C4A Deficiency Research Articles

130 Low Gene Copy Numbers (GCN) of complement C4 and C4A deficiency are highly significant genetic risk factors for idiopathic inflammatory myopathies and its major subgroups

130 Low Gene Copy Numbers (GCN) of complement C4 and C4A deficiency are highly significant genetic risk factors for idiopathic inflammatory myopathies and its major subgroups

Complement C4 gene copy number variations and polymorphisms, autoantibodies, and clinical manifestations of juvenile dermatomyositis– a multi-center study

Abstract Juvenile dermatomyositis (JDM) is an autoimmune myopathy characterized by rash and muscle weakness. Complement C4 gene copy number (GCN) variation is a known risk factor for JDM. JDM patients often develop autoantibodies. We investigated the relationship between C4 GCN, clinical features, and antibody development. METHODS: Subjects were recruited (n=255) from the US, Sweden, and the Czech Republic. C4 GCN was determined by real time PCR. Comparative analyses were performed via student’s t test or Mann Whitney U test. Correlation was assessed via linear regression or Fisher’s exact test. All studies were IRB approved, and informed consent was obtained. RESULTS: C4 GCN correlated with muscle pathology and extra-muscular disease at diagnosis. Lower C4A and C4L GCN were associated with a higher number of abnormal muscle enzymes (p=0.0062 and p=0.0029), while the opposite was true for C4S (p=0.024). Higher C4S GCN correlated with lower muscle strength scores (p=0.039). Lower C4L correlated with abnormal MRI (p= 0.042). C4B GCN correlated with dysphagia (p=0.035). Higher GCN of C4S was a risk factor for arthritis (p=0.032), systemic symptoms (p=0.020), and dysphagia (p=0.0078). C4 GCN was associated with myositis-specific (MSA) and myositis-associated (MAA) antibodies. Patients with homozygous C4A deficiency were more likely to test positive for anti-NXP2 (OR 20.0, p=0.011), and C4A GCN was lower in anti-NXP2 positive subjects (p=0.030). A higher C4B GCN correlated with positive MAA (p=0.043). DISCUSSION: Here, we show that low C4A/C4L GCN and high C4B/C4S GCN correlate with worse muscle disease, extra-muscular pathology, and autoantibodies. Our data suggest that complement C4 may play a key role in the ongoing pathogenesis of JDM. Supported by grants from NIH (R21 AR070509) and the CureJM Foundation

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

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. 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. 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. 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.

Low copy numbers of complement C4 and C4A deficiency are risk factors for myositis, its subgroups and autoantibodies

BackgroundIdiopathic inflammatory myopathies (IIM) are a group of autoimmune diseases characterised by myositis-related autoantibodies plus infiltration of leucocytes into muscles and/or the skin, leading to the destruction of blood vessels...

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.

RECURRENT ANGIOEDEMA IN SLE PATIENT

TOPIC: Allergy and Airway TYPE: Medical Student/Resident Case Reports INTRODUCTION: The pathophysiology of angioedema is broad. It may be hereditary, idiopathic, or a reaction to a range of medications or allergens; there have been a few case reports describing recurrent angioedema in patients with autoimmune conditions such as systemic lupus erythematosus (SLE). We present the case of a patient with SLE who suffered from recurrent angioedema of the face, including lips, tongue, and throat, without urticaria, with normal C1-Inhibitor levels. CASE PRESENTATION: A 69-year-old female with a history of SLE and recurrent angioedema presented with progressive swelling of eyes, lips, and tongue for one week. She did not consume angiotensin-converting enzyme inhibitors and reported no medication changes or exposure to unknown substances. Physical examination revealed facial, periorbital, and tongue edema with respiratory distress. Initial lab work was unremarkable. Histamine blockers and steroids were immediately administered, and she was intubated due to respiratory compromise. Racemic epinephrine was also initiated, and tracheostomy was performed for definitive airway management. Workup for infection was unrevealing. C1-Inhibitor, CH50, and C4 levels were not deficit. Once her respiratory status was stabilized, she was discharged to a long-term care facility. DISCUSSION: Angioedema, defined as localized subcutaneous or submucosal swelling due to the extravasation of fluid into interstitial tissues, maybe mast cell-mediated or kinin-mediated. The former is often associated with urticaria and is triggered by an allergic reaction or medications. At the same time, the latter occurs in the absence of a specific trigger and is associated with hereditary C1-Inhibitor deficiency or dysfunction. Our patient had normal C1 esterase inhibitor levels, rendering the diagnosis of hereditary angioedema less likely. Furthermore, she reported taking no medications known to cause angioedema and had not been exposed to any new allergens before the presentation. Literature states that patients with SLE characteristically exhibit reduced levels of components of the classical complement pathway. The defect can either originate from a lymphoproliferative disease that catabolizes C1-Inhibitor or circulating antibodies that inactivate C1-Inhibitor. However, our patient did not fulfill the above-mentioned criteria. Even though similar cases have been reported in the literature, the occurrence is still scarce. CONCLUSIONS: SLE has rarely been associated with recurrent angioedema. The pathophysiology of such is unclear. Our patient's late age of presentation and lack of family history of angioedema favored SLE as the independent risk factor for angioedema. Cl-inhibitor dysfunction in SLE is heterogeneous and distinct from that reported in either hereditary or acquired angioedema. Hence, more work needs to be done for early recognition and prompt prevention. REFERENCE #1: Dunckley H, Gatenby PA, Hawkins B, Naito S, Serjeantson SW. Deficiency of C4A is a genetic determinant of systemic lupus erythematosus in three ethnic groups. J Immunogenet. 1987 Aug-Oct;14(4-5):209-18. doi: 10.1111/j.1744-313x.1987.tb00383.x. PMID: 3502648. REFERENCE #2: Alsenz J, Bork K, Loos M. Autoantibody-mediated acquired deficiency of C1 inhibitor. N Engl J Med. 1987 May 28;316(22):1360-6. doi: 10.1056/NEJM198705283162202. PMID: 3494945. REFERENCE #3: Thong BY, Thumboo J, Howe HS, Feng PH. Life-threatening angioedema in systemic lupus erythematosus. Lupus. 2001;10(4):304-308. doi:10.1191/096120301680417011 DISCLOSURES: No relevant relationships by Asim Amjad, source=Web Response No relevant relationships by Zamara Hamid, source=Web Response No relevant relationships by Dan Kazmierski, source=Web Response No relevant relationships by Si Li, source=Web Response No relevant relationships by Pius Ochieng, source=Web Response No relevant relationships by NISHANT SHARMA, source=Web Response

A case report of complement C4B deficiency in a patient with steroid and IVIG-refractory anti-NMDA receptor encephalitis

BackgroundComplement C4A or C4B deficiency has never been reported in autoantibody-associated encephalitides patient. Here we present a case of anti-N-methyl- D-aspartate (NMDA) receptor encephalitis associated with homozygous C4B deficiency, who did not respond to intravenous immunoglobulin and pulse methylprednisolone but plasmapheresis and rituximab.Case presentationA fourteen-year-old boy presented to our unit with subacute onset of behavioral changes and confusion, and was later confirmed to be anti-NMDA receptor encephalitis. He was initially managed with intravenous immunoglobulin (IVIG) and pulse methylprednisolone but did not achieve any clinical improvement. Seven sessions of plasmapheresis was commenced with remarkable improvement after the second session, and was followed by four doses of rituximab. His neurological and cognitive functioning gradually returned to baseline. Immunological investigations demonstrated persistently low C4 levels below 8 mg/dL. A more in-depth complement analysis of the patient and his family showed that he has homozygous C4B deficiency. Genetic analysis revealed that the index patient has homozygous deficiency in complement C4B and he carries one non-functioning mutant C4B gene inherited from his mother.ConclusionsLow levels of serum C4 correlate with reduced functions of the classical and lectin pathways, leading to the impairment of immune-complexes removal. Plasmapheresis ameliorates complement deficiency and removes the offending immune-complexes leading to clinical improvement that was not achieved by IVIG and steroids. We postulate that serum C4 levels may serve as a biomarker for the need of plasmapheresis upfront rather than only after non-response to steroid and IVIG in treating anti-NMDA-receptor encephalitis.

Impact of Homozygous C4A Deficiency on Clinical Presentation of Systemic Lupus Erythematosus.

To investigate the association of C4A null allele (C4AQ0) with systemic lupus erythematosus (SLE) and determine the clinical presentation of SLE in relation to C4A null allele. Descriptive study. Armed Forces Institute of Pathology (AFIP), Rawalpindi, Immunology Department, from December 2018 to December 2019. Patients referred to AFIP, who fulfilled American College of Rheumatology (ACR) criteria of 1997 for diagnosis of SLE were included in the study. Approval from the Institutional Ethical Review Board was taken. C4A and C4B null alleles were determined in 66 SLE patients and 40 age- and gender-matched healthy controls by polymerase chain reaction (PCR) using sequence-specific primers (PCR-SSP). Various clinical features and laboratory findings in the SLE patients were analysed in relation with C4A null allele. The mean age of the study population was 30.56 ±10.08 years. C4A null allele was detected in 7 (10.6%) patients; whereas, C4B null allele was detected in only two (3%) patients. SLE patients with C4A null allele had increased incidence of arthritis (100%) and renal damage (85.7%); compared to those with normal C4A allele, 57.6% and 32%, respectively. Fisher's Exact test revealed strong association of C4A null allele with arthritis and renal damage, (p = 0.039 and 0.01, respectively). Homozygous absence of C4A alleles was encountered in 10.6% of Pakistani patients of SLE and is closely related with clinical features of arthritis and renal damage. Knowledge of C4A null allele in SLE patients at diagnosis can predict disease course. Key Words: SLE, C4A null alleles, C4AQ0, Homozygous C4A deficiency.

Opposite Profiles of Complement in Antiphospholipid Syndrome (APS) and Systemic Lupus Erythematosus (SLE) Among Patients With Antiphospholipid Antibodies (aPL).

APS is the association of antiphospholipid antibodies (aPL) with thromboses and/or recurrent pregnancy loss (RPL). Among patients with SLE, one-third have aPL and 10–15% have a manifestation of secondary APS. Animal studies suggested that complement activation plays an important role in the pathogenesis of thrombosis and pregnancy loss in APS. We performed a cross-sectional study on complement proteins and genes in 525 patients with aPL. Among them, 237 experienced thromboses and 293 had SLE; 111 had both SLE and thromboses, and 106 had neither SLE nor thrombosis. Complement protein levels were determined by radial immunodiffusion for C4, C3 and factor H; and by functional ELISA for mannan binding lectin (MBL). Total C4, C4A and C4B gene copy numbers (GCN) were measured by TaqMan-based realtime PCR. Two to six copies of C4 genes are frequently present in a diploid genome, and each copy may code for an acidic C4A or a basic C4B protein. We observed significantly (a) higher protein levels of total C4, C4A, C4B, C3, and anticardiolipin (ACLA) IgG, (b) increased frequencies of lupus anticoagulant and males, and (c) decreased levels of complement factor H, MBL and ACLA-IgM among patients with thrombosis than those without thrombosis (N = 288). We also observed significantly lower GCNs of total C4 and C4A among aPL-positive patients with both SLE and thrombosis than others. By contrast, aPL-positive subjects with SLE had significantly reduced protein levels of C3, total C4, C4A, C4B and ACLA-IgG, and higher frequency of females than those without SLE. Patients with thrombosis but without SLE (N = 126), and patients with SLE but without thrombosis (N = 182) had the greatest differences in mean protein levels of C3 (p = 2.6 × 10−6), C4 (p = 2.2 × 10−9) and ACLA-IgG (p = 1.2 × 10−5). RPL occurred in 23.7% of female patients and thrombotic SLE patients had the highest frequency of RPL (41.0%; p = 3.8 × 10−10). Compared with non-RPL females, RPL had significantly higher frequency of thrombosis and elevated C4 protein levels. Female patients with homozygous C4A deficiency all experienced RPL (p = 0.0001) but the opposite was true for patients with homozygous C4B deficiency (p = 0.017). These results provide new insights and biomarkers for diagnosis and management of APS and SLE.

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

IntroductionHomozygous 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 methodsThirty-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.ResultsNovel 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).ConclusionThis 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

ObjectivesLow 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.MethodsWe 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.ResultsHomozygous 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).ConclusionsOur 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.

Early Components of the Complement Classical Activation Pathway in Human Systemic Autoimmune Diseases.

The complement system consists of effector proteins, regulators, and receptors that participate in host defense against pathogens. Activation of the complement system, via the classical pathway (CP), has long been recognized in immune complex-mediated tissue injury, most notably systemic lupus erythematosus (SLE). Paradoxically, a complete deficiency of an early component of the CP, as evidenced by homozygous genetic deficiencies reported in human, are strongly associated with the risk of developing SLE or a lupus-like disease. Similarly, isotype deficiency attributable to a gene copy-number (GCN) variation and/or the presence of autoantibodies directed against a CP component or a regulatory protein that result in an acquired deficiency are relatively common in SLE patients. Applying accurate assay methodologies with rigorous data validations, low GCNs of total C4, and heterozygous and homozygous deficiencies of C4A have been shown as medium to large effect size risk factors, while high copy numbers of total C4 or C4A as prevalent protective factors, of European and East-Asian SLE. Here, we summarize the current knowledge related to genetic deficiency and insufficiency, and acquired protein deficiencies for C1q, C1r, C1s, C4A/C4B, and C2 in disease pathogenesis and prognosis of SLE, and, briefly, for other systemic autoimmune diseases. As the complement system is increasingly found to be associated with autoimmune diseases and immune-mediated diseases, it has become an attractive therapeutic target. We highlight the recent developments and offer a balanced perspective concerning future investigations and therapeutic applications with a focus on early components of the CP in human systemic autoimmune diseases.

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

ObjectiveComplement-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...

Complement activation and regulation in preeclamptic placenta.

Preeclampsia (PE) is a common disorder of pregnancy originating in the placenta. We examined whether excessive activation or poor regulation of the complement system at the maternal–fetal interface could contribute to the development of PE. Location and occurrence of complement components and regulators in placentae were analyzed. Cryostat sections of placentae were processed from 7 early-onset PE (diagnosis <34 weeks of gestation), 5 late-onset PE, 10 control pregnancies, and immunostained for 6 complement activators and 6 inhibitors. Fluorescence was quantified and compared between PE and control placentae. Gene copy numbers of complement components C4A and C4B were assessed by a quantitative PCR method. Maternal C4 deficiencies (≥1 missing or non-functional C4) were most common in the early-onset PE group (71%), and more frequent in late-onset PE compared to healthy controls (60 vs. 38%). Complement C1q deposition differed significantly between control and patient groups: controls and early-onset PE patients had more C1q than late-onset PE patients (mean p = 0.01 and p = 0.005, respectively). C3 activation was analyzed by staining for C3b/iC3b and C3d. C3d was mostly specific to the basal syncytium and C3b/iC3b diffuse in other structures, but there were no clear differences between the study groups. Activated C4 and membrane-bound regulators CD55, CD46, and CD59 were observed abundantly in the syncytiotrophoblast. Syncytial knots, structures enriched in PE, stained specifically for the classical pathway inhibitor C4bp, whereas the key regulator alternative pathway, factor H (FH) showed a wider distribution in the placenta. Differences in C1q deposition between late- and early-onset PE groups may be indicative of the different etiology of PE symptoms in these patients. Irregular distribution of the complement regulators C4bp and FH in the PE placenta and a higher frequency of C4A deficiencies suggest a disturbed balance between complement activation and regulation in PE.

Complement C4 Deficiency – A Plausible Risk Factor for Non-Tuberculous Mycobacteria (NTM) Infection in Apparently Immunocompetent Patients

BackgroundNon-tuberculous mycobacteria (NTM) are ubiquitous in the environment and they infect mainly persons with underlying pulmonary diseases but also previously healthy elderly women. Defects in host resistance that lead to pulmonary infections by NTM are relatively unknown. A few genetic defects have been associated with both pulmonary and disseminated mycobacterial infections. Rare disseminated NTM infections have been associated with genetic defects in T-cell mediated immunity and in cytokine signaling in families. We investigated whether there was an association between NTM infections and deficiencies of complement components C4A or C4B that are encoded by major histocompatibility complex (MHC).Methods50 adult patients with a positive NTM culture with symptoms and findings of a NTM disease were recruited. Patients' clinical history was collected and symptoms and clinical findings were categorized according to 2007 diagnostic criteria of The American Thoracic Society (ATS). To investigate the deficiencies of complement, C4A and C4B gene copy numbers and phenotype frequencies of the C4 allotypes were analyzed. Unselected, healthy, 149 Finnish adults were used as controls.ResultsNTM patients had more often C4 deficiencies (C4A or C4B) than controls (36/50 [72%] vs 83/149 [56%], OR = 2.05, 95%CI = 1.019–4.105, p = 0.042). C4 deficiencies for female NTM patients were more common than for controls (29/36 [81%] vs 55/100 [55%], OR = 3.39, 95% CI = 1.358–8.460, p = 0.007). C4 deficiences seemed not to be related to any specific underlying disease or C4 phenotype.Conclusions C4 deficiency may be a risk factor for NTM infection in especially elderly female patients.

THU0187 Complement C4 gene polymorphisms, serum protein levels and protein functional activity in relationship with clinical manifestations in patients with systemic lupus erythematosus

BackgroundThe complement component C4 and its associated polymorphisms have a strong association with systemic lupus erythematosus (SLE). A low gene copy number (GCN) of C4 and its two isotypes (C4A...

Copy Number Analysis of Complement C4A, C4B and C4A Silencing Mutation by Real-Time Quantitative Polymerase Chain Reaction

Low protein levels and copy number variation (CNV) of the fourth component of human complement (C4A and C4B) have been associated with various diseases. High-throughput methods for analysing C4 CNV are available, but they commonly do not detect the most common C4A mutation, a silencing CT insertion (CTins) leading to low protein levels. We developed a SYBR® Green labelled real-time quantitative polymerase chain reaction (qPCR) with a novel concentration range approach to address C4 CNV and deficiencies due to CTins. This method was validated in three sample sets and applied to over 1600 patient samples. CTins caused C4A deficiency in more than 70% (76/105) of the carriers. Twenty per cent (76/381) of patients with a C4A deficiency would have been erroneously recorded as having none, if the CTins had not been assessed. C4A deficiency was more common in patients than a healthy reference population, (OR = 1.60, 95%CI = 1.02–2.52, p = 0.039). The number of functional C4 genes can be straightforwardly analyzed by real-time qPCR, also with SYBR® Green labelling. Determination of CTins increases the frequency of C4A deficiency and thus helps to elucidate the genotypic versus phenotypic disease associations.

Increased frequency of complement C4B deficiency in rheumatoid arthritis

To assess the copy number variation of complement C4A and C4B genes in patients with rheumatoid arthritis (RA). DNA samples were obtained from 299 patients and controls and analyzed for copy number variation of total complement C4, C4A, and C4B genes. The results were compared by chi-square analysis, and odds ratios (ORs) and 95% confidence intervals (95% CIs) were calculated. Chi-square analysis revealed similar distribution patterns of total C4 alleles in RA patients (n = 160), non-RA patients (n = 88), and healthy controls (n = 51). There was no trend toward C4A deficiency as in lupus. Significant differences in C4B distribution were observed in RA patients, in whom an ∼2-fold increase in the frequency of homozygous and/or heterozygous C4B deficiency (0 or 1 allele) (40%) was present relative to non-RA patients or healthy controls (both 21.6%). C4B deficiency was more frequent in seropositive RA patients than in seronegative RA patients (44% versus 31%). The odds of C4B deficiency were 2.99 (95% CI 1.58-5.65) (P = 0.0006) in seropositive RA patients relative to non-RA controls. These findings were confirmed in a larger healthy control cohort, yielding an OR of 1.83 (95% CI 1.21-2.76) (P = 0.0056). The association of the shared epitope with C4B deficiency was significantly greater in seropositive RA patients than in non-seropositive RA controls (96% versus 54.5%) (P < 0.0001), suggesting that C4B deficiency interacts with the shared epitope in the development of seropositive RA. Our findings indicate a relationship between C4B copy number variation and RA that approximates that seen between C4A copy number variation and lupus. The concurrence of C4B deficiency and the shared epitope in seropositive RA may have broad implications for our understanding of RA pathogenesis.

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.

Genetic Deficiency of Complement Isoforms C4A or C4B Predicts Improved Survival of Metastatic Renal Cell Carcinoma

Autoimmune phenomena during immunotherapy are associated with favorable outcomes in patients with metastatic renal cell carcinoma. We have reported improved survival in patients with stage IV renal cell carcinoma who carry autoimmunity associated HLA class II haplotypes. We propose that the clinical benefit is mediated by products of other autoimmunity associated genes linked to these haplotypes. A candidate gene is complement C4, which replicates as part of the RCCX module, can be present in multiple copies and exists as C4A and C4B isoforms. Deficiencies of either isoform are associated with autoimmunity. In the current study we tested the hypothesis that C4A or C4B deficiency predicts improved survival of patients with RCC. The total C4 copy number was determined by simultaneous amplification of RP1 and TNXA/RP2 to quantitate RCCX modules. C4A and C4B alleles were distinguished by PshAI restriction fragment length polymorphism. Genetic complotypes were determined in 61 patients. Individuals with a solitary copy of either C4 isoform experienced longer survival. Median survival from the diagnosis of metastatic disease in patients with a solitary copy of C4A or C4B was 7.75 years vs 1.25 in the comparison group (p = 0.001). This was independent of the benefit derived from autoimmune class II genotypes. Improved survival is seen in patients with C4A or C4B deficiency and renal cell carcinoma treated with cytokine therapy with or without surgery. These data support our hypothesis that patients with renal cell carcinoma who have autoimmune genotypes have favorable outcomes resulting from autoimmune mechanisms directed to the tumor.