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.

Human Complement C4B Allotypes and Deficiencies in Selected Cases With Autoimmune Diseases.

Human complement C4 is one of the most diverse but heritable effectors for humoral immunity. To help understand the roles of C4 in the defense and pathogenesis of autoimmune and inflammatory diseases, we determined the bases of polymorphisms including the frequent genetic deficiency of C4A and/or C4B isotypes. We demonstrated the diversities of C4A and C4B proteins and their gene copy number variations (CNVs) in healthy subjects and patients with autoimmune disease, such as type 1 diabetes, systemic lupus erythematosus (SLE) and encephalitis. We identified subjects with (a) the fastest migrating C4B allotype, B7, or (b) a deficiency of C4B protein caused by genetic mutation in addition to gene copy-number variation. Those variants and mutants were characterized, sequenced and specific techniques for detection developed. Novel findings were made in four case series. First, the amino acid sequence determinant for C4B7 was likely the R729Q variation at the anaphylatoxin-like region. Second, in healthy White subject MS630, a C-nucleotide deletion at codon-755 led to frameshift mutations in his single C4B gene, which was a private mutation. Third, in European family E94 with multiplex lupus-related mortality and low serum C4 levels, the culprit was a recurrent haplotype with HLA-A30, B18 and DR7 that segregated with two defective C4B genes and identical mutations at the donor splice site of intron-28. Fourth, in East-Asian subject E133P with anti-NMDA receptor encephalitis, the C4B gene had a mutation that changed tryptophan-660 to a stop-codon (W660x), which was present in a haplotype with HLA-DRB1*04:06 and B*15:27. The W660x mutation is recurrent among East-Asians with a frequency of 1.5% but not detectable among patients with SLE. A meticulous annotation of C4 sequences revealed clusters of variations proximal to sites for protein processing, activation and inactivation, and binding of interacting molecules.

Clinical features of patients with homozygous complement C4A or C4B deficiency.

INTRODUCTION: Homozygous deficiencies of complement C4A or C4B are detected in 1-10% of populations. In genome-wide association studies C4 deficiencies are missed because the genetic variation of C4 is complex. There are no studies where the clinical presentation of these patients is analyzed. This study was aimed to characterize the clinical features of patients with homozygous C4A or C4B deficiency. MATERIAL AND METHODS: Thirty-two patients with no functional C4A, 87 patients with no C4B and 120 with normal amount of C4 genes were included. C4A and C4B numbers were assessed with genomic quantitative real-time PCR. Medical history was studied retrospectively from patients' files. RESULTS: Novel associations between homozygous C4A deficiency and lymphoma, coeliac disease and sarcoidosis were detected. These conditions were present in 12.5%, (4/32 in patients vs. 0.8%, 1/120, in controls, OR = 17.00, 95%CI = 1.83-158.04, p = 0.007), 12.5% (4/32 in patients vs. 0%, 0/120 in controls, OR = 1.14, 95%CI = 1.00-1.30, p = 0.002) and 12.5%, respectively (4/32 in patients vs. 2.5%, 3/120 in controls, OR = 5.571, 95%CI = 1.79-2.32, p = 0.036). In addition, C4A and C4B deficiencies were both associated with adverse drug reactions leading to drug discontinuation (34.4%, 11/32 in C4A-deficient patients vs. 14.2%, 17/120 in controls, OR = 3.174, 95%CI = 1.30-7.74, p = 0.009 and 28.7%, 25/87 in C4B-deficient patients, OR = 2.44, 95%CI = 1.22-4.88, p = 0.010). CONCLUSION: This reported cohort of homozygous deficiencies of C4A or C4B suggests that C4 deficiencies may have various unrecorded disease associations. C4 gene should be considered as a candidate gene in studying these selected disease associations.

Low copy numbers of complement C4 and homozygous deficiency of C4A may predispose to severe disease and earlier disease onset in patients with systemic lupus erythematosus.

Objectives Low copy numbers and deletion of complement C4 genes are potent risk factors for systemic lupus erythematosus (SLE). However, it is not known whether this genetic association affects the clinical outcome. We investigated C4 copy number variation and its relationship to clinical and serological features in a Northern European lupus cohort. Methods We genotyped the C4 gene locus using polymerase chain reaction (PCR)-based TaqMan assays in 169 patients with SLE classified according to the 1997 revised American College of Rheumatology (ACR) criteria and in 520 matched controls. In the patient group the mean C4 serum protein concentrations nephelometrically measured during a 12-month period prior to genetic analysis were compared to C4 gene copy numbers. Severity of disease was classified according to the intensity of the immunosuppressive regimens applied and compared to C4 gene copy numbers, too. In addition, we performed a TaqMan based analysis of three lupus-associated single-nucleotide polymorphisms (SNPs) located inside the major histocompatibility complex (MHC) to investigate the independence of complement C4 in association with SLE. Results Homozygous deficiency of the C4A isotype was identified as the strongest risk factor for SLE (odds ratio (OR) = 5.329; p = 7.7 × 10-3) in the case-control comparison. Moreover, two copies of total C4 were associated with SLE (OR = 3.699; p = 6.8 × 10-3). C4 serum levels were strongly related to C4 gene copy numbers in patients, the mean concentration ranging from 0.110 g/l (two copies) to 0.256 g/l (five to six copies; p = 4.9 × 10-6). Two copies of total C4 and homozygous deletion of C4A were associated with a disease course requiring cyclophosphamide therapy (OR = 4.044; p = 0.040 and OR = 5.798; p = 0.034, respectively). Homozygous deletion of C4A was associated with earlier onset of SLE (median 24 vs. 34 years; p = 0.019) but not significant after correction for multiple testing. SNP analysis revealed a significant association of HLA-DRB1*0301 with SLE (OR = 2.231; p = 1.33 × 10-5). Conclusions Our findings confirm the important role of complement C4 genes in the development of SLE. Beyond the impact on the susceptibility for lupus, C4 copy numbers may be related to earlier onset and a more severe course of the disease. The association of homozygous deletion of C4A and SLE is accompanied by the presence of HLA-DRB1*0301 without a proven pathophysiological mechanism.

Effects of Complement C4 Gene Copy Number Variations, Size Dichotomy, and C4A Deficiency on Genetic Risk and Clinical Presentation of Systemic Lupus Erythematosus in East Asian Populations.

OBJECTIVE: Human complement C4 is complex, with multiple layers of diversity. The aims of this study were to elucidate the copy number variations (CNVs) of C4A and C4B in relation to disease risk in systemic lupus erythematosus (SLE), and to compare the basis of race-specific C4A deficiency between East Asians and individuals of European descent. METHODS: The East Asian study population included 999 SLE patients and 1,347 healthy subjects. Variations in gene copy numbers (GCNs) of total C4, C4A, and C4B, as well as C4-Long and C4-Short genes, were determined and validated using independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein occurring concurrently with C4A deficiency. RESULTS: In East Asians, high GCNs of total C4 and C4A were strongly protective against SLE, whereas low and medium GCNs of total C4 and C4A, and the absence of C4-Short genes, were risk factors for SLE. Homozygous C4A deficiency was infrequent in East Asian subjects, but had an odds ratio (OR) of 12.4 (P = 0.0015) for SLE disease susceptibility. Low serum complement levels were strongly associated with low GCNs of total C4 (OR 3.19, P = 7.3 × 10(-7) ) and C4B (OR 2.53, P = 2.5 × 10(-5) ). Patients with low serum complement levels had high frequencies of anti-double-stranded DNA antibodies (OR 4.96, P = 9.7 × 10(-17) ), hemolytic anemia (OR 3.89, P = 3.6 × 10(-10) ), and renal disease (OR 2.18, P = 8.5 × 10(-6) ). The monomodular-Short haplotype found to be prevalent in European Americans with C4A deficiency, which was in linkage disequilibrium with HLA-DRB1*0301, was scarce in East Asians. Instead, most East Asian subjects with C4A deficiency were found to have a recombinant haplotype with bimodular C4-Long and C4-Short genes, encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA sequencing revealed an E920K polymorphism in C4B96. CONCLUSION: C4 CNVs and deficiency of C4A both play an important role in the risk and manifestations of SLE in East Asian and European populations.

Gene copy-number variations (CNVs) of complement C4 and C4A deficiency in genetic risk and pathogenesis of juvenile dermatomyositis.

OBJECTIVE: Complement-mediated vasculopathy of muscle and skin are clinical features of juvenile dermatomyositis (JDM). We assess gene copy-number variations (CNVs) for complement C4 and its isotypes, C4A and C4B, in genetic risks and pathogenesis of JDM. METHODS: The study population included 105 patients with JDM and 500 healthy European Americans. Gene copy-numbers (GCNs) for total C4, C4A, C4B and HLA-DRB1 genotypes were determined by Southern blots and qPCRs. Processed activation product C4d bound to erythrocytes (E-C4d) was measured by flow cytometry. Global gene-expression microarrays were performed in 19 patients with JDM and seven controls using PAXgene-blood RNA. Differential expression levels for selected genes were validated by qPCR. RESULTS: Significantly lower GCNs and differences in distribution of GCN groups for total C4 and C4A were observed in JDM versus controls. Lower GCN of C4A in JDM remained among HLA DR3-positive subjects (p=0.015). Homozygous or heterozygous C4A-deficiency was present in 40.0% of patients with JDM compared with 18.2% of controls (OR=3.00 (1.87 to 4.79), p=8.2×10(-6)). Patients with JDM had higher levels of E-C4d than controls (p=0.004). In JDM, C4A-deficient subjects had higher levels of E-C4d (p=0.0003) and higher frequency of elevated levels of multiple serum muscle enzymes at diagnosis (p=0.0025). Microarray profiling of blood RNA revealed upregulation of type I interferon-stimulated genes and lower abundance of transcripts for T-cell and chemokine function genes in JDM, but this was less prominent among C4A-deficient or DR3-positive patients. CONCLUSIONS: Complement C4A deficiency appears to be an important factor for the genetic risk and pathogenesis of JDM, particularly in patients with a DR3-positive background.

C4A deficiency in children and adolescents with recurrent respiratory infections.

Increased susceptibility to recurrent viral and bacterial respiratory infections in children and young adults is not well understood. To evaluate the role of complement factor C4 in the defense against respiratory infections, we studied complement factor C4 allotypes C4A and C4B and copy numbers of C4A and C4B genes in 84 children and young adults with recurrent acute otitis media, sinusitis, or pneumonia and in 74 healthy controls. The occurrence of C4A gene deficiency was significantly higher in patients compared with controls (26% vs 14%, p = 0.048). Girls predominated in the group of patients with C4A deficiency (73% girls and 27% boys, p = 0.004). The lectin pathway of complement was more often functionally impaired in patients with C4A deficiency than in patients with no C4A deficiency (41% vs 13%, p = 0.033). Classical and alternative pathways were normal in individuals with C4 null alleles. C4A deficiency is 1 of the minor defects of the innate immunity that may predispose children and young adults to recurrent respiratory infections. C4 gene testing should be added to the list of investigations when the cause for recurrent acute otitis media, maxillary sinusitis, or pneumonia in children and young adults is sought.

Genetically determined partial complement C4 deficiency states are not independent risk factors for SLE in UK and Spanish populations.

Systemic lupus erythematosus (SLE) is a chronic, multisystem autoimmune disease. Complete deficiency of complement component C4 confers strong genetic risk for SLE. Partial C4 deficiency states have also shown association with SLE, but despite much effort over the last 30 years, it has not been established whether this association is primarily causal or secondary to long-range linkage disequilibrium. The complement C4 locus, located in the major histocompatibility complex (MHC) class III region, exhibits copy-number variation (CNV) and C4 itself exists as two paralogs, C4A and C4B. In order to determine whether partial C4 deficiency is an independent genetic risk factor for SLE, we investigated C4 CNV in the context of HLA-DRB1 and MHC region SNP polymorphism in the largest and most comprehensive complement C4 study to date. Specifically, we genotyped 2,207 subjects of northern and southern European ancestry (1,028 SLE cases and 1,179 controls) for total C4, C4A, and C4B gene copy numbers, and the loss-of-function C4 exon 29 CT indel. We used multiple logistic regression to determine the independence of C4 CNV from known SNP and HLA-DRB1 associations. We clearly demonstrate that genetically determined partial C4 deficiency states are not independent risk factors for SLE in UK and Spanish populations. These results are further corroborated by the lack of association shown by the C4A exon 29 CT insertion in either cohort. Thus, although complete homozygous deficiency of complement C4 is one of the strongest genetic risk factors for SLE, partial C4 deficiency states do not independently predispose to the disease.

HLA-DRB1 allele frequencies and C4 copy number variation in Finnish sarcoidosis patients and associations with disease prognosis.

Sarcoidosis is a multiorgan immune-mediated disease of unknown etiology with varying clinical pictures. We studied 3 genes in the major histocompatibility complex region (HLA-DRB1 and complement C4A and C4B) in patients with resolved disease after a 2-year follow-up (n = 90) and in patients whose disease was still active at that time point (n = 98) and compared them with controls (n = 150). Our primary aim was to detect genetic differences between the patient groups. We observed that the susceptibility allele for sarcoidosis was HLA-DRB1*15:01 (p = 0.011; odds ratio [OR] = 1.67) and the protective allele was HLA-DRB1*01:01 (p = 0.001; OR = 0.43). HLA-DRB1*03:01 was associated with resolving disease when compared with the persistent group (p = 0.011; OR = 2.22). The probability of having resolving disease was even greater if the patient had HLA-DRB1*03:01 and did not have extrapulmonary lesions (p = 0.001; OR = 3.39). By evaluating amino acid variants of the HLA-DRB1 gene, we determined that specific amino acids in pockets 4, 7, and 9 were associated with the prognosis of sarcoidosis. Our results support the importance of HLA-DRB1 as a predisposing gene for sarcoidosis. Particularly, HLA-DRB1*03:01 and polymorphisms of DRB1 pocket residues were associated with a favorable prognosis. Thus, accurate categorization of disease phenotype and HLA-DRB1 sequencing offer a basis for disease course estimation of sarcoidosis.

[Masked deficiency of C4 component of complement in patients with chronic gastrointestinal tract diseases of different etiology].

AIM: Frequency of occurrence detection of C4A and C4B complement system deficiency in patients with chronic gastrointestinal tract (GIT) diseases including gastric ulcer (GU) and duodenal ulcer (DU). MATERIALS AND METHODS: 74 patients with chronic GIT diseases were examined. Endoscopy with stomach mucosa condition evaluation based on histobacterioscopic examination of gastroduodenal biopsy samples was used. Intestine microbiocenosis evaluation was performed by using microflora degree of manifestation according to Federal Standard of Russian Ministry of Health No 231 -91500.11.0004-2003. C4A and C4B isotypes in blood sera of patients were measured by using enzyme immunoassay. RESULTS: Chronic gastroduodenitis was diagnosed in 35.1%, pangastritis B--in 41.9%, GU and DU--in 23% of patients. Histological evaluation of biopsy samples revealed marked inflammatory changes in stomach and duodenum mucosa in 77% of patients. Stomach mucosa infection rate by Helicobacter pylori reached 85%. Microbiological disorders manifestation in microflora of patients matched endoscopic and histobacteriscopic changes in it and was the highest for GU and DU. In 76.0% of cases C4A and C4B isotype deficiency in blood sera matches the development of erosive-ulcerous process in stomach and duodenum mucosa with marked background dysbiotic GIT microbiota disorders. CONCLUSION: Patients with functional deficiency of C4A and C4B isotypes have a genetic burden to susceptibility to chronic GIT diseases whereas H.pylori infection deteriorate the disease.

[Enzyme immunoassay of masked complement component C4 deficiency in patients with urogenital Chlamydia infection].

AIM: Development of new method of C4B isotype functional activity evaluation in enzyme immunoassay by using pharmaceutical preparation derinat as a classical pathway complement activator and its use for blood sera isotyping in confirmed urogenital tract chlamydia infection. MATERIALS AND METHODS: Enzyme immunoassay was used to detect C4A and C4B isotype functional deficiency in blood sera of patients. Chlamydia etiology urogenital infection diagnosis was based on results of standard clinical-instrumental examination methods: vaginal clinical smear analysis, scrape sample light microscopy with consequent treatment by fluorescent monoclonal antibodies against Chlamydia trachomatis and PCR. RESULTS: In acute form of the disease C4A deficiency frequency of occurrence was 0.36, and C4B deficiency - 0.55. In chronic form of the disease deficiency frequency of occurrence was 0.38 for both isotypes. In the group of healthy people isotype deficiency was 0.08 and 0.25, respectively. CONCLUSION: Innate masked C4 deficiency interfere with the normal immune defense of organism against chlamydia infection, and antigen carbohydrate pathogenicity may possibly be more significant for the development of immune response to which C4B isotype activity is necessary.

Partial C4 deficiency in juvenile idiopathic arthritis patients.

OBJECTIVES: C4 is encoded by 2 distinct but closely linked loci within the major histocompatibility complex locus on human chromosome 6. C4A deficiencies have been associated with autoimmune disease and C4B with increased frequency of infection. C4 deficiencies have rarely been associated with juvenile idiopathic arthritis (JIA). Our aim was to investigate the prevalence of deficiencies in C4 allotypes in JIA patients. METHODS: We evaluated 61 patients [35 JIA patients, 15 systemic lupus erythematosus patients, 9 rheumatoid arthritis patients, and 2 mixed connective tissue disease (CTD) patients] for C4 deficiency. Genomic DNA was isolated from whole blood and subjected to polymerase chain reaction using sequence-specific primers for C4 allotypes. RESULTS: We found 5 JIA patients with C4 deficiencies. Two IgM rheumatoid factor-positive JIA polyarthritis patients had C4 deficiencies, one with complete C4A deficiency and another with partial C4A and complete C4B deficiency. Two oligoarthritis patients displayed partial C4B deficiencies, and complete C4B deficiency was revealed in 1 IgM rheumatoid factor-negative polyarthritis patient. Three patients had histories of recurrent infections and 2 demonstrated a more severe disease course. Disease controls showed 8 systemic lupus erythematosus patients had partial C4 deficiencies, whereas no deficiencies were revealed in the rheumatoid arthritis or mixed CTD patients. CONCLUSIONS: Defects in the complement system have been implicated in the pathogenesis of CTD. However, the specific role of C4 in JIA is not clear. We demonstrate partial C4 deficiencies in 5 JIA patients. Our findings suggest an association between C4 deficiency and another CTD, JIA, as well as with disease severity and recurrent infections.

[Deficiencies in C4A and C4B isotypes of human complement revealed by isoelectrofocusing and chemical reactivity of activated forms].

An approach is proposed to detect deficiencies in isotypes A and B of the C4 component of human complement, based on the calculation of the ratio of their IEA activities and the ratio of their quantities determined by isoelectrofocusing of their desialated forms with chemiluminescent detection in an immunoblot. The ratios of the quantities and activities of C4A/C4B practically coincided when determined in blood serum of 20 patients, many of which had inherited deficiencies in the C4 component isotypes.

Capillary leak syndrome in children with C4A-deficiency undergoing cardiac surgery with cardiopulmonary bypass: a double-blind, randomised controlled study.

BACKGROUND: Capillary leak syndrome is a life-threatening complication after cardiopulmonary bypass (CPB), with an incidence of about 4-37% in children worldwide. On the basis of previous results, we undertook a randomised controlled study to investigate the priming with plasma rich in the C4A isotype of complement component 4 on the incidence of capillary leak syndrome in children with C4A deficiency. METHODS: In a hospital in Wuhan, China, we randomly assigned 116 neonates, infants, and children lacking complement component C4A to receive C4A-free or C4A-rich plasma priming (n=58 each, 20 mL/kg). The primary outcome was capillary leak syndrome, identified as an increased transvascular escape rate of Evans blue dye from plasma. Concentrations of activated complement components C4 and C3, inflammatory mediators interleukin 6, interleukin 8, tumour necrosis factor (TNF) alpha, plasma protein, and PaO2/F(I)O2 ratios (ratio of the partial arterial pressure of oxygen to the fractional concentration of oxygen in inspired air) were measured before and 4 h after CPB. Analysis was by intention to treat. FINDINGS: Three (5%) patients given C4A-rich plasma priming had capillary leak syndrome compared with 56 (97%) given C4A-free plasma (p<0.0001). At 4 h after CPB, activated C4, interleukin 6, interleukin 8, and TNFalpha concentrations were higher, whereas PaO2/F(I)O2 ratios and plasma protein concentrations were significantly lower in the C4A-free group than changes in the C4A-rich group. Activated C3 rose equally in both groups. Activated C4 significantly correlated with interleukin 6, interleukin 8, and TNFalpha concentrations; PaO2/F(I)O2 ratios; and the escape rate of Evans blue dye at 4 h after CPB. Two patients in the C4A-free group died of respiratory and renal failure on day 3 after CPB. INTERPRETATION: In paediatric patients with C4A deficiency, C4A-rich plasma priming reduces the incidence of CPB-related capillary leak syndrome by blocking the activated C4 increase and attenuating the systemic inflammatory response after CPB.

Complete deficiencies of complement C4A and C4B including 2-bp insertion in codon 1213 are genetic risk factors of systemic lupus erythematosus in Thai populations.

The complement component C4 is encoded by two genes: C4A and C4B on human chromosome 6p in the major histocompatibility complex (MHC). Most studies have linked the deficiencies in C4 with systemic lupus erythematosus (SLE) in Angio-Irish, North American, Black American, Mexican American, Australian and Japanese populations. Null alleles at either locus (C4AQ0 or C4BQ0) are relatively common in Americans occurring at the C4A and C4B loci in approximately 10% and 16% of normal individuals, respectively. In the present study, we extensively examined the possible association between homozygous C4Q0 and SLE in a large cohort of Thai populations diagnosed as SLE and further attempted to identify the genetic basis of C4Q0. One hundred and eighteen cases of SLE patients and 145 matched controls were genotyped by touchdown PCR. The results confirmed the previous studies that 5.93% (7/118) of C4 null genes: 2.54% (3/118) of C4AQ0 and 3.39% (4/118) of C4BQ0 were found in SLE patients. In contrast to other studies, we found no cases of C4 null genes in normal control (0 from 145 samples). To further investigate the genetic basis of C4 deficiency, all genomic DNAs were also analyzed for 2-bp (TC) insertion at codon 1213 in exon 29 which is a common mutation in many C4A null genes and a novel 1-bp deletion (C) at codon 522 in exon 13 that is common in most C4B null genes. Both mutation results in a flame-shift mutation and premature stop codon using sequence specific primers PCR (SSP-PCR) and direct sequencing. The results showed that there was 2-bp insertion in exon 29 of mutant C4B gene in one SLE patient carrying C4AQ0. There was no 2-bp insertion in exon 29 of both C4A and C4B genes in normal individual and the rest of SLE patients. All patients with C4AQ0 exhibited more than 5 ACR criteria including malar rash, oral ulcers, renal disorder, immunological disorder, anti-nuclear antibody, without hematological disorder. In contrast, all of C4BQ0 SLE patients showed 5 or 6 ACR criteria including hematological disorder, malar rash, oral ulcers, renal disorder, immunological disorder and anti-nuclear antibody. A patient who possesses C4AQ0 and 2-bp insertion in exon 29 of mutant C4B showed 9 ACR criteria but no discoid rash and hematological disorder. In conclusion, both C4AQ0 and C4BQ0 are the strong predisposing factors for SLE in Thais. It was supported by the absence of either C4A or C4B deletion in healthy control. We suggested that the different racial and genetic backgrounds could alter the thresholds for requirement of C4A or C4B protein levels in immune tolerance and regulation.

Complete complement components C4A and C4B deficiencies in human kidney diseases and systemic lupus erythematosus.

Although a heterozygous deficiency of either complement component C4A or C4B is common, and each has a frequency of approximately 20% in a Caucasian population, complete deficiencies of both C4A and C4B proteins are extremely rare. In this paper the clinical courses for seven complete C4 deficiency patients are described in detail, and the molecular defects for complete C4 deficiencies are elucidated. Three patients with homozygous HLA A24 Cw7 B38 DR13 had systemic lupus erythematosus, mesangial glomerulonephritis, and severe skin lesions or membranous nephropathy. Immunofixation, genomic restriction fragment length polymorphisms, and pulsed field gel electrophoresis experiments revealed the presence of monomodular RP-C4-CYP21-TNX (RCCX) modules, each containing a solitary, long C4A mutant gene. Sequencing of the mutant C4A genes revealed a 2-bp, GT deletion in exon 13 that leads to protein truncation. The other four patients with homozygous HLA A30 B18 DR7 had SLE, severe kidney disorders including mesangial or membranoproliferative glomerulonephritis, and/or Henoch Schoenlein purpura. Molecular genetic analyses revealed an unusual RCCX structure with two short C4B mutant genes, each followed by an intact gene for steroid 21-hydroxylase. Nine identical, intronic mutations were found in each mutant C4B. In particular, the 8127 g-->a mutation present at the donor site of intron 28 may cause an RNA splice defect. Analyses of 12 complete C4 deficiency patients revealed two hot spots of deleterious mutations: one is located at exon 13, the others within a 2.6-kb genomic region spanning exons 20-29. Screening of these mutations may facilitate epidemiologic studies of C4 in infectious, autoimmune, and kidney diseases.

Involvement of C4 allotypes in the pathogenesis of human diseases.

The complement system is an important humoral defense mechanism that plays a relevant role against microbial agents, inflammatory response control, and immunocomplex clearance. Classical complement pathway activation is antibody-dependent. The C4 component participates in the initial step of activation, and C4 expression is determined by 2 pairs of allotypes: C4A and C4B. Deficiencies in C4 allotypes have been associated with several diseases. The aim of the present review is evaluate the reported data in the literature regarding specific C4A and C4B deficiencies and characterize their clinical relevance. We searched the MEDLINE and LILACS databases. Papers referring to total C4 deficiency without allotype evaluation and case reports of primary C4 deficiency were not included. Deficiencies in C4 allotypes have been associated with Mycobacterium leprae infection, erythema nodosum, systemic sclerosis with anti-topoisomerase I antibodies, intermediate congenital adrenal hyperplasia with DR5 genotype, diabetes mellitus type 1 with DR3,4 genotype, and diabetes mellitus with antibodies against islet cells. C4 allotype deficiency is also related to C4B deficiency and autoimmune-associated diseases, such as systemic lupus erythematosus, or diseases with an autoimmune component, such as autism. Some reports associate C4A with thyroiditis after delivery as well as limited and systemic sclerosis without anti-topoisomerase I antibodies. However, the studies with C4A and C4B have been concentrated in isolated populations, and some of the studies could not be reproduced by other authors.

Patients with systemic lupus erythematosus are deficient in complement-dependent prevention of immune precipitation.

OBJECTIVE: A functional deficiency of complement has been implicated but not conclusively demonstrated in the pathogenesis of systemic lupus erythematosus (SLE). To test this, we studied several aspects of complement in 44 patients with SLE, 46 patients with rheumatoid arthritis and 102 blood donors. METHODS: Prevention of immune precipitation (PIP) was measured by an enzyme immunoassay, levels of C1q, C4 and C3 by rocket immunoelectrophoresis, C4A, C4B and C3d by enzyme-linked immunosorbent assay (ELISA), complement haemolysis (CH50) by standard methods and C4 allotypes by high-voltage agarose electrophoresis and sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: PIP was significantly reduced in SLE (P<0.001); the defect was revealed by a sensitive assay measuring this function of complement but not by the other tests employed. The patients were clinically well at the time of study, and levels of C3d, which have been shown to correlate with disease activity, were normal. The defect was more common in patients with early disease (P = 0.009), supporting a role in aetiology or early pathophysiology. PIP was positively correlated with levels of C4 (P = 3 x 10(-5)) and in particular the C4A isotype (P = 9 x 10(-10)) whereas C4B was redundant. CONCLUSIONS: Our results reveal a defect in prevention of immune precipitation in SLE that is apparent at an early stage in the disease and correlates with low levels of C4A. These results indicate that subtle deficiencies of complement may predispose to SLE.

C4A deficiency and elevated level of immune complexes: the mechanism behind increased susceptibility to systemic lupus erythematosus.

OBJECTIVE: Studies of an Icelandic cohort showed that susceptibility to systemic lupus erythematosus (SLE) in individuals with C4A deficiency was increased only in the presence of increased concentrations of immune complexes. We investigated the interaction of C4A deficiency with elevated concentrations of immune complexes in healthy individuals; i.e., how different levels of C4A affected the activation of C4 and C3 and subsequent binding of increased immune complex load to human red blood cells (RBC). METHODS: Forty-five healthy individuals having different levels of C4A were studied, 8 with homozygous C4AQ0, 12 with heterozygous C4A deficiency, and 25 with normal C4A. For comparison to the complement status present after prolonged disease activity, 5 patients with SLE homozygous for C4AQ0 were also studied. RESULTS: The results showed that intact immune complex-RBC binding is dependent on the levels of immune complex-bound C3 fragments, which correlate to the levels of IC-bound C4Ad (R = 0.677, p = 0.02), but not on levels of IC-bound total C4d (R = 0.451, p = 0.16). Immune complex binding to RBC was also evaluated in increasing immune complex load. C4A deficient sera had less ability to bind the increased immune complex load to RBC than sera with normal C4A. These results are consistent with the presence of increased amounts of poorly opsonized immune complexes in C4A deficiency, leading to increased precipitation in tissues and initiation of a self-perpetuating cycle. CONCLUSION: Susceptibility to SLE is increased in individuals with C4A deficiency as C3 opsonization of immune complexes becomes insufficient at elevated immune complex concentrations.

Hipocomplementemia de causa poco habitual / An infrecuent cause of hypocomplementaemia

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