The complement system and human autoimmune diseases

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

Gene-resolution analysis of DNA copy number variation using oligonucleotide expression microarrays

<p>Abstract</p> <p>Background</p> <p>Array-based comparative genomic hybridization (aCGH) is a high-throughput method for measuring genome-wide DNA copy number changes. Current aCGH methods have limited resolution, sensitivity and reproducibility. Microarrays for aCGH are available only for a few organisms and combination of aCGH data with expression data is cumbersome.</p> <p>Results</p> <p>We present a novel method of using commercial oligonucleotide expression microarrays for aCGH, enabling DNA copy number measurements and expression profiles to be combined using the same platform. This method yields aCGH data from genomic DNA without complexity reduction at a median resolution of approximately 17,500 base pairs. Due to the well-defined nature of oligonucleotide probes, DNA amplification and deletion can be defined at the level of individual genes and can easily be combined with gene expression data.</p> <p>Conclusion</p> <p>A novel method of gene resolution analysis of copy number variation (graCNV) yields high-resolution maps of DNA copy number changes and is applicable to a broad range of organisms for which commercial oligonucleotide expression microarrays are available. Due to the standardization of oligonucleotide microarrays, graCNV results can reliably be compared between laboratories and can easily be combined with gene expression data using the same platform.</p

Biomarkers of lupus nephritis determined by serial urine proteomics

Lupus nephritis is a frequent and serious complication of systemic lupus erythematosus (SLE), the treatment of which often requires the use of immunosuppressives that can have severe side effects. Here we determined the low-molecular weight proteome of serial lupus urine samples to uncover novel and predictive biomarkers of SLE renal flare. Urine from 25 flare cycles of 19 patients with WHO Class III, IV, and V SLE nephritis were obtained at baseline, pre-flare, flare and post-flare. Each sample was first fractionated to remove proteins larger than 30kDa, then applied onto weak cation exchanger protein chips for analysis by SELDI-TOF mass spectrometry. We found 176 protein ions of which 27 were differentially expressed between specific flare intervals. On-chip peptide sequencing by integrated tandem mass spectrometry positively identified the 20 and 25 amino-acid isoforms of hepcidin, as well as fragments of α1-antitrypsin and albumin among the selected differentially expressed protein ions. Hepcidin 20 increased 4 months before renal flare and returned to baseline at renal flare, whereas hepcidin 25 decreased at renal flare and returned to baseline 4 months after the flare. These studies provide a beginning proteomic analysis aimed at predicting impending renal relapse, relapse severity, and the potential for recovery after SLE nephritis flare

Association of genetic variants in complement factor H and factor H-related genes with systemic lupus erythematosus susceptibility

Systemic lupus erythematosus (SLE), a complex polygenic autoimmune disease, is associated with increased complement activation. Variants of genes encoding complement regulator factor H (CFH) and five CFH-related proteins (CFHR1-CFHR5) within the chromosome 1q32 locus linked to SLE, have been associated with multiple human diseases and may contribute to dysregulated complement activation predisposing to SLE. We assessed 60 SNPs covering the CFH-CFHRs region for association with SLE in 15,864 case-control subjects derived from four ethnic groups. Significant allelic associations with SLE were detected in European Americans (EA) and African Americans (AA), which could be attributed to an intronic CFH SNP (rs6677604, in intron 11, Pmeta = 6.6×10-8, OR = 1.18) and an intergenic SNP between CFHR1 and CFHR4 (rs16840639, Pmeta = 2.9×10-7, OR = 1.17) rather than to previously identified disease-associated CFH exonic SNPs, including I62V, Y402H, A474A, and D936E. In addition, allelic association of rs6677604 with SLE was subsequently confirmed in Asians (AS). Haplotype analysis revealed that the underlying causal variant, tagged by rs6677604 and rs16840639, was localized to a ~146 kb block extending from intron 9 of CFH to downstream of CFHR1. Within this block, the deletion of CFHR3 and CFHR1 (CFHR3-1Δ), a likely causal variant measured using multiplex ligation-dependent probe amplification, was tagged by rs6677604 in EA and AS and rs16840639 in AA, respectively. Deduced from genotypic associations of tag SNPs in EA, AA, and AS, homozygous deletion of CFHR3-1Δ (Pmeta = 3.2×10-7, OR = 1.47) conferred a higher risk of SLE than heterozygous deletion (Pmeta = 3.5×10-4, OR = 1.14). These results suggested that the CFHR3-1Δ deletion within the SLE-associated block, but not the previously described exonic SNPs of CFH, might contribute to the development of SLE in EA, AA, and AS, providing new insights into the role of complement regulators in the pathogenesis of SLE

The gene structure and the polymorphism of the human complement component C4

1. The DNA sequence of the human complement C4A gene from a cosmid clone Cos 3A3 was determined and the complete exon-intron structure elucidated. The 5' flanking region of the C4 gene contains three TATA sequences and a transcriptional enhancer core sequence, which are &gt;200 nucleotides (nt) and 60-70 nt upstream from the CAP site, respectively. The gene consists of 42 exons coding for a precursor protein of 1745 residues. The first exon codes for a 51 nt 5' untranslated sequence, a leader peptide of 19 residues, and the N-terminus of the β chain. The β-α and the α-γ chain junctions are encoded by exons 17 and 34, respectively. The anaphylatoxin C4a and the thiolester site are encoded by phase 1-1 symmetrical exons. Most of the amino acids encoded at the splice junctions are polar or charged. Between exons 10 and 11 is a 6-7 kb intron that is flanked by direct long terminal repeats and may be absent in some C4 genes located at the second C4 locus. The last exon codes for the C-terminus of the γ chain and a 140 bp 3' untranslated sequence. The intergenic region between the C4 gene and its neighbouring 21-hydroxylase (210Hase) gene is ~3028 bp. 2. Eighteen polymorphic amino acids on C4 have been identified through genomic DNA, cDNA and protein sequencing. Fourteen of them are located on the* chain (C4a: 2 changes; C4d: 12 changes). The rest are scattered on the β and the γ chains. There are potential size variations by one residue on the β chain, and by a tripeptide that contains a sulphation site on the α chain. 3. Four common and rare C4 alleles have been cloned from individuals whose C4 proteins were chemically and serologically characterised. Analysis of the sequences at the C4d regions has allowed the identification of the C4A/C4B isotypic residues at positions 1101-6: C4A has the sequence PCPVLD, while C4B has the sequence LSPVIH. Presumably these isotypic residues are the cause of the class-specific, differential chemical reactivates. Moreover, the probable locations for the two Eodgers (Kg) and the six Chido (Ch) antigenic determinants were deduced. The C4B isotypic residues may be involved in the expression of the Ch2 and the Ch4 epitopes, while the C4A isotypic residues may not be related to either of the Eg determinants. 4. Definitive restriction fragment length polymorphisms (RFLPs) representing the exact locations responsible for the isotypicity between C4A and C4B, and for their generally associated Rg1 and Ch1 antigenic determinants, have been designed. In combination with the Taq I polymorphic patterns specific for the C4 and for the 210Hase gene loci, it has been shown that the null allele of the HLA haplotype B44 DR6 C4A 3 C4B QO is not a C4B allele, but probably encodes another C4A 3 allotype at the second C4 locus.</p

An RNA Metabolism and Surveillance Quartet in the Major Histocompatibility Complex

At the central region of the mammalian major histocompatibility complex (MHC) is a complement gene cluster that codes for constituents of complement C3 convertases (C2, factor B and C4). Complement activation drives the humoral effector functions for immune response. Sandwiched between the genes for serine proteinase factor B and anchor protein C4 are four less known but critically important genes coding for essential functions related to metabolism and surveillance of RNA during the transcriptional and translational processes of gene expression. These four genes are NELF-E (RD), SKIV2L (SKI2W), DXO (DOM3Z) and STK19 (RP1 or G11) and dubbed as NSDK. NELF-E is the subunit E of negative elongation factor responsible for promoter proximal pause of transcription. SKIV2L is the RNA helicase for cytoplasmic exosomes responsible for degradation of de-polyadenylated mRNA and viral RNA. DXO is a powerful enzyme with pyro-phosphohydrolase activity towards 5&prime; triphosphorylated RNA, decapping and exoribonuclease activities of faulty nuclear RNA molecules. STK19 is a nuclear kinase that phosphorylates RNA-binding proteins during transcription. STK19 is also involved in DNA repair during active transcription and in nuclear signal transduction. The genetic, biochemical and functional properties for NSDK in the MHC largely stay as a secret for many immunologists. Here we briefly review the roles of (a) NELF-E on transcriptional pausing; (b) SKIV2L on turnover of deadenylated or expired RNA 3&prime;&rarr;5&prime; through the Ski-exosome complex, and modulation of inflammatory response initiated by retinoic acid-inducible gene 1-like receptor (RLR) sensing of viral infections; (c) DXO on quality control of RNA integrity through recognition of 5&prime; caps and destruction of faulty adducts in 5&prime;&rarr;3&prime; fashion; and (d) STK19 on nuclear protein phosphorylations. There is compelling evidence that a dysregulation or a deficiency of a NSDK gene would cause a malignant, immunologic or digestive disease

Muscle MRI at the time of questionable disease flares in Juvenile Dermatomyositis (JDM)

Abstract Background The course of JDM has improved substantially over the last 70 years with early and aggressive treatments. Yet it remains difficult to detect disease flares as symptoms may be mild; signs of rash and muscle weakness vary widely and are often equivocal; laboratory tests of muscle enzyme levels are often normal; electromyography and muscle biopsy are invasive. Alternative tools are needed to help decide if more aggressive treatment is needed. Our objective is to determine the effectiveness of muscle Magnetic Resonance Imaging (MRI) in detecting JDM flares, and how an MRI affects physician’s decision-making regarding treatment. Methods This study was approved by the Institutional Review Board of Nationwide Children’s Hospital. JDM patients were consulted between 1/2005 and 6/2015. MRIs were performed on both lower extremities without contrast sequentially: axial T1, axial T2 fat saturation, axial and coronal inversion recovery, and axial diffusion weighted. The physician decision that a JDM patient was in a flare was considered the gold standard. MRI results were compared with physician’s decisions on whether a relapse had occurred, and if there was a concordance between the assessment methods. Results Forty-five JDM patients were studied. Eighty percent had weakness at diagnosis, 100% typical rash, and 73% typical nail-fold capillary changes. At diagnosis, muscle enzymes were compatible with JDM generally (CK 52%, LDH 62%, aldolase 72%, AST 54% abnormal). EMG was abnormal in 3/8, muscle biopsy typical of JDM in 10/11, and MRI abnormal demonstrating myositis in 31/40. Thirteen patients had a repeat MRI for possible flares with differing indications. Three repeat MRI’s were abnormal, demonstrating myositis. There was moderate agreement about flares between MRI findings and physician’s treatment decisions (kappa = 0.59). In each abnormal MRI case the physician decided to increase treatment (100% probability for flares). MRI was negative for myositis in 10 patients, by which 7/10 the physicians chose to continue or to taper the medications (70% probability for non-flares). Conclusion A muscle MRI would facilitate objective assessments of JDM flares. When an MRI shows myositis, physicians tend to treat 100% of the time. When an MRI shows no myositis, physicians continued the same medications or tapered medications 70% of the time. Further studies would help confirm the utility and cost-effectiveness of MRI to determine JDM flares