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Introduction

Inherited deficiencies of several complement components strongly predispose to systemic lupus erythematosus (SLE) while deficiencies of complement inhibitors are found in kidney diseases such as atypical hemolytic uremic syndrome (aHUS).

Methods

The exons of complement inhibitor genes CD46 and CFH (factor H) were fully sequenced using the Sanger method in SLE patients with nephritis originating from two cohorts from southern and mid Sweden (n = 196). All identified mutations and polymorphisms were then analyzed in SLE patients without nephritis (n = 326) and in healthy controls (n = 523).

Results

We found nonsynonymous, heterozygous mutations in CFH in 6.1% patients with nephritis, in comparison with 4.0% and 5.4% in patients without nephritis and controls, respectively. No associations of SLE or nephritis with common variants in CFH (V62I/Y402H/E936D) were found. Furthermore, we found two nonsynonymous heterozygous mutations in CD46 in SLE patients but not in controls. The A353V polymorphism, known to affect function of CD46, was found in 6.6% of nephritis patients versus 4.9% and 6.1% of the non-nephritis SLE patients and controls. The presence of mutations in CD46 and CFH did not predispose to SLE or nephritis but was associated with earlier onset of nephritis. Furthermore, we found weak indications that there is one protective and one risk haplotype predisposing to nephritis composed of several polymorphisms in noncoding regions of CD46, which were previously implicated in aHUS.

Conclusions

SLE nephritis is not associated with frequent mutations in CFH and CD46 as found in aHUS but these may be modifying factors causing earlier onset of nephritis.

Dysregulation of the complement system has been linked to pathogenesis of hypertension. However, whether genetic changes of complement factor H (CFH) and its related genes are associated with hypertension is unknown. We genotyped three SNPs in the CFH gene cluster that are closely linked to age-related macular degeneration, namely rs1061170 (Y402H), rs2274700 (A473A) and rs7542235 (CFHR1–3Δ), and tested for their associations with blood pressure and hypertension risk in a population-based cohort including 3,210 unrelated Chinese Hans (50–70 years of age) from Beijing and Shanghai. We found that rs2274700 (A473A) and rs7542235 (CFHR1–3Δ) were both significantly associated with diastolic blood pressure (DBP) (β = 0.632–1.431, P≤0.038) and systolic blood pressure (SBP) (β = 1.567–4.445, P≤0.008), and rs2274700 (A473A) was associated with hypertension risk (OR [95%CI]: 1.175 [1.005–1.373], P = 0.048). Notably, the associations of rs2274700 (A473A) with DBP (P = 2.1×10−3), SBP (P = 8×10−5) and hypertension risk (P = 7.9×10−3) were significant only in the individuals with low CRP levels (<2.0 mg/l), but not in those with CRP levels ≥2.0 mg/l (P≥0.0807) (P for interaction ≤0.0467). However, no significant association between rs1061170 (Y402H) and blood pressure or hypertension risk was observed (P≥0.259). In conclusion, our results suggest that genetic variations in CFH and its related genes may contribute to hypertension risk in Chinese Hans.

Background

Variants in the complement factor H gene (CFH) are associated with age-related macular degeneration (AMD). CFH and five CFH-related genes (CFHR1-5) lie within the regulators of complement activation (RCA) locus on chromosome 1q32.

Aims and Methods

In this study, we refined the structural and evolutionary relationships between these genes and AMD using a combined molecular and immunohistochemical approach.

Results

We identify and characterize a large, common deletion that encompasses both the CFHR1 and CFHR3 genes. CFHR1, an abundant serum protein, is absent in subjects homozygous for the deletion. Analysis of AMD cases and controls from two cohorts demonstrates that deletion homozygotes comprise 1.1% of cases and 5.7% of the controls ( 2=32.8; P=1.6 E-09). CFHR1 and CFHR3 transcripts are abundant in liver, but undetectable in the ocular RPE/choroid complex. AMD-associated CFH/CFHR1/CFHR3 haplotypes are widespread in human populations.

Conclusion

The absence of CFHR1 and/or CFHR3 may account for the protective effects conferred by some CFH haplotypes. Moreover, the high frequencies of the 402H allele and the delCFHR1/CFHR3 alleles in African populations suggest an ancient origin for these alleles. The considerable diversity accumulated at this locus may be due to selection, which is consistent with an important role for the CFHR genes in innate immunity.

Introduction

Membranoproliferative glomerulonephritis type II or dense deposit disease (MPGN II/DDD) causes chronic renal dysfunction that progresses to end stage renal disease in about half of patients within 10 years of diagnosis. Deficiency of and mutations in the complement factor H (CFH) gene are associated with the development of MPGN II/DDD, suggesting that dysregulation of the alternative pathway of the complement cascade is important in disease pathophysiology.

Subjects

Patients with MPGN II/DDD were studied to determine whether specific allele variants of CFH and CFHR5 segregate preferentially with the MPGN II/DDD disease phenotype. The control group was compromised of 131 people in whom age related macular degeneration had been excluded.

Results

Allele frequencies of four single nucleotide polymorphisms in CFH and three in CFHR5 were significantly different between MPGN II/DDD patients and controls.

Conclusion

We have identified specific allele variants of CFH and CFHR5 associated with the MPGN II/DDD disease phenotype. While our data can be interpreted to further implicate complement in the pathogenesis of MPGN II/DDD, these associations could also be unrelated to disease pathophysiology. Functional studies are required to resolve this question.

Highlights

► Hypothesis tested: common sequence variants in complement are associated with aHUS. ► SNPs in complement genes were genotyped in two independent aHUS cohorts. ► Only SNPs within CFH, CD46 and the CFHRs showed a replicable association with aHUS. ► Complement regulators variability has the main role in determining the disease.

It is well established that common genetic variants in CFH, CD46 and the CFHRs are additional risk factors for the development of aHUS. To examine the hypothesis that common variants in other complement genes have a similar effect we genotyped 501 SNPs in 47 complement genes in 94 aHUS patients from Newcastle, 126 aHUS patients from Paris, 374 UK controls and 165 French controls. We replicated the associations in CFH, CD46 and the CFHRs but found no association with any other complement gene. The strongest associations replicated in both cohorts were found for four SNPs within CD46 (p-value < 10−3) and five SNPs within CFH (p-value <5 × 10−3). Significant replicable associations with single SNPs in CFHR2, CFHR4 and an intergenic SNP (CR1–CD46) were also found. Analysis of the Paris cohort showed that the association with CD46 SNPs was only present in those patients with complement mutations. Haplotype analysis showed at-risk and protective haplotypes in both CD46 and CFH. The CD46 haplotype was only disease-associated in those patients with mutations.

Introduction

Complement activation is involved in rheumatoid arthritis (RA), systemic lupus erythematosus (SLE) and atypical hemolytic uremic syndrome (aHUS). Autoantibodies to complement inhibitor factor H (FH), particularly in association with deletions of the gene coding for FH-related protein 1 (CFHR1), are associated with aHUS.

Methods

Autoantibodies against FH, factor I (FI) and C4b-binding protein (C4BP) were measured by ELISA, while CFHR1 homozygous deletion was determined with Western blotting of sera. Epitopes for FH autoantibodies were mapped using recombinant fragments of FH.

Results

FH autoantibodies were detected in SLE (6.7%, n = 60, RA patients (16.5%, n = 97 in the Swedish cohort and 9.2%, n = 217 in the Dutch cohort) and thrombosis patients positive for the lupus anticoagulants (LA+) test (9.4%, n = 64) compared with aHUS patients (11.7%, n = 103). In the control groups (n = 354), an average of 4% of individuals were positive for FH autoantibodies. The frequencies observed in both RA cohorts and LA+ patients were statistically significantly higher than in controls. We also found that an average of 15.2% of the FH-autoantibody positive individuals in all studied disease groups had homozygous deficiency of CFHR1 compared with 3.8% of the FH autoantibody negative patients. The levels of FH autoantibodies varied in individual patients over time. FH autoantibodies found in LA+, SLE and RA were directed against several epitopes across FH in contrast to those found in aHUS, which bound mainly to the C-terminus. Autoantibodies against FI and C4BP were detected in some patients and controls but they were not associated with any of the diseases analyzed in this study.

Conclusions

Autoantibodies against FH are not specific for aHUS but are present at a significant frequency in rheumatic diseases where they could be involved in pathophysiological mechanisms.

Systemic lupus erythematosus (SLE) is an inflammatory autoimmune disease with a strong genetic component. African-Americans (AA) are at increased risk of SLE, but the genetic basis of this risk is largely unknown. To identify causal variants in SLE loci in AA, we performed admixture mapping followed by fine mapping in AA and European-Americans (EA). Through genome-wide admixture mapping in AA, we identified a strong SLE susceptibility locus at 2q22–24 (LOD = 6.28), and the admixture signal is associated with the European ancestry (ancestry risk ratio ∼1.5). Large-scale genotypic analysis on 19,726 individuals of African and European ancestry revealed three independently associated variants in the IFIH1 gene: an intronic variant, rs13023380 [Pmeta = 5.20×10−14; odds ratio, 95% confidence interval = 0.82 (0.78–0.87)], and two missense variants, rs1990760 (Ala946Thr) [Pmeta = 3.08×10−7; 0.88 (0.84–0.93)] and rs10930046 (Arg460His) [Pdom = 1.16×10−8; 0.70 (0.62–0.79)]. Both missense variants produced dramatic phenotypic changes in apoptosis and inflammation-related gene expression. We experimentally validated function of the intronic SNP by DNA electrophoresis, protein identification, and in vitro protein binding assays. DNA carrying the intronic risk allele rs13023380 showed reduced binding efficiency to a cellular protein complex including nucleolin and lupus autoantigen Ku70/80, and showed reduced transcriptional activity in vivo. Thus, in SLE patients, genetic susceptibility could create a biochemical imbalance that dysregulates nucleolin, Ku70/80, or other nucleic acid regulatory proteins. This could promote antibody hypermutation and auto-antibody generation, further destabilizing the cellular network. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.

Author Summary

African-Americans (AA) are at increased risk of systemic lupus erythematosus (SLE), but the genetic basis of this risk increase is largely unknown. We used admixture mapping to localize disease-causing genetic variants that differ in frequency across populations. This approach is advantageous for localizing susceptibility genes in recently admixed populations like AA. Our genome-wide admixture scan identified seven admixture signals, and we followed the best signal at 2q22–24 with fine-mapping, imputation-based association analysis and experimental validation. We identified two independent coding variants and a non-coding variant within the IFIH1 gene associated with SLE. Together with molecular modeling, our results establish a distinct role for IFIH1 in apoptosis, inflammation, and autoantibody production, and explain the molecular basis of these three risk alleles for SLE pathogenesis.

Atypical hemolytic uremic syndrome (aHUS) is a heterogeneous disease that is caused by defective complement regulation in over 50% of cases. Mutations have been identified in genes encoding both complement regulators [complement factor H (CFH), complement factor I (CFI), complement factor H-related proteins (CFHR), and membrane cofactor protein (MCP)], as well as complement activators [complement factor B (CFB) and C3]. More recently, mutations have also been identified in thrombomodulin (THBD), an anticoagulant glycoprotein that plays a role in the inactivation of C3a and C5a. Inhibitory autoantibodies to CFH account for an additional 5–10% of cases and can occur in isolation or in association with mutations in CFH, CFI, CFHR 1, 3, 4, and MCP. Plasma therapies are considered the mainstay of therapy in aHUS secondary to defective complement regulation and may be administered as plasma infusions or plasma exchange. However, in certain cases, despite initiation of plasma therapy, renal function continues to deteriorate with progression to end-stage renal disease and renal transplantation. Recently, eculizumab, a humanized monoclonal antibody against C5, has been described as an effective therapeutic strategy in the management of refractory aHUS that has failed to respond to plasma therapy. Clinical trials are now underway to further evaluate the efficacy of eculizumab in the management of both plasma-sensitive and plasma-resistant aHUS.

Electronic supplementary material

The online version of this article (doi:10.1007/s00467-010-1556-4) contains supplementary material, which is available to authorized users.

Purpose

To investigate the frequency of variants in three major age-related macular degeneration (AMD)-associated loci in patients with polypoidal choroidal vasculopathy (PCV) of European-American descent.

Design

Cross-sectional case-control association study.

Participants

Fifty-five patients with PCV, 368 patients with advanced AMD and 368 age- and ethnically-matched unaffected controls of European-American descent.

Methods

Association analysis of allele and genotype frequencies, determined by TaqMan assays, was performed for the following haplotype-tagging single nucleotide polymorphisms (htSNPs): risk alleles in the complement factor H (CFH) gene (Y402H and IVS14) in the ARMS2/HTRA1 locus on 10q26 (A69S) and protective alleles in CFH (IVS1 and IVS6) and in the complement factor B/complement component C2 (CFB/C2) locus (IVS10 and H9L).

Main Outcome Measures

Allele and genotype frequencies of the htSNPs in the CFH, CFB/C2, and ARMS2/HTRA1 loci.

Results

Four AMD-associated haplotype-tagging alleles (rs547154, rs1061170, rs1410996, rs10490924) in the three major loci, CFH, CFB/C2 and ARMS2/HTRA1, were statistically significantly associated also with the PCV phenotype (P<0.05). Three other alleles from the same loci (rs4151667, rs529825, rs3766404) showed a trend towards association (P<0.2), but did not reach statistical significance possibly because of the combined effects of a relatively small sample size and low minor allele frequency in the screened populations.

Conclusion

The PCV phenotype in Caucasian patients is associated with the major alleles/genotypes in the AMD-associated loci, suggesting that PCV and AMD are genetically similar in the tested loci.

Gene variants in the alternative pathway of the complement system strongly associate with atypical hemolytic uremic syndrome (aHUS), presumably by predisposing to increased complement activation within the kidney. Complement factor H (CFH) is the major regulator of complement activation through the alternative pathway. Factor H-deficient mice transgenically expressing a mutant CFH protein (Cfh−/−.FHΔ16–20) that functionally mimics the CFH mutations reported in aHUS patients spontaneously develop thrombotic microangiopathy. To investigate the role of complement C5 activation in this aHUS model, we generated C5-deficient Cfh−/−.FHΔ16–20 mice. Both C5-sufficient and C5-deficient Cfh−/−.FHΔ16–20 mice had abnormal C3 deposition within the kidney, but spontaneous aHUS did not develop in any of the C5-deficient mice. Furthermore, although Cfh−/−.FHΔ16–20 animals demonstrated marked hypersensitivity to experimentally triggered renal injury, animals with concomitant C5 deficiency did not. These data demonstrate a critical role for C5 activation in both spontaneous aHUS and experimentally triggered renal injury in animals with defective complement factor H function. This study provides a rationale to investigate therapeutic inhibition of C5 in human aHUS.

Borrelia burgdorferi evades complement-mediated killing by interacting with complement regulators through distinct complement regulator-acquiring surface proteins (CRASPs). Here, we extend our analyses to the contribution of CRASP-4 in mediating complement resistance of B. burgdorferi and its interaction with human complement regulators. CRASP-4 (also known as ErpC) was immobilized onto magnetic beads and used to capture proteins from human serum. Following Western blotting, factor H (CFH), CFH-related protein 1 (CFHR1), CFHR2, and CFHR5 were identified as ligands of CRASP-4. To analyze the impact of native CRASP-4 on mediating survival of serum-sensitive cells in human serum, a B. garinii strain was generated that ectopically expresses CRASP-4. CRASP-4-producing bacteria bound CFHR1, CFHR2, and CFHR5 but not CFH. In addition, transformed spirochetes deposited significant amounts of lethal complement components on their surface and were susceptible to human serum, thus indicating that CRASP-4 plays a subordinate role in complement resistance of B. burgdorferi.

Background

Sequence analysis of the regulators of complement activation (RCA) cluster of genes at chromosome position 1q32 shows evidence of several large genomic duplications. These duplications have resulted in a high degree of sequence identity between the gene for factor H (CFH) and the genes for the five factor H-related proteins (CFHL1–5; aliases CFHR1–5). CFH mutations have been described in association with atypical haemolytic uraemic syndrome (aHUS). The majority of the mutations are missense changes that cluster in the C-terminal region and impair the ability of factor H to regulate surface-bound C3b. Some have arisen as a result of gene conversion between CFH and CFHL1. In this study we tested the hypothesis that nonallelic homologous recombination between low-copy repeats in the RCA cluster could result in the formation of a hybrid CFH/CFHL1 gene that predisposes to the development of aHUS.

Methods and Findings

In a family with many cases of aHUS that segregate with the RCA cluster we used cDNA analysis, gene sequencing, and Southern blotting to show that affected individuals carry a heterozygous CFH/CFHL1 hybrid gene in which exons 1–21 are derived from CFH and exons 22/23 from CFHL1. This hybrid encodes a protein product identical to a functionally significant CFH mutant (c.3572C>T, S1191L and c.3590T>C, V1197A) that has been previously described in association with aHUS.

Conclusions

CFH mutation screening is recommended in all aHUS patients prior to renal transplantation because of the high risk of disease recurrence post-transplant in those known to have a CFH mutation. Because of our finding it will be necessary to implement additional screening strategies that will detect a hybrid CFH/CFHL1 gene.

Tim Goodship and colleagues have identified a heterozygousCFH/CFHL1 hybrid gene which encodes a protein product identical to one previously described in association with atypical hemolytic uremic syndrome.

Editors' Summary

Background.

Atypical hemolytic uremic (aHUS) syndrome is a rare, chronic disease that can run in families. People with the condition are prone to developing kidney failure and high blood pressure, and are likely to have a shorter life span than healthy people. Previous work done by a group of researchers in Newcastle-on-Tyne, UK looked at the genetic underpinnings of aHUS in three families suffering from the condition. They found a region of the genome that was linked with the disease in all three families. That region was known to contain a gene for a protein called “factor H,” as well as a number of other genes for proteins that are involved in the same pathway as factor H in controlling an ancient defence system called complement. This system helps antibodies to kill invaders by marking any cell that is not protected by proteins such as factor H. Our own cells would be under constant threat without protective proteins such as factor H. Later studies found simple genetic mutations in people with aHUS, in the genes coding for factor H. However, other work suggested that in some families with aHUS, simple genetic mutations might not be the cause; instead more complicated rearrangements of the genome might occur which would then result in an abnormal factor H that incorporated part of the gene for another protective protein called factor H related protein 1.

Why Was This Study Done?

The researchers knew that it was important to understand the exact genetic mutations linked with aHUS in different families. This was because the exact type of mutation would help them predict whether a kidney transplant is likely to be successful in treating an individual with aHUS who has developed kidney failure. In people with mutations affecting proteins produced by the kidney, a kidney transplant would be likely to work; but in people with mutations affecting factor H, which is produced by the liver, the disease would probably recur after a kidney transplant.

What Did the Researchers Do and Find?

In this study, the researchers went back to one of the three families with aHUS they had previously studied. The researchers had shown before that in this family, the disease was linked with the genome region containing factor H, but no precise mutation in that region had been found. This time, the researchers screened the genome of the family members and looked in particular for a specific rearrangement of the genome that they suspected might be involved. They found that the genomes in this family had been shuffled in the factor H region, resulting in an abnormal version of factor H being produced.

What Do These Findings Mean?

The mutation these researchers identified is likely to result in development of aHUS that does not get better after a kidney transplant, because the abnormal factor H would still be produced in the liver after a transplant had been done. Therefore, the researchers suggest that patients with aHUS be checked for this particular mutation before it is decided whether to go ahead with a transplant.

Additional Information.

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030431.

US National Institutes of Health Office of Rare Diseases information about atypical hemolytic uremic syndrome

The Online Mendelian Inheritance in Man (OMIM) contains an entry on hemolytic uremic syndrome. OMIM is a database of human genes and genetic disorders developed by the US National Center for Biotechnology Information

The US National Kidney and Urologic Diseases has a page about hemolytic uremic syndrome

The Wikipedia has a page about HUS (note that Wikipedia is a free online encyclopedia that anyone can edit)

Copy Number Variants (CNVs) are now recognized as playing a significant role in complex disease etiology. Age-related macular degeneration (AMD) is the most common cause of irreversible vision loss in the western world. While a number of genes and environmental factors have been associated with both risk and protection in AMD, the role of CNVs has remained largely unexplored. We analyzed the two major AMD risk-associated regions on chromosome 1q32 and 10q26 for CNVs using Multiplex Ligation-dependant Probe Amplification. The analysis targeted nine genes in these two key regions, including the Complement Factor H (CFH) gene, the 5 CFH-related (CFHR) genes representing a known copy number “hotspot”, the F13B gene as well as the ARMS2 and HTRA1 genes in 387 cases of late AMD and 327 controls. No copy number variation was detected at the ARMS2 and HTRA1 genes in the chromosome 10 region, nor for the CFH and F13B genes at the chromosome 1 region. However, significant association was identified for the CFHR3-1 deletion in AMD cases (p = 2.38×10−12) OR = 0.31, CI-0.95 (0.23–0.44), for both neovascular disease (nAMD) (p = 8.3×10−9) OR = 0.36 CI-0.95 (0.25–0.52) and geographic atrophy (GA) (p = 1.5×10−6) OR = 0.36 CI-0.95 (0.25–0.52) compared to controls. In addition, a significant association with deletion of CFHR1-4 was identified only in patients who presented with bilateral GA (p = 0.02) (OR = 7.6 CI-0.95 1.38–41.8). This is the first report of a phenotype specific association of a CNV for a major subtype of AMD and potentially allows for pre-diagnostic identification of individuals most likely to proceed to this end stage of disease.

Objective

Interferon-α (IFNα) is a heritable risk factor for systemic lupus erythematosus (SLE). Genetic variation near IRF7 is implicated in SLE susceptibility. SLE-associated autoantibodies can stimulate IFNα production through the Toll-like receptor/IRF7 pathway. This study was undertaken to determine whether variants of IRF7 act as risk factors for SLE by increasing IFNα production and whether autoantibodies are important to this phenomenon.

Methods

We studied 492 patients with SLE (236 African American, 162 European American, and 94 Hispanic American subjects). Serum levels of IFNα were measured using a reporter cell assay, and single-nucleotide polymorphisms (SNPs) in the IRF7/PHRF1 locus were genotyped.

Results

In a joint analysis of European American and Hispanic American subjects, the rs702966 C allele was associated with the presence of anti–double-stranded DNA (anti-dsDNA) antibodies (odds ratio [OR] 1.83, P = 0.0069). The rs702966 CC genotype was only associated with higher serum levels of IFNα in European American and Hispanic American patients with anti-dsDNA antibodies (joint analysis P = 4.1 × 10−5 in anti-dsDNA–positive patients and P = 0.99 in anti-dsDNA–negative patients). In African American subjects, anti-Sm antibodies were associated with the rs4963128 SNP near IRF7 (OR 1.95, P = 0.0017). The rs4963128 CT and TT genotypes were associated with higher serum levels of IFNα only in African American patients with anti-Sm antibodies (P = 0.0012). In African American patients lacking anti-Sm antibodies, an effect of anti-dsDNA–rs702966 C allele interaction on serum levels of IFNα was observed, similar to the other patient groups (overall joint analysis P = 1.0 × 10−6). In European American and Hispanic American patients, the IRF5 SLE risk haplotype showed an additive effect with the rs702966 C allele on IFNα level in anti-dsDNA–positive patients.

Conclusion

Our findings indicate that IRF7/PHRF1 variants in combination with SLE-associated autoantibodies result in higher serum levels of IFNα, providing a biologic relevance for this locus at the protein level in human SLE in vivo.

Jane Salmon and colleagues studied 250 pregnant patients with SLE and/or antiphospholipid antibodies and found an association of risk variants in complement regulatory proteins in patients who developed preeclampsia, as well as in preeclampsia patients lacking autoimmune disease.

Background

Pregnancy in women with systemic lupus erythematosus (SLE) or antiphospholipid antibodies (APL Ab)—autoimmune conditions characterized by complement-mediated injury—is associated with increased risk of preeclampsia and miscarriage. Our previous studies in mice indicate that complement activation targeted to the placenta drives angiogenic imbalance and placental insufficiency.

Methods and Findings

We use PROMISSE, a prospective study of 250 pregnant patients with SLE and/or APL Ab, to test the hypothesis in humans that impaired capacity to limit complement activation predisposes to preeclampsia. We sequenced genes encoding three complement regulatory proteins—membrane cofactor protein (MCP), complement factor I (CFI), and complement factor H (CFH)—in 40 patients who had preeclampsia and found heterozygous mutations in seven (18%). Five of these patients had risk variants in MCP or CFI that were previously identified in atypical hemolytic uremic syndrome, a disease characterized by endothelial damage. One had a novel mutation in MCP that impairs regulation of C4b. These findings constitute, to our knowledge, the first genetic defects associated with preeclampsia in SLE and/or APL Ab. We confirmed the association of hypomorphic variants of MCP and CFI in a cohort of non-autoimmune preeclampsia patients in which five of 59 were heterozygous for mutations.

Conclusion

The presence of risk variants in complement regulatory proteins in patients with SLE and/or APL Ab who develop preeclampsia, as well as in preeclampsia patients lacking autoimmune disease, links complement activation to disease pathogenesis and suggests new targets for treatment of this important public health problem.

Study Registration

ClinicalTrials.gov NCT00198068

Please see later in the article for the Editors' Summary

Editors' Summary

Background

Most pregnancies culminate in the birth of a healthy baby but, sadly, about a quarter of women lose their babies during pregnancy. A common pregnancy-related medical problem that threatens the life of both baby and mother is preeclampsia. Mild and severe preeclampsia affects up to 10% and 1%–2% of pregnancies, respectively. Preeclampsia occurs because of a problem with the function of the placenta, the organ that transfers nutrients and oxygen from mother to baby and removes waste products from the baby. Although preeclampsia begins early in pregnancy, it is diagnosed by the onset of high blood pressure (hypertension) and the appearance of protein in the urine (proteinuria) after 20 weeks of pregnancy. Other warning signs include headaches and swelling of the hands and face. The only cure for preeclampsia is delivery, and labor is usually induced early to prevent eclampsia (seizures), stroke, liver and kidney failure, and breathing and blood vessel problems developing in the mother. Although delivery before 37 weeks of pregnancy is not generally recommended, in cases of preeclampsia it may be too dangerous for both the baby and the mother to allow the pregnancy to continue. Unfortunately when severe preeclampsia occurs in the second trimester, babies weighing only 500 grams may be delivered and they may not survive.

Why Was This Study Done?

Because the exact cause of preeclampsia is unknown, it is difficult to develop treatments for the condition or to find ways to prevent it. Many experts think that immune system problems—in particular, perturbations in complement activation—may be involved in preeclampsia. The complement system is a set of blood proteins that attacks invading bacteria and viruses. The activation of complement proteins is usually tightly regulated (overactivation of the complement system causes tissue damage) and, because preeclampsia may run in families, one hypothesis is that mutations (genetic changes) in complement regulatory proteins might predispose women to preeclampsia. In this study, the researchers test this hypothesis by sequencing genes encoding complement regulatory proteins in pregnant women with the autoimmune diseases systemic lupus erythematosus (SLE) and/or antiphospholipid antibodies (APL Ab) who developed preeclampsia. In autoimmune diseases, the immune system attacks healthy human cells instead of harmful invaders. Both SLE and APL Ab are characterized by complement-mediated tissue injury and are associated with an increased risk of preeclampsia and miscarriage.

What Did the Researchers Do and Find?

Two hundred fifty women with SLE and/or APL Ab were enrolled into the PROMISSE study (a multi-center observational study to identify predictors of pregnancy outcome in women with SLE and/or APL Ab) when they were 12 weeks pregnant and followed through pregnancy. Thirty patients developed preeclampsia during the study and ten more had had preeclampsia during a previous pregnancy. The researchers sequenced the genes for complement regulatory proteins: membrane cofactor protein (MCP), factor I, and factor H in these 40 patients. Seven women (18%) had mutations in one copy of one of these genes (there are two copies of most genes in human cells). Five mutations were alterations in MCP or factor I that are gene variants that increase the risk of hemolytic uremic syndrome, a disease characterized by blood vessel damage. The sixth mutation was a new MCP mutation that impaired MCP's ability to regulate complement component C4b. The final mutation was a factor H mutation that did not have any obvious functional effect. No mutations in complement regulatory proteins were found in 34 matched participants in PROMISSE without preeclampsia but, among a group of non-autoimmune women who developed preeclampsia during pregnancy, 10% had mutations in MCP or factor I.

What Do These Findings Mean?

These findings identify MCP and factor I mutations as genetic defects associated with preeclampsia in pregnant women with SLE and/or APL Ab. Importantly, they also reveal an association between similar mutations and preeclampsia in women without any underlying autoimmune disease. Taken together with evidence from previous animal experiments, these findings suggest that dysregulation of complement activation is involved in the development of preeclampsia. Although further studies are needed to confirm and extend these findings, these results suggest that proteins involved in the regulation of complement activation could be new targets for the treatment of preeclampsia and raise the possibility that tests could be developed to identify women at risk of developing preeclampsia.

Additional Information

Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001013.

Tommy's, a UK charity that funds scientific research into the causes and prevention of miscarriage, premature birth, and stillbirth, has information on preeclampsia

The March of Dimes Foundation, a nonprofit organization for pregnancy and baby health, has information on preeclampsia

The UK National Health Services Choices website also has information about preeclampsia

Wikipedia has pages on the complement system, on autoimmune disease, and on preeclampsia (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)

More information on the PROMISSE study is available

Background

Mutations in complement factor H (CFH), factor I (CFI), factor B (CFB), thrombomodulin (THBD), C3 and membrane cofactor protein (MCP), and autoantibodies against factor H (αFH) with or without a homozygous deletion in CFH-related protein 1 and 3 (∆CFHR1/3) predispose development of atypical hemolytic uremic syndrome (aHUS).

Methods

Different mutations in genes encoding complement proteins in 45 pediatric aHUS patients were retrospectively linked with clinical features, treatment, and outcome.

Results

In 47% of the study participants, potentially pathogenic genetic anomalies were found (5xCFH, 4xMCP, and 4xC3, 3xCFI, 2xCFB, 6xαFH, of which five had ∆CFHR1/3); four patients carried combined genetic defects or a mutation, together with αFH. In the majority (87%), disease onset was preceeded by a triggering event; in 25% of cases diarrhea was the presenting symptom. More than 50% had normal serum C3 levels at presentation. Relapses were seen in half of the patients, and there was renal graft failure in all except one case following transplant.

Conclusions

Performing adequate DNA analysis is essential for treatment and positive outcome in children with aHUS. The impact of intensive initial therapy and renal replacement therapy, as well as the high risk of recurrence of aHUS in renal transplant, warrants further understanding of the pathogenesis, which will lead to better treatment options.

Spirochetes belonging to the Borrelia burgdorferi sensu lato complex differ in resistance to complement-mediated killing by human serum. Here, we characterize complement sensitivity of a panel of B. lusitaniae isolates derived from ticks collected in Germany and Portugal as well as one patient-derived isolate, PoHL. All isolates are highly susceptible to complement-mediated lysis in human serum and activate complement predominantly by the alternative pathway, leading to an increased deposition of complement components C3, C6, and the terminal complement complex. Interestingly, serum-sensitive B. lusitaniae isolates were able to bind immune regulator factor H (CFH), and some strains also bound CFH-related protein 1 (CFHR1) and CFHR2. Moreover, CFH bound to the surface of B. lusitaniae was inefficient in mediating C3b conversion. Furthermore, the identification and characterization of a potential CFH-binding protein, OspE, revealed that this molecule possesses a significantly reduced binding capacity for CFH compared to that of CFH-binding OspE paralogs expressed by various serum-resistant Borrelia species. This finding suggests that a reduced binding capability of CFH is associated with an increased serum sensitivity of B. lusitaniae to human complement.

Background

Age-related maculopathy (ARM) is a common cause of visual impairment in the elderly populations of industrialized countries and significantly affects the quality of life of those suffering from the disease. Variants within two genes, the complement factor H (CFH) and the poorly characterized LOC387715 (ARMS2), are widely recognized as ARM risk factors. CFH is important in regulation of the alternative complement pathway suggesting this pathway is involved in ARM pathogenesis. Two other complement pathway genes, the closely linked complement component receptor (C2) and complement factor B (CFB), were recently shown to harbor variants associated with ARM.

Methods/Principal Findings

We investigated two SNPs in C2 and two in CFB in independent case-control and family cohorts of white subjects and found rs547154, an intronic SNP in C2, to be significantly associated with ARM in both our case-control (P-value 0.00007) and family data (P-value 0.00001). Logistic regression analysis suggested that accounting for the effect at this locus significantly (P-value 0.002) improves the fit of a genetic risk model of CFH and LOC387715 effects only. Modeling with the generalized multifactor dimensionality reduction method showed that adding C2 to the two-factor model of CFH and LOC387715 increases the sensitivity (from 63% to 73%). However, the balanced accuracy increases only from 71% to 72%, and the specificity decreases from 80% to 72%.

Conclusions/Significance

C2/CFB significantly influences AMD susceptibility and although accounting for effects at this locus does not dramatically increase the overall accuracy of the genetic risk model, the improvement over the CFH-LOC387715 model is statistically significant.

Objective

A haplotype of the interferon regulatory factor 5 (IRF5) gene has been associated with the risk of developing systemic lupus erythematosus (SLE), and our previous studies have demonstrated that high levels of serum interferon-α (IFNα) activity are a heritable risk factor for SLE. The aim of this study was to determine whether the IRF5 SLE risk haplotype mediates the risk of SLE by predisposing patients to the development of high levels of serum IFNα activity.

Methods

IFNα levels in 199 SLE patients of European and Hispanic ancestry were measured with a sensitive functional reporter cell assay. The rs2004640, rs3807306, rs10488631, and rs2280714 single-nucleotide polymorphisms (SNPs) in IRF5 were genotyped in these patients. Haplotypes were categorized as SLE risk, neutral, or protective based on published data.

Results

SLE patients with risk/risk and risk/neutral IRF5 genotypes had higher serum IFNα activity than did those with protective/protective and neutral/protective genotypes (P = 0.025). This differential effect of IRF5 genotype on serum IFNα levels was driven largely by SLE patients who were positive for either anti–RNA binding protein (anti-RBP) or anti–double-stranded DNA (anti-dsDNA) autoantibodies (P = 0.012 for risk/risk or risk/neutral versus protective/protective or neutral/protective). The rs3807306 genotype was independently associated with high serum IFNα in this autoantibody group. We found no difference in IFNα activity according to IRF5 genotype in patients lacking either type of autoantibody or in patients positive for both classes of autoantibody.

Conclusion

The IRF5 SLE risk haplotype is associated with higher serum IFNα activity in SLE patients, and this effect is most prominent in patients positive for either anti-RBP or anti-dsDNA autoantibodies. This study demonstrates the biologic relevance of the SLE risk haplotype of IRF5 at the protein level.

Atypical HUS (aHUS) is a severe renal disorder that is associated with mutations in the genes encoding proteins of the complement alternative pathway. Previously, we identified pathogenic variations in genes encoding complement regulators (CFH, CFI, and MCP) in our aHUS cohort. In this study, we screened for mutations in the alternative pathway regulator CFHR5 in 65 aHUS patients by means of PCR on genomic DNA and sequence analysis. Potential pathogenicity of genetic alterations was determined by published data on CFHR5 variants, evolutionary conservation, and in silico mutation prediction programs. Detection of serum CFHR5 was performed by western blot analysis and ELISA.

A potentially pathogenic sequence variation was found in CFHR5 in three patients (4.6%). All variations were located in SCRs that might be involved in binding to C3b, heparin, or CRP. The identified CFHR5 mutations require functional studies to determine their relevance to aHUS, but they might be candidates for an altered genetic profile predisposing to the disease.

Purpose

Variations in the complement factor H (CFH) gene are tightly associated with age-related macular degeneration (AMD) across diverse populations. Of the many nonsynonymous coding variants in CFH, two are most strongly associated with increased risk of AMD: isoleucine 62 to valine (I62V) and tyrosine 402 to histidine (Y402H). Detection of these variations in a patient’s blood is important for a risk assessment of AMD and disease prognosis. However, traditional methods of genetic analysis cannot be used for measuring CFH allotypes in some sources of human plasma and other biological fluids not containing DNA. The purpose was to develop a protein-based method of detecting CFH allotypes.

Methods

A combination of a single-step affinity enrichment of CFH, gel separation, and mass spectrometry identification of the CFH peptides spanning amino acids at positions 62 and 402 was used to identify individual CFH allotypes.

Results

The CFH isoforms V62, I62, H402, and Y402 were reliably detected based on identification of tryptic peptides with masses of 1148.59 Da, 1162.60 Da, 2031.88 Da, and 2057.88 Da, respectively, using MALDI-TOF-TOF. The presence or absence pattern of these peptides in mass spectra of different CFH samples robustly correlated with all nine genotypes of CFH, as a result of variations at positions 62 and 402.

Conclusions

A rapid and sensitive method has been developed for detection of V62, I62, H402, and Y402 variants of CFH in human plasma samples using mass spectrometry. This method can be used in clinical laboratories equipped with a basic inexpensive mass spectrometer capable of performing peptide fingerprinting.

Acquisition of complement regulator factor H (CFH) and factor H-like protein 1 (CFHL1) from human serum enables Borrelia spielmanii, one of the etiological agents of Lyme disease, to evade complement-mediated killing by the human host. Up to three distinct complement regulator-acquiring surface proteins (CRASPs) may be expressed by serum-resistant B. spielmanii, each exhibiting an affinity for CFH and/or CFHL1. Here, we describe the functional characterization of the 15-kDa CRASPs of B. spielmanii, members of the polymorphic Erp (OspE/F-related) protein family, that bind two distinct host complement regulators, CFH and factor H-related protein 1 (CFHR1), but not CFHL1. CFH bound to the B. spielmanii CRASPs maintained cofactor activity for factor I-mediated C3b inactivation. Three naturally occurring alleles of this protein bound CFH and CFHR1 while a fourth natural allele could not. Comparative sequence analysis of these protein alleles identified a single amino acid, histidine-79, as playing a significant role in CFH/CFHR1 binding, with substitution by an arginine completely abrogating ligand binding. The mutation of His-79 to Arg did not inhibit binding of plasminogen, another known ligand of this group of borrelial outer-surface proteins.

Background

Functional polymorphisms of the solute carrier family 22, member 4 (SLC22A4), runt related transcription factor 1 (RUNX1) and small ubiquitin‐like modifier 4 (SUMO4) genes have been shown to be associated with several autoimmune diseases.

Objective

To test the possible role of these variants in susceptibility to or severity of systemic lupus erythematosus (SLE), on the basis that common genetic bases are shared by autoimmune disorders.

Methods

597 SLE patients and 987 healthy controls of white Spanish origin were studied. Two additional cohorts of 228 SLE patients from Sweden and 122 SLE patients from Colombia were included. A case–control association study was carried out with six single nucleotide polymorphisms (SNP) spanning the SLC22A4 gene, one SNP in RUNX1 gene, and one additional SNP in SUM04 gene.

Results

No significant differences were observed between SLE patients and healthy controls when comparing the distribution of the genotypes or alleles of any of the SLC22A4, RUNX1, or SUMO4 polymorphisms tested. Significant differences were found in the distribution of the SUMO4 genotypes and alleles among SLE patients with and without nephritis, but after multiple testing correction, the significance of the association was lost. The association of SUMO4 with nephritis could not be verified in two independent SLE cohorts from Sweden and Colombia.

Conclusions

These results suggest that the SLC22A4, RUNX1, and SUMO4 polymorphisms analysed do not play a role in the susceptibility to or severity of SLE.

Background:

Two biologically related factors, complement factor H (CFH) and C-reactive protein (CRP) have been associated with age-related macular degeneration (AMD). The Y402H variant of CFH is located within the binding site of CFH for CRP. Although plasma CRP levels have been related to AMD, and plasma CRP levels are partly determined by genetic variation, there is no information on whether genetic variants in CRP are associated with AMD.

Methods:

We performed a prospective analysis among 111 men who eventually developed AMD and 401 men who remained free of AMD, all participants in the Physicians’ Health Study. We determined genotypes for the common T>C single nucleotide polymorphism (SNP) in exon 9 of CFH (rs1061170; protein Y402H) as well as seven previously described CRP SNPs (rs3093059, rs2794521, rs3091244, rs1417938, rs1800947, rs1130864, rs1205). We used logistic regression analysis to evaluate individual SNPs as well as six CRP haplotypes for association with AMD.

Results:

The high-risk C allele of CFH was present in 45% of cases and 34% of controls. We observed an odds ratio for AMD of 1.46 (95% CI=1.05-2.04) for TC heterozygotes and 2.13 (95% CI=1.10-4.16) for CC homozygotes assuming a multiplicative (log-additive) model, and calculated an attributable fraction of 25% (95% CI=1% to 44%). For CRP, single-marker or haplotype-based analysis failed to reveal any significant associations with risk of AMD.

Conclusion:

These prospective data confirm an association between the Y402H variant of CFH and risk of AMD. In contrast, although biologically plausible, genetic variation in CRP does not appear to be associated with risk of AMD. Further prospective study of larger numbers of subjects is needed to substantiate available information on the genetic epidemiology of AMD.

The complement system is a critical component of innate immunity that requires regulation to avoid inappropriate activation. This regulation is provided by many proteins, including complement factor H (CFH), a critical regulator of the alternative pathway of complement activation. Given its regulatory function, mutations in CFH have been implicated in diseases such as age-related macular degeneration and membranoproliferative glomerulonephritis, and central nervous system diseases such as Alzheimer’s disease, Parkinson’s disease, and a demyelinating murine model, experimental autoimmune encephalomyelitis (EAE). There have been few investigations on the transcriptional regulation of CFH in the brain and CNS. Our studies show that CFH mRNA is present in several CNS cell types. The murine CFH (mCFH) promoter was cloned and examined through truncation constructs and we show that specific regions throughout the promoter contain enhancers and repressors that are positively regulated by inflammatory cytokines in astrocytes. Database mining of these regions indicated transcription factor binding sites conserved between different species, which led to the investigation of specific transcription factor binding interactions in a 241 base pair (bp) region at −416 bp to −175 bp that showed the strongest activity. Through supershift analysis it was determined that c-Jun and c-Fos interact with the CFH promoter in astrocytes in this region. These results suggest a relationship between cell cycle and complement regulation, and how these transcription factors and CFH affect disease will be a valuable area of investigation.