Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Objective To determine the roles of complement C4A and C4B gene copy-number variations and their plasma protein concentrations in residual insulin secretion and loss of pancreatic β-cell function in new-onset type 1 diabetes (T1D) patients. Methods We studied 34 patients of European ancestry with new-onset T1D, aged between 3 and 17 yr (10.7 ± 3.45), at Nationwide Children's Hospital in Columbus, Ohio. Gene copy-number and size variations of complement C4A and C4B were determined by genomic Southern blot analyses. C4A and C4B protein phenotypes were elucidated by immunofixation and radial immunodiffusion. Two-digit human leukocyte antigen ( HLA) -DRB1 genotypes were determined by sequence-specific polymerase chain reaction. At 1- and 9-month post diagnosis, stimulated C-peptide levels were measured after a standardized mixed-meal tolerance test. Results The diploid gene copy-numbers of C4A varied from 0 to 4, and those of C4B from 0 to 3. Patients with higher copy-number of C4A or higher C4A plasma protein concentrations at diagnosis had higher C-peptide levels at 1-month post diagnosis (p = 0.008; p = 0.008). When controlled by the Z-score of body mass index, C4A copy-numbers, C4A protein concentrations, the age of disease onset, and the number of HLA-DR3 but not DR4 alleles were significant parameters in determining C-peptide levels. At 9-month post diagnosis, 42.3% of patients remained in partial remission, and these patients were characterized by lower total C4B copy-numbers or lower C4B protein concentrations (p = 0.02; p = 0.0004). Conclusions C4A appears to associate with the protection of residual β-cell function in new-onset T1D; C4B is correlated with the end of disease remission at 9-month post diagnosis. [ABSTRACT FROM AUTHOR]

Class-switch recombination (CSR) involves replacement of the Cμ constant region with another downstream C H region. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated DNA breaks that are targeted to transcriptionally active switch (S) regions. S region promoters (Prs) direct synapsis by associating with the Eμ and 3′Eα enhancers that jointly anchor a chromatin loop. We report that asymmetric loop extrusion allows 3′Eα to track along the locus and form Pr-Pr-E interactions that mediate CSR between downstream S regions, followed by switching to donor Sμ. This alternative pathway bypasses sequential switching and creates immunoglobulin (Ig)E+ B cells in the absence of IgG1 expression. Based on the analysis of diagnostic CSR products in B cell subsets, we identify a BCR-negative cell intermediate that is pivotal to efficient CSR. [Display omitted] • Loop extrusion drives germline transcript promoter-enhancer contacts during CSR • Promoter-promoter-3′ enhancer contacts mediate reverse sequential switching • Use of the three CSR pathways is mouse strain- and Ig isotype-specific • During CSR B cells become surface BCRneg then re-express IgM or a switched isotype Shen et al. report that 3′Eα tracks along the Igh locus via unidirectional loop extrusion to form germline transcript promoter (Pr)-Pr-E interactions that mediate an alternative CSR pathway. B cell intermediates of CSR are identified, which are AID-dependent, surface BCR-negative, and in the G 1 phase of the cell cycle. [ABSTRACT FROM AUTHOR]

Within the human MHC region larger stretches of conserved DNA, called conserved ancestral haplotypes exist. However, many MHC haplotypes contain only fragments of an ancestral haplotype. Little is known, however, on relative distribution of the ancestral haplotypes to their fragments. Therefore we determined the frequency of carriers of the whole ancestral haplotype 8.1 (AH8.1) and its fragments in 127 healthy Hungarian people, 101 healthy Ohioian females, and in nine Hungarian families. The HLA-DQ2, HLA-DR3(17), RAGE -429C allele, the mono-S-C4B genotype, the HSP70-2 1267G allele and the TNF -308A (TNF2) allele were used as markers of the AH8.1. Frequency of carriers of the whole AH8.1 and its fragments was similar in the both populations. 18% of the subjects carried the whole AH8.1 in at least one chromosome, while 17-20%, 36-39%, and 24-29%, respectively carried two or three constituents of the haplotype, only one constituent or none of them. Similar results were obtained in the family study. In addition, marked differences were found in the relationship of the constituents' alleles to the whole AH8.1. In both populations, 29%, 50-59%, 52-56% and 76-96%, respectively of the carriers of HSP70-2 1267G, RAGE-429C, TNF2, and mono-S carriers carried the whole 8.1 haplotype. These findings may have important implications for studies of the disease associations with different MHC ancestral haplotypes. [ABSTRACT FROM AUTHOR]

The development of high-affinity antibodies in response to infection is an iterative process in which B cells cycle between proliferation/somatic hypermutation and antigen-driven selection. These processes occur within specific regions of the secondary lymphoid structures known as germinal centers (GCs) and the environmental and signaling cues provided by these regions guide the GC reactions that drive B cell maturation and antibody production, ultimately determining B cell fate. In this issue of the JCI, Nakagawa and colleagues examine the role of miR-155, a microRNA that is required for GC development and the production of high-affinity antibodies. They show that miR-155 is highly expressed in positively selected B cells and promotes survival of these cells by orienting the Myc transcription program toward survival rather than apoptosis through the inhibition of the transcriptional regulator JARID2. These findings illustrate the fine balance between apoptosis and proliferation that is required for the development of high-affinity antibodies. [ABSTRACT FROM AUTHOR]

Abstract: Previously we reported on strong linkage disequilibrium (LD) between the mono-S-C4B-RCCX module (mono-S) and the TNF2 allele (both known constituents of the 8.1 ancestral haplotype (8.1 AH)) in two Caucasian populations. The gene for the receptor of advanced glycation endproducts (RAGE) is encoded between the RCCX module and the HLA class II genes in the central MHC region. In order to assess the relationship between the promoter polymorphisms of the RAGE gene and the 8.1 AH, we performed a family study in eight informative families affected with type 1 diabetes mellitus; haplotypes of a RAGE promoter SNP (−429T>C) with the HLA-DQ2, -DR-3(17) and TNF2 alleles, as well as the mono-S genotype were determined. A similar analysis was performed in 82 unrelated patients with type 1 diabetes mellitus, and in unrelated healthy individuals of three different Caucasian populations (Hungarians, Ohioian females, Icelandics). In the diabetic patients clinical correlations were also investigated. Out of the 32 paternal and maternal chromosome 6 from the eight families, 15 different MHC haplotypes were found. Haplotypes containing at least three of the known constituents of the 8.1 AH (HLA-DQ2, -DR17, mono-S, TNF2) were always linked to the RAGE −429C allele. The RAGE −429C allele exhibited highly significant (p <0.0001) LD coefficients to known constituents of the 8.1 AH both in healthy persons and patients with type 1 diabetes. In the group of patients with diabetes we found significantly (p =0.013) higher maximal hemoglobinA1C concentration in the carriers of the RAGE −429C allele, this trait, however was not linked to the 8.1 AH. Our present findings indicate that the RAGE −429C allele can be considered as a candidate member of the 8.1 AH. The results also reveal a spectrum of recombinant MHC haplotypes in addition to the conserved ancestral haplotypes. [Copyright &y& Elsevier]

Background: Idiopathic 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 and muscle fibres, chronic weakness and fatigue. While complement-mediated destruction of capillary endothelia is implicated in paediatric and adult dermatomyositis, the complex diversity of complement C4 in IIM pathology was unknown., Methods: We elucidated the gene copy number (GCN) variations of total C4 , C4A and C4B, long and short genes in 1644 Caucasian patients with IIM, plus 3526 matched healthy controls using real-time PCR or Southern blot analyses. Plasma complement levels were determined by single radial immunodiffusion., Results: The large study populations helped establish the distribution patterns of various C4 GCN groups. Low GCNs of C4T ( C4T =2+3) and C4A deficiency ( C4A =0+1) were strongly correlated with increased risk of IIM with OR equalled to 2.58 (2.28-2.91), p=5.0×10 -53 for C4T , and 2.82 (2.48-3.21), p=7.0×10 -57 for C4A deficiency. Contingency and regression analyses showed that among patients with C4A deficiency, the presence of HLA-DR3 became insignificant as a risk factor in IIM except for inclusion body myositis (IBM), by which 98.2% had HLA-DR3 with an OR of 11.02 (1.44-84.4). Intragroup analyses of patients with IIM for C4 protein levels and IIM-related autoantibodies showed that those with anti-Jo-1 or with anti-PM/Scl had significantly lower C4 plasma concentrations than those without these autoantibodies., Conclusions: C4A deficiency is relevant in dermatomyositis, HLA-DRB1*03 is important in IBM and both C4A deficiency and HLA-DRB1*03 contribute interactively to risk of polymyositis., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY. Published by BMJ.)

Objective: Systemic lupus erythematosus (SLE) features high frequency of cardiovascular disease (CVD) and fluctuating complement levels. The clinical trial Atherosclerosis Prevention in Pediatric Lupus Erythematosus (APPLE) aimed to evaluate whether atorvastatin treatment reduced the progression of atherosclerosis in 221 patients with childhood-onset SLE (cSLE), using carotid intima media thickness (CIMT) as surrogates. We leveraged APPLE biorepository and trial data to investigate the relationship between complement and CVD in cSLE., Methods: Gene copy numbers (GCNs) for total C4 , C4A and C4B were measured by TaqMan-based real-time PCR and Southern blotting, and analysed with laboratory and clinical parameters through Student's t-test and χ 2 analyses. Effects of total C4 , C4A and C4B GCNs on the response to placebo or atorvastatin treatment and progression of CIMT were examined by regression analyses., Results: At baseline, C4 protein levels strongly correlated with GCNs of total C4 (p=1.8×10 -6 ). Each copy of C4 gene increased mean serum C4 by 3.28 mg/dL. Compared with those without hypertension (N=142), individuals with hypertension demonstrated significantly elevated serum levels for C4 and C3 at baseline and serially (C4: P=5.0×10 -25 ; C3: P=5.84×10 -20 ). Individuals with ≥2 C4B genes had 2.5 times the odds of having hypertension (p=0.016) and higher diastolic blood pressure (p=0.015) compared with those with C4B deficiency. At the study end, subjects with ≥2 C4B and atorvastatin treatment had significantly slower increase in CIMT compared with those treated with placebo (p=0.018)., Conclusions: cSLE with hypertension had elevated serum levels of C4 and C3 and higher GCN of C4B ; cSLE with ≥2 C4B genes would benefit from statins therapy to prevent atherosclerosis., Competing Interests: Competing interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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.

Noncoding transcripts originating upstream of the immunoglobulin constant region (I transcripts) are required to direct activation-induced deaminase to initiate class switching in B cells. Differential regulation of Iε and Iγ1 transcription in response to interleukin 4 (IL-4), hence class switching to IgE and IgG1, is not fully understood. In this study, we combine novel mouse reporters and single-cell RNA sequencing to reveal the heterogeneity in IL-4-induced I transcription. We identify an early population of cells expressing Iε but not Iγ1 and demonstrate that early Iε transcription leads to switching to IgE and occurs at lower activation levels than Iγ1. Our results reveal how probabilistic transcription with a lower activation threshold for Iε directs the early choice of IgE versus IgG1, a key physiological response against parasitic infestations and a mediator of allergy and asthma., (© 2017 Wu et al.)

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., (© 2016, American College of Rheumatology.)

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.

Cytidine deaminases are single stranded DNA mutators diversifying antibodies and restricting viral infection. Improper access to the genome leads to translocations and mutations in B cells and contributes to the mutation landscape in cancer, such as kataegis. It remains unclear how deaminases access double stranded genomes and whether off-target mutations favor certain loci, although transcription and opportunistic access during DNA repair are thought to play a role. In yeast, AID and the catalytic domain of APOBEC3G preferentially mutate transcriptionally active genes within narrow regions, 110 base pairs in width, fixed at RNA polymerase initiation sites. Unlike APOBEC3G, AID shows enhanced mutational preference for small RNA genes (tRNAs, snoRNAs and snRNAs) suggesting a putative role for RNA in its recruitment. We uncover the high affinity of the deaminases for the single stranded DNA exposed by initiating RNA polymerases (a DNA configuration reproduced at stalled polymerases) without a requirement for specific cofactors.

Breast cancer genomes have revealed a novel form of mutation showers (kataegis) in which multiple same-strand substitutions at C:G pairs spaced one to several hundred nucleotides apart are clustered over kilobase-sized regions, often associated with sites of DNA rearrangement. We show kataegis can result from AID/APOBEC-catalysed cytidine deamination in the vicinity of DNA breaks, likely through action on single-stranded DNA exposed during resection. Cancer-like kataegis can be recapitulated by expression of AID/APOBEC family deaminases in yeast where it largely depends on uracil excision, which generates an abasic site for strand breakage. Localized kataegis can also be nucleated by an I-SceI-induced break. Genome-wide patterns of APOBEC3-catalyzed deamination in yeast reveal APOBEC3B and 3A as the deaminases whose mutational signatures are most similar to those of breast cancer kataegic mutations. Together with expression and functional assays, the results implicate APOBEC3B/A in breast cancer hypermutation and give insight into the mechanism of kataegis. DOI:http://dx.doi.org/10.7554/eLife.00534.001.

Genetic variants resulting in non-expression of complement C4A and C4B genes are common in healthy European populations and have shown association with a number of diseases, most notably the autoimmune disease, systemic lupus erythematosus. The most frequent cause of a C4 "null" allele, following that of C4 gene copy number variation (CNV), is a non-sense mutation arising from a 2 bp CT insertion into codon 1232 of exon 29. Previous attempts to accurately genotype this polymorphism have not been amenable to high-throughput typing, and have been confounded by failure to account for CNV at this locus, as well as by inability to distinguish between paralogs. We have developed a novel, high-throughput, paralog-specific assay to detect the presence and copy number of this polymorphism. We have genotyped healthy cohorts from the United Kingdom (UK) and Spain. Overall, 30/719 (4.17%) individuals from the UK cohort and 8/449 (1.78%) individuals from the Spanish cohort harboured the CT insertion in a C4A gene. A single Spanish individual possessed a C4B CT insertion. There is weak correlation between the C4 CT insertion and flanking MHC polymorphism. Therefore it is important to note that, as with C4 gene CNV, disease-association due to this variant will be missed by current SNP-based genome-wide association strategies.

Recent comparative genome hybridization studies revealed that hundreds to thousands of human genomic loci can have interindividual copy number variations (CNVs). One of such CNV loci in the HLA codes for the immune effector protein complement component C4. Sensitive, specific, and accurate assays to interrogate the C4 CNV and its associated polymorphisms by using submicrogram quantities of genomic DNA are needed for high throughput epidemiologic studies of C4 CNVs in autoimmune, infectious, and neurological diseases. Quantitative real-time PCR (qPCR) assays were developed using TaqMan chemistry and based on sequences specific for C4A and C4B genes, structural characteristics corresponding to the long and short forms of C4 genes, and the breakpoint region of RP-C4-CYP21-TNX (RCCX) modular duplication. Assignments for gene copy numbers were achieved by relative standard curve methods using cloned C4 genomic DNA covering 6 logs of DNA concentrations for calibrations. The accuracies of test results were cross-confirmed internally in each sample, as the sum of C4A plus C4B equals to the sum of C4L plus C4S or the total copy number of RCCX modules. These qPCR assays were applied to determine C4 CNVs from samples of 50 consanguineous subjects who were mostly homozygous in HLA genotypes. The results revealed eight HLA haplotypes with single C4 genes in monomodular RCCX that are associated with multiple autoimmune and infectious diseases and 32 bimodular, 4 trimodular, and one quadrimodular RCCX. These C4 qPCR assays are proven to be robust, sensitive, and reliable, as they have contributed to the elucidation of C4 CNVs in >1000 human samples with autoimmune and neurological diseases.

Interindividual gene copy-number variation (CNV) of complement component C4 and its associated polymorphisms in gene size (long and short) and protein isotypes (C4A and C4B) probably lead to different susceptibilities to autoimmune disease. We investigated the C4 gene CNV in 1,241 European Americans, including patients with systemic lupus erythematosus (SLE), their first-degree relatives, and unrelated healthy subjects, by definitive genotyping and phenotyping techniques. The gene copy number (GCN) varied from 2 to 6 for total C4, from 0 to 5 for C4A, and from 0 to 4 for C4B. Four copies of total C4, two copies of C4A, and two copies of C4B were the most common GCN counts, but each constituted only between one-half and three-quarters of the study populations. Long C4 genes were strongly correlated with C4A (R=0.695; P<.0001). Short C4 genes were correlated with C4B (R=0.437; P<.0001). In comparison with healthy subjects, patients with SLE clearly had the GCN of total C4 and C4A shifting to the lower side. The risk of SLE disease susceptibility significantly increased among subjects with only two copies of total C4 (patients 9.3%; unrelated controls 1.5%; odds ratio [OR] = 6.514; P=.00002) but decreased in those with > or =5 copies of C4 (patients 5.79%; controls 12%; OR=0.466; P=.016). Both zero copies (OR=5.267; P=.001) and one copy (OR=1.613; P=.022) of C4A were risk factors for SLE, whereas > or =3 copies of C4A appeared to be protective (OR=0.574; P=.012). Family-based association tests suggested that a specific haplotype with a single short C4B in tight linkage disequilibrium with the -308A allele of TNFA was more likely to be transmitted to patients with SLE. This work demonstrates how gene CNV and its related polymorphisms are associated with the susceptibility to a human complex disease.

The serial changes of serum complement proteins C4 and C3 in SLE were characterized in 33 pediatric SLE patients with defined C4 genotypes. Three distinct groups of C4 protein profiles were observed. The first group was characterized by persistently low C4 levels (<10 mg/dL) throughout the course of the study. Patients with this profile had mild disease manifestations and low to medium copy numbers of C4 genes. The second group featured periodic fluctuations of serum C4 protein concentrations above and below 10 mg/dL, paralleled with ups and downs of SLE disease activities. Most patients with the second profile had unequal copy numbers of C4A and C4B genes and relatively severe disease. The third group had normal serum C4 levels (>15 mg/dL) most of the time and occasionally low C4 and C3 levels that were mostly coincident with disease flares prior to effective medical treatment. Most patients in this group

This unit describes methods that can accurately determine the genotypes and phenotypes of human complement components C4A and C4B. Specifically, they allow investigators to determine how many C4 genes are present in a diploid genome of a human subject and to quantify how many of them encode C4A proteins and how many of them encode C4B proteins. In addition, methods to determine how many long and short C4 genes are present in a diploid genome of a subject are described together with experimental strategies to determine haplotypes and order or configuration of these genes in the MHC. Finally, methods to assess the degree of polymorphism in C4A and C4B proteins and whether low protein levels of plasma C4 may be caused by low C4 gene dosages and/or by mutant C4 genes.