Preferential binding to Elk-1 by SLE-associated IL10 risk allele upregulates IL10 expression.

Immunoregulatory cytokine interleukin-10 (IL-10) is elevated in sera from patients with systemic lupus erythematosus (SLE) correlating with disease activity. The established association of IL10 with SLE and other autoimmune diseases led us to fine map causal variant(s) and to explore underlying mechanisms. We assessed 19 tag SNPs, covering the IL10 gene cluster including IL19, IL20 and IL24, for association with SLE in 15,533 case and control subjects from four ancestries. The previously reported IL10 variant, rs3024505 located at 1 kb downstream of IL10, exhibited the strongest association signal and was confirmed for association with SLE in European American (EA) (P = 2.7×10⁻8, OR = 1.30), but not in non-EA ancestries. SNP imputation conducted in EA dataset identified three additional SLE-associated SNPs tagged by rs3024505 (rs3122605, rs3024493 and rs3024495 located at 9.2 kb upstream, intron 3 and 4 of IL10, respectively), and SLE-risk alleles of these SNPs were dose-dependently associated with elevated levels of IL10 mRNA in PBMCs and circulating IL-10 protein in SLE patients and controls. Using nuclear extracts of peripheral blood cells from SLE patients for electrophoretic mobility shift assays, we identified specific binding of transcription factor Elk-1 to oligodeoxynucleotides containing the risk (G) allele of rs3122605, suggesting rs3122605 as the most likely causal variant regulating IL10 expression. Elk-1 is known to be activated by phosphorylation and nuclear localization to induce transcription. Of interest, phosphorylated Elk-1 (p-Elk-1) detected only in nuclear extracts of SLE PBMCs appeared to increase with disease activity. Co-expression levels of p-Elk-1 and IL-10 were elevated in SLE T, B cells and monocytes, associated with increased disease activity in SLE B cells, and were best downregulated by ERK inhibitor. Taken together, our data suggest that preferential binding of activated Elk-1 to the IL10 rs3122605-G allele upregulates IL10 expression and confers increased risk for SLE in European Americans.

Increased frequency of complement C4B deficiency in rheumatoid arthritis.

OBJECTIVE: To assess the copy number variation of complement C4A and C4B genes in patients with rheumatoid arthritis (RA). METHODS: 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. RESULTS: 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. CONCLUSION: 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.

Sex-specific association of X-linked Toll-like receptor 7 (TLR7) with male systemic lupus erythematosus.

Systemic lupus erythematosus (SLE) is a multisystem, autoimmune disease that predominantly affects women. Previous findings that duplicated Toll-like receptor 7 (Tlr7) promotes lupus-like disease in male BXSB mice prompted us to evaluate TLR7 in human SLE. By using a candidate gene approach, we identified and replicated association of a TLR7 3'UTR SNP, rs3853839 (G/C), with SLE in 9,274 Eastern Asians (P(combined) = 6.5 x 10(-10)), with a stronger effect in male than female subjects [odds ratio, male vs. female = 2.33 (95% CI = 1.64-3.30) vs. 1.24 (95% CI = 1.14-1.34); P = 4.1 x 10(-4)]. G-allele carriers had increased TLR7 transcripts and more pronounced IFN signature than C-allele carriers; heterozygotes had 2.7-fold higher transcripts of G-allele than C-allele. These data established a functional polymorphism in type I IFN pathway gene TLR7 predisposing to SLE, especially in Chinese and Japanese male subjects.

Gene CNVs and protein levels of complement C4A and C4B as novel biomarkers for partial disease remissions in new-onset type 1 diabetes patients.

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 ß-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 ß-cell function in new-onset T1D; C4B is correlated with the end of disease remission at 9-month post diagnosis.

Association of a functional IRF7 variant with systemic lupus erythematosus.

OBJECTIVE: A previous genome-wide association study conducted in a population of European ancestry identified rs4963128, a KIAA1542 single-nucleotide polymorphism (SNP) 23 kb telomeric to IRF7 (the gene for interferon regulatory factor 7 [IRF-7]), to be strongly associated with systemic lupus erythematosus (SLE). This study was undertaken to investigate whether genetic polymorphism within IRF7 is a risk factor for the development of SLE. METHODS: We genotyped one KIAA1542 SNP (rs4963128) and one IRF7 SNP (rs1131665 [Q412R]) in an Asian population (1,302 cases, 1,479 controls), to assess their association with SLE. Subsequently, rs1131665 was further genotyped in independent panels of Chinese subjects (528 cases, 527 controls), European American subjects (446 cases, 461 controls), and African American subjects (159 cases, 115 controls) by TaqMan genotyping assay, to seek confirmation of association in various ethnic groups. A luciferase reporter assay was used to assess the effect of Q412R polymorphism on the activation of IRF-7. RESULTS: Consistent association of rs1131665 (Q412R) with SLE was identified in Asian, European American, and African American populations (total 2,435 cases and 2,582 controls) (P(meta) = 6.18 × 10(-6) , odds ratio 1.42 [95% confidence interval 1.22-1.65]). Expression of the IRF7 412Q risk allele resulted in a 2-fold increase in interferon-stimulated response element transcriptional activity compared with expression of IRF7 412R (P = 0.0003), suggesting that IRF7 412Q confers elevated IRF-7 activity and may therefore affect a downstream interferon pathway. CONCLUSION: These findings show that the major allele of a nonsynonymous SNP, rs1131665 (412Q) in IRF7, confers elevated activation of IRF-7 and predisposes to the development of SLE in multiple ethnic groups. This result provides direct genetic evidence that IRF7 may be a risk gene for human SLE.

A dinucleotide deletion in CD24 confers protection against autoimmune diseases.

It is generally believed that susceptibility to both organ-specific and systemic autoimmune diseases is under polygenic control. Although multiple genes have been implicated in each type of autoimmune disease, few are known to have a significant impact on both. Here, we investigated the significance of polymorphisms in the human gene CD24 and the susceptibility to multiple sclerosis (MS) and systemic lupus erythematosus (SLE). We used cases/control studies to determine the association between CD24 polymorphism and the risk of MS and SLE. In addition, we also considered transmission disequilibrium tests using family data from two cohorts consisting of a total of 150 pedigrees of MS families and 187 pedigrees of SLE families. Our analyses revealed that a dinucleotide deletion at position 1527 approximately 1528 (P1527(del)) from the CD24 mRNA translation start site is associated with a significantly reduced risk (odds ratio = 0.54 with 95% confidence interval = 0.34-0.82) and delayed progression (p = 0.0188) of MS. Among the SLE cohort, we found a similar reduction of risk with the same polymorphism (odds ratio = 0.38, confidence interval = 0.22-0.62). More importantly, using 150 pedigrees of MS families from two independent cohorts and the TRANSMIT software, we found that the P1527(del) allele was preferentially transmitted to unaffected individuals (p = 0.002). Likewise, an analysis of 187 SLE families revealed the dinucleotide-deleted allele was preferentially transmitted to unaffected individuals (p = 0.002). The mRNA levels for the dinucleotide-deletion allele were 2.5-fold less than that of the wild-type allele. The dinucleotide deletion significantly reduced the stability of CD24 mRNA. Our results demonstrate that a destabilizing dinucleotide deletion in the 3' UTR of CD24 mRNA conveys significant protection against both MS and SLE.

T cells and in situ cryoglobulin deposition in the pathogenesis of lupus nephritis.

We discuss a 53-year-old woman with systemic lupus erythematosus who presented with vasculitis, hypocomplementemia and nephritis. Although her serum complement 4 (C4) levels were zero, she had four copies of C4 gene. Renal biopsy revealed membranoproliferative glomerulonephritis and the presence of cryoglobulins, detected by electron microscopy, and significant numbers of T cells in the interstitium. Cryoglobulins were considered responsible for the complete consumption of C4 in the serum the levels of which improved gradually after treatment. T cells in the kidney were found to express CD44 and phosphorylated ezrin/radixin/moiesin which explain why they homed to the kidney inappropriately. The contribution of cryoglobulins and T cells in the expression of kidney pathology is discussed.

Cloning and functional analysis of the swine eNOS promoter.

We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5' flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5' RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5' deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from -227 to -82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from -227 to -1290, while negative cis-regulatory elements may be present from -1290 to -1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at -68/-59 was involved in the formation of these complexes.

A polymorphism in the type one complement receptor (CR1) involves an additional cysteine within the C3b/C4b binding domain that inhibits ligand binding.

The type one complement receptor (CR1) contains a variable number of binding domains for C3b and C4b, formed through a nearly identical set of repeating units known as short consensus repeats (SCRs). Each SCR contains four cysteines that, by forming two disulfide bonds, impart a conformation critical for function. In this study, we identified a CR1 single nucleotide polymorphism (1597C>T) that results in an additional cysteine (483R>C) in SCR 8 of the N-terminal C3b/C4b binding domain, and occurring sporadically in corresponding SCRs of other repeated C3b/C4b binding domains. The normal carrier frequency for 483-C was 6.3% in 175 African Americans, and 2.4% in 153 Caucasians. In expression constructs containing one C3b/C4b binding domain, the 483-C residue reduced binding to C3b, C3bi, and C4b by over 80% (each p<0.0001), versus the wildtype construct. Full-length CR1 from 483-C carriers also exhibited reduced binding to C3b and C4b, although the effect was influenced by the total number of binding domains present. Race-matched comparisons between SLE patients (86 African Americans, 228 Caucasians) and the normal cohort showed that 483-C carrier status alone is not a risk factor for SLE or lupus nephritis. The physiological role of this polymorphism remains to be determined.

The HLA 8.1 ancestral haplotype is strongly linked to the C allele of -429T>C promoter polymorphism of receptor of the advanced glycation endproduct (RAGE) gene. Haplotype-independent association of the -429C allele with high hemoglobinA1C levels in diabetic patients.

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.

Three distinct profiles of serum complement C4 proteins in pediatric systemic lupus erythematosus (SLE) patients: tight associations of complement C4 and C3 protein levels in SLE but not in healthy subjects.

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

Dancing with complement C4 and the RP-C4-CYP21-TNX (RCCX) modules of the major histocompatibility complex.

The number of the complement component C4 genes varies from 2 to 8 in a diploid genome among different human individuals. Three quarters of the C4 genes in Caucasian populations have the endogenous retrovirus, HERV-K(C4), in the ninth intron. The remainder does not. The C4 serum proteins are highly polymorphic and their concentrations vary from 100 to approximately 1000 microg/ml. There are two distinct classes of C4 protein, C4A and C4B, which have diversified to fulfill (a) the opsonization/immunoclearance purposes and (b) the well-known complement function in the killing of microbes by lysis and neutralization, respectively. Many infectious and autoimmune diseases are associated with complete or partial deficiency of C4A and/or C4B. The adverse effects of high C4 gene dosages, however, are just emerging, as the concepts of human C4 genetics are revised and accurate techniques are applied to distinguish partial deficiencies from differential expression caused by unequal C4A and C4B gene dosages and gene sizes. This review attempts to dissect the sophisticated genetics of complement C4A and C4B. The emphases are on the qualitative and quantitative diversities of C4 genotypes and phenotypes. The many allotypic variants and the processed products of human and mouse C4 proteins are described. The modular variation of C4 genes together with the serine/threonine nuclear kinase gene RP, the steroid 21-hydroxylase CYP21, and extracellular matrix protein TNX (RCCX modules) are investigated for the effects on homogenization of C4 protein polymorphisms, and on the unequal genetic crossovers that knocked out the functions of CYP21 and/or TNX. Furthermore, the influence of the endogenous retrovirus HERV-K(C4) on C4 gene expression and the dispersal of HERV-K(C4) family members in the human genome are discussed.

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.

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

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

The intricate role of complement component C4 in human systemic lupus erythematosus.

It was observed about 50 years ago that low serum complement activity or low protein concentrations of complement C4 concurred with disease activities of systemic lupus erythematosus (SLE). Complete deficiencies of complement components C4A and C4B, albeit rare in human populations, are among the strongest genetic risk factors for SLE or lupus-like disease, across HLA haplotypes and racial backgrounds. However, whether heterozygous or partial deficiency of C4A (C4AQ0) or C4B (C4BQ0) is a predisposing factor for SLE has been a highly controversial topic. In this review we critically analyzed past epidemiologic studies on deficiency of C4A or C4B in human SLE. Cumulative results from more than 35 different studies revealed that heterozygous and homozygous deficiencies of C4A were present in 40-60% of SLE patients from almost all ethnic groups or races investigated, which included northern and central Europeans, Anglo-Saxons, Caucasians in the US, African Americans, Asian Chinese, Koreans and Japanese. In addition, French SLE and control populations had relatively low frequencies of C4AQ0, but the difference between the patient and control groups was statistically significant. The relative risk of C4AQ0 in SLE varied between 2.3 and 5.3 among different ethnic groups. In Caucasian and African SLE patients, the two major causes for C4AQ0 are (1) the presence of a mono-S RCCX (RP-C4-CYP21-TNX) module with a single, short C4B gene in the major histocompatibility complex; and (2) a 2-bp insertion into the sequence for codon 1213 at exon 29 of the mutant C4A gene. Both mono-S structures and 2-bp insertion in exon 29 are absent or extremely rare in the C4AQ0 of Oriental SLE patients. The highly significant association of C4AQ0 with SLE across multiple HLA haplotypes and ethnic groups, and the presence of different mechanisms leading to a C4A protein deficiency among SLE patients suggested that deficiency or low expression level of C4A protein is a primary risk factor for SLE disease susceptibility per se. On the other hand, Spanish, Mexican, Australian Aborigine SLE patients had increased frequencies of C4B deficiency instead of C4A deficiency. Such observations underscore the importance of both C4A and C4B proteins in the fine control of autoimmunity. Different racial and genetic backgrounds could change the thresholds for the requirement of C4A or C4B protein levels in immune tolerance and immune regulation. Most past epidemiological studies of C4 in human SLE did not consider the polygenic and gene size variations of C4A and C4B. In addition, many studies were overly dependent on phenotypic observations or methods that did not distinguish differential C4A and C4B protein expression caused by unequal gene number or different gene size from the absence of a functional C4A or C4B gene. For further longitudinal studies on clinical manifestations of SLE, it would be informative to stratify the patients with accurately defined C4A and C4B genotypes. Likewise, elucidation of epistatic genetic factors interacting with C4AQ0 would provide important insights into the intricate roles of C4 in SLE disease susceptibility and pathogenesis.

Relationship between complement components C4A and C4B diversities and two TNFA promoter polymorphisms in two healthy Caucasian populations.

The RP-C4-CYP21-TNX (RCCX) modules and the tumor necrosis factor (TNF) gene cluster are probably the most polymorphic genomic regions in the human central major histocompatibility complex (MHC). Using definitive methods for genotypic and phenotypic analyses of complement components C4A and C4B, determination of the RCCX length variants, and SSP-PCR/RFLP analyses of TNFA promoter polymorphisms at positions -308 and -238, we studied the complex relationships between the C4 and TNFA polymorphisms in two normal Caucasian populations. The patterns of the RCCX modular structures and the allelic frequency of -308A TNFA (TNF2) were similar between the Budapest (n = 125) and the Ohio (n = 80) Caucasians. However, the frequency of the -238A allele was significantly higher in the Ohio (11.3%) than in the Budapest (1.6%) study population (p < 0.0001). Marked features were found in the RCCX length variants in the TNF2 carriers and noncarriers. Strong associations were found between the C4AQ0 B1 haplotype from the monomodular short (mono-S) RCCX structure and the TNF2 allele, and between the C4A6 B1 haplotype from the bimodular long-short (LS) structure of the RCCX and the TNFA -238A allele. However, 36%-46% of the TNF2 carriers did not associate with a mono-S in both study cohorts, and 57.1% of the TNFA -238A carriers in Ohio did not associate with C4A6, which has a defective complement C5 convertase activity. The carriers of TNF2 allele had significantly lower C4A serum concentration (0.17 +/- 0.08 g/l) than noncarriers (0.23 +/- 0.09 g/l) (p < 0.001). The lowest C4A serum levels were found in TNF2 carriers with mono-S structures (0.14 +/- 0.06 g/l). In essence, our results demonstrated the heterogeneities of the TNFA promoter polymorphisms, and the linkage disequilibrium of TNFA -308A and -238A alleles with complement C4A deficiency and impaired C4A protein function, respectively.

Great genotypic and phenotypic diversities associated with copy-number variations of complement C4 and RP-C4-CYP21-TNX (RCCX) modules: a comparison of Asian-Indian and European American populations.

Inter-individual gene copy-number variations (CNVs) probably afford human populations the flexibility to respond to a variety of environmental challenges, but also lead to differential disease predispositions. We investigated gene CNVs for complement component C4 and steroid 21-hydroxylase from the RP-C4-CYP21-TNX (RCCX) modules located in the major histocompatibility complex among healthy Asian-Indian Americans (AIA) and compared them to European Americans. A combination of definitive techniques that yielded cross-confirmatory results was used. The medium gene copy-numbers for C4 and its isotypes, acidic C4A and basic C4B, were 4, 2 and 2, respectively, but their frequencies were only 53-56%. The distribution patterns for total C4 and C4A are skewed towards the high copy-number side. For example, the frequency of AIA-subjects with three copies of C4A (30.7%) was 3.92-fold of those with a single copy (7.83%). The monomodular-short haplotype with a single C4B gene and the absence of C4A, which is in linkage-disequilibrium with HLA DRB1*0301 in Europeans and a strong risk factor for autoimmune diseases, has a frequency of 0.012 in AIA but 0.106 among healthy European Americans (p=6.6x10(-8)). The copy-number and the size of C4 genes strongly determine the plasma C4 protein concentrations. Parallel variations in copy-numbers of CYP21A (CYP21A1P) and TNXA with total C4 were also observed. Notably, 13.1% of AIA-subjects had three copies of the functional CYP21B, which were likely generated by recombinations between monomodular and bimodular RCCX haplotypes. The high copy-numbers of C4 and the high frequency of RCCX recombinants offer important insights to the prevalence of autoimmune and genetic diseases.

Biomarker discovery in human SLE nephritis.

The treatment of systemic lupus erythematosus (SLE) nephritis, while effective, is associated with significant morbidity and mortality. These side effects could be mitigated if the onset, severity, and response of renal flare could be predicted, and therapy modified accordingly. In this review, an approach to derive prediction equations of SLE nephritis flare is discussed. Integral to generating such prediction equations is the identification of biomarkers of lupus nephritis that can serve as input variables for modeling flare. The use of urine as a source of SLE nephritis biomarkers is described, and the results of urine biomarker discovery studies using candidate and proteomic approaches are presented.

Sensitive and specific real-time polymerase chain reaction assays to accurately determine copy number variations (CNVs) of human complement C4A, C4B, C4-long, C4-short, and RCCX modules: elucidation of C4 CNVs in 50 consanguineous subjects with defined HLA genotypes.

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.

Frequency of carriers of 8.1 ancestral haplotype and its fragments in two Caucasian populations.

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.