Regulator Of Complement Activation Gene Cluster Research Articles

Regulatory Architecture of the RCA Gene Cluster Captures an Intragenic TAD Boundary, CTCF-Mediated Chromatin Looping and a Long-Range Intergenic Enhancer.

The Regulators of Complement Activation (RCA) gene cluster comprises several tandemly arranged genes with shared functions within the immune system. RCA members, such as complement receptor 2 (CR2), are well-established susceptibility genes in complex autoimmune diseases. Altered expression of RCA genes has been demonstrated at both the functional and genetic level, but the mechanisms underlying their regulation are not fully characterised. We aimed to investigate the structural organisation of the RCA gene cluster to identify key regulatory elements that influence the expression of CR2 and other genes in this immunomodulatory region. Using 4C, we captured extensive CTCF-mediated chromatin looping across the RCA gene cluster in B cells and showed these were organised into two topologically associated domains (TADs). Interestingly, an inter-TAD boundary was located within the CR1 gene at a well-characterised segmental duplication. Additionally, we mapped numerous gene-gene and gene-enhancer interactions across the region, revealing extensive co-regulation. Importantly, we identified an intergenic enhancer and functionally demonstrated this element upregulates two RCA members (CR2 and CD55) in B cells. We have uncovered novel, long-range mechanisms whereby autoimmune disease susceptibility may be influenced by genetic variants, thus highlighting the important contribution of chromatin topology to gene regulation and complex genetic disease.

Laboratory-Developed MLPA® Probes Enable CNV Detection in the CFHR4 Gene to Aid aHUS Genetic Evaluation

Large copy number variants (CNVs) such as deletions in the regulator of complement activation (RCA) gene cluster (CFH, CFHR3, CFHR1, CFHR4, CFHR2, CFHR5) are associated with elevated risk for atypical hemolytic uremic syndrome (aHUS), a complement regulation disorder (Zipfel 2007). Of particular interest are compound heterozygous CFHR3-CFHR1 and CFHR1-CFHR4 deletions, linked to aHUS due to the resulting composite homozygous CFHR1 deletions (Moore 2010). Detection of CFHR4 CNVs is important as it enables identification of said CFHR3-CFHR1 deletion in trans with the CFHR1-CFHR4 deletion, as well as rare reported CFHR4 duplications (Sudmant 2010). Large heterozygous deletions or duplications involving CFHR3 and CFHR1 or CFHR1 and CFHR4 are undetectable by Sanger sequencing and difficult to detect with amplicon-based Next-Generation Sequencing (NGS). When CNVs are detected by NGS, confirmation with an independent reference method is required by regulatory agencies including the New York State Department of Health and College of American Pathology.Multiplex Ligation-Dependent Probe Amplification (MLPA®) has been used to reliably detect large CNVs for CFH, CFHR3, CFHR1, CFHR2 and CFHR5, and a commercial research use only (RUO) probe mix is available for this purpose from MRC-Holland (Amsterdam, NL).For the CFHR4 region, however, MLPA® probes are not available from any commercial source. Furthermore, CFHR4 MLPA® probe design is complicated by high internal and external homology with adjacent RCA regions (including 91.3-96.2% homology within CFHR4 exons, as well as 90-100% homology with CFHR3 exons). Using a combination of MLPA® probe design freeware, commercial software, literature sources and in-house modifications, a multiplex MLPA® laboratory-developed test (LDT) targeting 5 regions of interest (ROIs) in the CFHR4 gene was designed and validated.The multiplex CFHR4 MLPA® LDT was customized to include an internally-designed ROI MLPA® probe mix targeting CFHR4 intron 1, exon 2, exon 5, intron 6, intron 9, as well as commercially available MLPA® quality control and reference probes. Exon 2 probe sequences were obtained from the literature (Kubista 2011). The remaining MLPA® ROI probes were designed using AlleleID® software (Premier Biosoft) or online freeware MAPD-MLPA (courtesy Stony Brook University Bioinformatics). The MLPA® CFHR4 ROI probes were optimized and challenged to achieve complete specificity for and coverage across the CFHR4 region, as well as to prevent overlap with quality control and reference probes.The CFHR4 MLPA® LDT was validated with 10 genomic DNA samples, comprising a mixture of healthy donors or patients (BloodCenter of Wisconsin archives) and 6 samples from Coriell Repositories (Camden, NJ), all of which were known to harbor large RCA CNVs. 100% concordance with expected findings was observed.Full aHUS genetic evaluation comprising NGS analysis of 15 genes and MLPA® analysis of the RCA gene cluster has now been performed for 161 patients. In 95 (59%) of these patients, no large CNV was detected. Forty patients (25%) tested negative for any large CFHR4 variant but did test positive for the CFHR3-CFHR1 heterozygous deletion. 28% of the population are reported heterozygous for this CFHR3-CFHR1 deletion (Moore 2010). Fifteen patients (9%) were positive for homozygous CFHR1 deletions, which are associated with increased risk for development of autoantibody to complement factor H (CFH), as reported by Moore 2010. Using the CFHR4 MLPA® LDT, the latter homozygous CFHR1 deletions were attributed to compound heterozygosity for the CFHR3-CFHR1 and CFHR1-CFHR4 deletions in 3 of the aforesaid 15 patients. The other 12 patients were found to harbor homozygous deletions of CFHR3 and CFHR1. In 3 (1.9%) patients, the CFHR4 MLPA® LDT revealed a CFHR1-CFHR4 heterozygous deletion, similar to the previously reported frequency of 1.4% in aHUS patients (Moore 2010). Finally, 2 patients tested positive for a heterozygous CFHR3 deletion and a CFHR4 duplication, consistent with the presence of a CFHR3-CFHR1 deletion on one allele and CFHR1-CFHR4 duplication on the other allele resulting in a normal CFHR1 copy number.Overall, these data show the importance of CFHR4 MLPA® CNV testing to aid genetic evaluation of suspected aHUS cases, and this testing should be part of the aHUS genetic panel. DisclosuresFriedman:Alexion: Speakers Bureau; NovoNordisk: Consultancy; Shire: Consultancy; CSL Behring: Consultancy.

Pathogenic microRNAs Common to Brain and Retinal Degeneration; Recent Observations in Alzheimer's Disease and Age-Related Macular Degeneration.

Pathogenic microRNAs Common to Brain and Retinal Degeneration; Recent Observations in Alzheimer's Disease and Age-Related Macular Degeneration.

Disease-associated Sequence Variations Congregate in a Polyanion Recognition Patch on Human Factor H Revealed in Three-dimensional Structure

Mutations and polymorphisms in the regulator of complement activation, factor H, have been linked to atypical hemolytic uremic syndrome (aHUS), membranoproliferative glomerulonephritis, and age-related macular degeneration. Many aHUS patients carry mutations in the two C-terminal modules of factor H, which normally confer upon this abundant 155-kDa plasma glycoprotein its ability to selectively bind self-surfaces and prevent them from inappropriately triggering the complement cascade via the alternative pathway. In the current study, the three-dimensional solution structure of the C-terminal module pair of factor H has been determined. A binding site for a fully sulfated heparin-derived tetrasaccharide has been delineated using chemical shift mapping and the C3d/C3b-binding site inferred from sequence comparisons and computational docking. The resultant information allows assessment of the likely consequences of aHUS-associated amino acid substitutions in this critical region of factor H. It is striking that, excepting those likely to perturb the three-dimensional structure, aHUS-associated missense mutations congregate in the polyanion-binding site delineated in this study, thus potentially disrupting a vital mechanism for control of complement on self-surfaces in the microvasculature of the kidney. It is intriguing that a single nucleotide polymorphism predisposing to age-related macular degeneration occupies another region of factor H that harbors a polyanion-binding site.

Regulator of Complement Activation (RCA) Locus in Chicken: Identification of Chicken RCA Gene Cluster and Functional RCA Proteins

A 150-kb DNA fragment, which contains the gene of the chicken complement regulatory protein CREM (formerly named Cremp), was isolated from a microchromosome by screening bacterial artificial chromosome library. Within 100 kb of the cloned region, three complete genes encoding short consensus repeats (SCRs, motifs with tandemly arranged 60 aa) were identified by exon-trap method and 3'- or 5'-RACE. A chicken orthologue of the human gene 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 2, which exists in close proximity to the regulator of complement activation genes in humans and mice, was located near this chicken SCR gene cluster. Moreover, additional genes encoding SCR proteins appeared to be present in this region. Three distinct transcripts were detected in RNA samples from a variety of chicken organs and cell lines. Two novel genes named complement regulatory secretory protein of chicken (CRES) and complement regulatory GPI-anchored protein of chicken (CREG) besides CREM were identified by cloning corresponding cDNA. Based on the predicted primary structures and properties of the expressed molecules, CRES is a secretory protein, whereas CREG is a GPI-anchored membrane protein. CREG and CREM were protected host cells from chicken complement-mediated cytolysis. Likewise, a membrane-bound form of CRES, which was artificially generated, also protected host cells from chicken complement. Taken together, the chicken possesses an regulator of complement activation locus similar to those of the mammals, and the gene products function as complement regulators.

Altered expression of host-encoded complement regulators on human cytomegalovirus-infected cells.

Human cytomegalovirus (HCMV)-infected cells persist in the presence of anti-HCMV antibody, suggesting that HCMV has evolved mechanisms to evade host immune defenses. Insofar as no virus-encoded complement inhibitors have been identified for HCMV, we hypothesized that HCMV infection may alter the expression of host-encoded cell surface complement inhibitors. Herein, we report that cell surface expression of two complement regulator proteins, CD55 and CD46, which are members of the regulators of complement activation (RCA) gene cluster, increased up to eightfold following infection of fibroblasts or glioblastoma cells with HCMV, but not after infection with HSV-1 or adenovirus. However, the cell surface expression of a third complement regulator, CD59, which is not a member of the RCA gene cluster, was not altered during HCMV infection. Functional studies using purified complement components demonstrated that up-regulation of CD55 suppressed the activity of cell-associated C3 convertases on HCMV-infected cells. Furthermore, increased CD55 expression protected infected cells from complement-mediated lysis, an effect which directly correlated with the length of HCMV infection. Increased expression of host-encoded complement regulator proteins may provide protection of HCMV-infected cells from the host immune response in vivo, through increasing the resistance of infected cells to complement-mediated lysis and decreasing the deposition of C3-derived products on the cell surface.

Ordering of the human regulator of complement activation gene cluster on 1q32 by two-colour FISH.

The regulator of complement activation (RCA) gene cluster and PTPRC and REN genes have been mapped to the 1q31-->q32 band interval. We have used two-colour in situ hybridization to determine the chromosomal position of these genes. We report here that HF1/F13B are proximal to the C4BP/MCP genes. We also propose that PTPRC and REN genes map to a region of the RCA gene cluster between the HF1/F13B and C4BP/MCP regions. In summary, we propose the following gene order: centromere-HF1/F13B-PTPRC-REN-C4BP/MCP-telomere.

C4BPAL2: a second duplication of the C4BPA gene in the human RCA gene cluster

We report here the characterization of the C4BPAL2 gene, a new member of the human regulator of complement activation (RCA) gene cluster that arose from the duplication of the gene coding for the A chain of the complement component C4b-binding protein (C4BPA). We postulate that C4BPAL2 is the human homolog of the pig ApoR gene. 21 refs., 3 figs., 1 tab.

C4BPAL1, a Member of the Human Regulator of Complement Activation (RCA) Gene Cluster That Resulted from the Duplication of the Gene Coding for the α-Chain of C4b-Binding Protein

The regulator of complement activation (RCA) gene cluster evolved by multiple gene duplications to produce a family of genes coding for proteins that collectively control the activation of the complement system. We report here the characterization of C4BPAL1, a member of the human RCA gene cluster that arose from the duplication of the C4BPA gene after the separation of the rodent and primate lineages. C4BPAL1 maps 20 kb downstream of the C4BPA gene and is the same 5' to 3' orientation found for all RCA genes characterized thus far. It includes nine exon-like regions homologous to exons 2-8, 11, and 12 of the C4BPA gene. Analysis of the C4BPAL1 sequence suggests that it is currently a pseudogene in humans. However, comparisons between C4BPAL1 and the human and murine C4BPA genes show sequence conservation, which strongly suggests that, for a long period of time, C4BPAL1 has been a functional gene coding for a protein with structural requirements similar to those of the alpha-chain of C4b-binding protein.

Genes for C4b-binding protein ?- and ?-chains (C4BPA andC4BPB) are located on chromosome 1, band 1q32, in humans and on chromosome 13 in rats

C4b-binding protein is involved in the regulation of the complement system. It is a multimeric protein composed of seven identical alpha-chains and a single copy of a unique beta-chain. The latter was identified only recently and its structure determined by cDNA cloning. Both subunits in C4b-binding protein belong to the same superfamily of proteins composed predominantly of tandemly arranged short consensus repeats (SCR) approximately 60 amino acid residues in length. The gene for the human alpha-chain is known to be located in a gene cluster on chromosome 1, band 1q32, which is called the regulators of complement activation (RCA) gene cluster. We have used cDNA probes for both alpha- and beta-chains of human C4b-binding protein to localize their genes with an in situ hybridization technique. We find the genes for both chains to be located on chromosome 1, band 1q32, in the human. This suggests that the beta-chain gene is also a member of the RCA gene cluster and that the alpha- and beta-chain genes are located close to each other. The cDNA probes for the alpha- and beta-chains also were used to screen mouse-rat somatic cell hybrids using Southern blotting to localize their genes in the rat. Both the alpha- and beta-chain genes were shown to be located on chromosome 13 in the rat. These are the second and third genes to be located on rat chromosome 13, and the results suggest that the genes for the alpha- and beta-chains together with the gene for coagulation factor V represent a conserved chromosomal region in rat and man.

A physical map of the human regulator of complement activation gene cluster linking the complement genes CR1, CR2, DAF, and C4BP.

We report the organization of the human genes encoding the complement components C4-binding protein (C4BP), C3b/C4b receptor (CR1), decay accelerating factor (DAF), and C3dg receptor (CR2) within the regulator of complement activation (RCA) gene cluster. Using pulsed field gel electrophoresis analysis these genes have been physically linked and aligned as CR1-CR2-DAF-C4BP in an 800-kb DNA segment. The very tight linkage between the CR1 and the C4BP loci, contrasted with the relative long DNA distance between these genes, suggests the existence of mechanisms interfering with recombination within the RCA gene cluster.

Decay-accelerating factor. Genetic polymorphism and linkage to the RCA (regulator of complement activation) gene cluster in humans.

We have investigated the genetic relationships between the human decay-accelerating factor (DAF) and a group of complement components including the C3b/C4b receptor (CR1), C4-binding protein (C4bp), and factor H (H), to which DAF is structurally and functionally related. CR1, C4bp, and H were previously demonstrated to be encoded by a cluster of closely linked genes, which we have designated regulator of complement activation (RCA). Southern blot analysis of genomic DNA using a DAF cDNA probe unraveled the existence of restriction fragment length polymorphism (RFLP) for both Bam HI and Hind III restriction endonucleases. Segregation analysis of these polymorphic fragments in families informative for the segregation of alleles at the CR1, C4BP, and H loci (RCA-haplotypes), demonstrated that, in humans, the gene encoding DAF is located within the RCA gene cluster. No recombinants between DAF and C4BP/CR1 were encountered in 32 informative meioses. In addition, in two individuals showing recombination between the CR1/C4BP and H loci, DAF segregated with the CR1/C4BP segment. Thus, the DAF gene maps closer to the CR1/C4BP loci than to the H gene, from which it can be separated by genetic recombination.