secondary terminal complement deficiency are variable. Patients with low antigenic C5 concentrations require genetic testing, as the low level may reflect consumption secondary to regulatory defects in the pathway. Screening of siblings is important. Only half of the patients develop septicaemia, but all should have a clear management plan.

Complement factor H deficiency combined with smoking promotes retinal degeneration in a novel mouse model.

Age-related macular degeneration is the leading cause of blindness in the elderly. The Y402H polymorphism in complement factor H promotes disease-like pathogenesis, and a Cfh+/- murine model can replicate this phenotype, but only after two years. We reasoned that by combining CFH deficiency with cigarette smoke exposure, we might be able to accelerate disease progression to facilitate preclinical research in this disease. Wild-type and Cfh+/- mice were exposed to nose-only cigarette smoke for three months. Retinal tissue morphology and visual function were evaluated by optical coherence tomography, fundus photography and autofluorescence, and electroretinogram. Retinal pigment epithelial cell phenotype and ultrastructure were evaluated by immunofluorescence staining and transmission electron microscopy. Cfh+/- smoking mice showed a dome-like protruding lesion at the ellipsoid zone (drusen-like deposition), many retinal hyper-autofluorescence spots, and a marked decrease in A- and B-wave amplitudes. Compared with non-smoking mice, wild-type and Cfh+/- smoking mice showed sub-retinal pigment epithelium complement protein 3 deposition, activation of microglia, metabolic waste accumulation, and impairment of tight junctions. Microglia cells migrated into the photoreceptor outer segment layer in Cfh+/- smoking mice showed increased activation. Our results suggest that exposing Cfh+/- mice to smoking leads to earlier onset of age-related macular degeneration than in other animal models, which may facilitate preclinical research into the pathophysiology and treatment of this disease.

Extreme gonococcal susceptibility associated with acquired complement deficiency secondary to hypocomplementemic urticarial vasculitis and systemic lupus erythematosus.

Gonococcal infection is rarely associated with septic shock. We describe a recurrent case of septic shock related to disseminated gonococcemia in a patient with systemic lupus erythematosus and hypocomplementemic urticarial vasculitis, and discuss the implication of profound acquired complement deficiency secondary to these auto-immune diseases. This case raises the question of systematic antibioprophylaxis in patients with acquired complement deficiency.

Monogenic lupus with homozygous C4A deficiency presenting as bronchiectasis and immune-mediated thrombocytopenia.

Monogenic lupus is a subset of lupus caused by single-gene disorders, integrating the paradoxical combination of autoimmunity and immunodeficiency. Pulmonary manifestations with recurrent pneumonia and bronchiectasis have rarely been described as the predominant presentation of juvenile lupus and may suggest an alternate differential like primary immunodeficiency, especially in early childhood. We describe a case of 10-year girl who presented with a history of recurrent pneumonia, arthritis, alopecia, and poor weight gain for the past 2 years. On examination, she had respiratory distress, bilateral diffuse crackles and arthritis of the small joints of hands. Lab investigations showed pancytopenia, low complement levels and high titers of ANA and anti-dsDNA antibodies. The patient was diagnosed with juvenile lupus. Imaging studies revealed evidence of multiple lobar collapse and consolidation with bronchiectasis. She was started on steroids, HCQ and supportive measures for bronchiectasis. The child reported relief in initial symptoms of lupus on follow-up but developed recurrent thrombocytopenia requiring IVIG and escalating the doses of oral steroids. The young age and atypical presentation prompted a screening for monogenic lupus, and clinical exome sequencing revealed a novel homozygous missense variation in exon 20 of the C4Agene with clinically reduced C4 levels, consistent with the diagnosis of C4A deficiency.

Neisseria meningitidis Serogroup Z Meningitis in a Child With Complement C8 Deficiency and Potential Cross Protection of the MenB-4C Vaccine.

Complement deficient patients are susceptible to rare meningococcal serogroups. A 6-year-old girl presented with serogroup Z meningitis. This led to identification of a C8 deficiency. The MenB-4C vaccine induced cross-reactive antibodies to serogroup Z and increased in vitro opsonophagocytic killing and may thus protect complement deficient patients.